.■■ltfii:ii[Liiitut:::.i!lJl •f Demonstrating phagocytosis of various bacteria as observed in the determina- tion of the opsonic index. 1, Staphylococcus; 2, Micrococcus candicans; 3, Strepto- coccus pyogenes; 4, Colon bacillus; 5, Gonococcus; 6, Tubercle bacillus. {See p. 234.) APPLIED IMMUNOLOGY THE PRACTICAL APPLICATION OF SERA AND BACTERINS PROPHYLACTICALLY, DLVGNOSTICALLY AND THERAPEUTICALLY WITH AN APPENDIX ON SERUM TREATMENT OF HEMORRHAGE, ORGANOTHERAPY AND CHEMOTHERAPY BY B. A. THOMAS, A.M., M.D. PROFESSOR OF GENITO-URINART SURGERY IN THE POLYCLINIC HOSPITAL AND COLLEGE FOR GRADUATES IN MEDICINE ; INSTRUCTOR IN SURGERY IN THE UNIVEK81TT OF PENNSYLVANIA ; ASSOCIATE IN THE WILLIAM PEPPER LABORATORY OF CLINICAL MEDICINE AND R. H. IVY, M.D., D.D.S. ASSISTANT INSTRUCTOR IN SURGERY IN THE UNIVERSITY OP PENNSYLVANIA ; INSTRUC- TOR IN GENITO-URINARY SURGERY IN THE POLYCLINIC HOSPITAL AND COLLEGE FOR GRADUATES IN MEDICINE PHILADELPHIA 5 COLORED INSERTS AND 68 ILLUSTRATIONS IN TEXT PHILADELPHIA AND LONDON B. LIPPINCOTT COMPANY T5 BiOtOGX Copyright, 1915 By J. B. LiPPiNcoTT Company Printed by J. B. Lippincott Company The Washington Square Press, Philadelphia, U, S. A. To ALFRED STENGEL. M.D. PROFESSOR OF MEDICINE IN THE UNIVERSITY OF PENNSTLVANIA IN GRATEFUL RECOGNITION OF HIS WISE COUN- SEL AND KINDLY ADVICE THIS VOLUME IS BESPECTFULLY DEDICATED BY THE AUTHOBS Sf)Kf^.^i PREFACE Despite the enormous strides, experimentally and clinically, that have marked the progress in serological and bacteriological research in medicine for the past quarter of a century, leading to results of the highest clinical value diagnostically and therapeutically, three facts stand out in bold relief: First, that the average practitioner's knowledge of biological therapeusis is a " dangerous thing " because he does not " drink deep of the Pierian spring " ; second, insufficient practical instruction is given to students in our medical schools in view of the prevalent practice of this mode of diag- nosis and treatment; third, authors have sadly neg- lected to give a concise presentation of immunology in its entirety for the practical grasp and comprehension of students and practitioners. The vast majority of standard works on the subject of " immunity " are exhaustive treatises on the experimental and theoreti- cal phases of the subject and are often unintelligible and of little value to the average practicing physician. The result of the above has been that pharmaceuti- cal firms have assumed the function of the Department of Therapeutics in our medical colleges, not always impartially and to the best interests of medical science. vi PREFACE Obviously, licentiates to practice medicine should re- ceive instruction of a definite and practical nature in this important branch of modern medicine, or immu- nology should receive a place in the classification of specialties, a fact which the importance of the subject may command. In this book it has been the aim of the authors purposely to omit most of the experimental research and to present theories only in so far as they may assist in a more thorough comprehension of biological prophylaxis, diagnosis and therapeusis. The primary object has been to crystallize and de- tail the practical phases of serum and bacterin applica- tions in medicine, thereby enabling the student and general practitioner, with even a slight laboratory ex- perience, to appreciate the significance of, and more competently apply the principles underlying, immu- nology. In order to render the treatise more complete allusion has been made in places to certain allied sub- stances that have been utilized from time to time in attempts at immunization, with a consideration of their merits and demerits. The interest and close association of blood trans- fusion, organotherapy and administration of salvarsan and neosalvarsan to the main subject have prompted the authors to devote an appendix to their discussion. The reader who may have his enthusiasm aroused PREFACE vii and is ambitious to enter more deeply into the realms of immunity is referred to the systems of Kolle and Wassermann, and Kraus and Levaditi, or the works of Ehrlich and Bolduan, and Bordet and Gay on " Studies in Immunity "; Emery on " Immunity and Specific Therapy "; Wright on " Studies on Immuni- zation "; Simon on " Infection and Immunity "; Cit- ron on " Immunity," and Vaughan on " The Relation of Anaphylaxis to Immunity and Disease." B. A. Thomas R. H. Ivy William I*epper Laboratory of Clinical, Medicine, Philadelphia, 1915 CONTENTS CHAPTER I PAOB Introduction 1 Immunity and Immunization — Natural and Acquired Immunity — Active and Passive Immunization — Mechanism of the Production of Immunity — History and Development of Immunology. CHAPTER II Antigens and Antibodies 19 Toxins and Antitoxins — Agglutinins — Precipitins — Lysins — Isocytoly- sins — Opsonins — Antiferments — Auto-antibodies — Allergy and Ana- phylaxis. CHAPTER III Ehrlich's Side-chain Theory 24 CHAPTER IV Anaphylaxis or Hypersusceptibility 27 CHAPTER V Antisera 34 Preparation of Antisera — Indications for Therapeutic Use of Antisera. CHAPTER VI Antitoxic Sera 42 Antidiphtheritic Serum — Antitetanic Serum — Antigonococcic Serum — Antituberculosis Serum — Antidysenteric Serum — Antibotulism Serum — Antiphytotoxic Serum — Antivenin. CHAPTER VII Antibacterial Sera 55 Antistaphylococcic Serum — Antistreptococcic Serum — Antipneumo- coccic Serum — Antigonococcic Serum — Antimeningococcic Serum — Antityphoid Serum — Anticolonic ' Serum — Antidysenteric Serum — Anticholera Serum — Antiplague Serum — Anti-anthrax Serum — Anti- melitensic Serum. CHAPTER VIII Miscellaneous Sera and Extracts 64 Antirabic Serum — Antileprosy Serum — Antityphoid Extract of Jez — Leucocytic Extract — Antiferment — Anticarcinomatous Extracts — Pyocyanase — Antithyroid Serum and Extracts — Spangler's Crotalin — Phylacogens. CHAPTER IX Agglutinins 72 The Widal Phenomenon and Other Agglutination Reactions. ix X CONTENTS CHAPTER X Precipitins 82 Significance and Application of Precipitins — Technic of Reaction — Specific Identification of Blood and Other Proteins. CHAPTER XI Ltsins 86 Bacteriolysins and Cytolysins (Hsemolysins). CHAPTER XII Fixation of Complement 90 Principles of the Reaction — Bordet-Gengou Phenomenon — Wasser- mann-Neisser-Bruck Modification — Technic of the Wassermann Re- action in the Diagnosis of Syphilis — Modifications of the Wassermann Reaction — Hecht-Weinberg Modification — Clinical Application of the Wassermann Reaction — Effects of Treatment on the Wassermann Reaction. CHAPTER XIII Fixation of Complement (Continued) 141 Gonococcus Complement-fixation Test — Serum Diagnosis of Echino- coccus Disease — Complement-fixation Reaction in Typhoid Fever — Complement-fixation Reaction in Tuberculosis — The Complement- fixation Reaction as Applied in Protein Differentiation (Neisser-Sachs Reaction). CHAPTER XIV Miscellaneous Biochemical Reactions 157 Abderhalden's Biological Test for Pregnancy — Sero-enzyme Test for Syphilis — Abderhalden-Fauser Reaction in Mental Diseases — Meio- stagmin Reaction — Epiphanin Reaction. CHAPTER XV SPEaFic Bacterial Cutaneous Reactions 167 Allergic Phenomena — Tuberculin Tests — Luetin, Gonorrhoea! and Typhoid Tests — Schick's Diphtheria Toxin Skin Reaction. CHAPTER XVI Tuberculin Therapy 193 Prophylaxis — Therapeutic Administration of Tuberculin — Available Preparations — Modes of Administration and Dosage — Control of Tuberculin Treatment — Limitations and Contra-indications — Indica- tions and Results. CHAPTER XVII Phagocytosis 211 CHAPTER XVIII Recovery from Bacterial Infections 21S CONTENTS xi CHAPTER XIX Bacterial Inoculation 216 Principles Underlying Inoculation Therapy — Preparation of Bacterins — Autogenous versus Heterogeneous Preparations — Clinical Symptoms versus Opsonic Index in Control of Treatment. CHAPTER XX The Opsonic Index 228 Definition of Opsonins and the Opsonic Index — Technic of Determina- tion of the Opsonic Index — Interpretation, Value and Limitations of the Opsonic Index. CHAPTER XXI Practical Application of Bacterial Inoculation in Medicine, Prophylactically and Therapeutically 242 General Considerations — Induced Auto-inoculation — Duration of Ac- tive Immunity — Modes and Technic of Administration of Bacterins — Dosage — Contra-indications, Limitations and Causes of Failure in Bacterin Therapy — Application and Results of Bacterial Inoculations in Special Diseases — Diseases of the Skin and Soft Parts — Diseases of the Genito-urinary System — Diseases of Bones and Joints — Diseases of the Eye, Ear, Nose and Throat — Diseases of the Lungs — Diseases of the Alimentary System — Diseases of the Cardiovascular, Lymphatic and Nervous Systems, also Other Acute Specific Fevers — Malignant Neoplasmata — Yeast and Sour Milk. APPENDIX Part A. Serum Treatment of Hemorrhage 306 Normal Fresh Serum — Precipitated Horse Serum — Transfusion of Blood. Part B. Organotherapy 312 Thyroid Gland — Adrenal Gland — Pituitary Body — Ovary — Corpus Luteum — Thymus Gland. Part C. Chemotherapy 318 Administration of Salvarsan and Neosalvarsan, Intravenously, Intra- muscularly and Intraspinally — Autosalvarsanized and Artificially Sal- varsanized Serum. ILLUSTRATIONS PLATES PAGE I. Demonstrating Phagocytosis of Various Bacteria as Observed in the Determination of the Opsonic Index Frontispiece II Graphic Portrayal of the "Wassermann Reaction/' Demonstrating the Results in the Case to be Tested, the Positive Control, and the Negative Control 125 III. Von Pirquet's Cutaneous Tuberculin Test (Positive Reaction) . . 182 IV. Luetin Cutaneous Reaction, Demonstrating the Papular Charac- ter of the Reaction on an Erythematous, Indurated Base 187 V. Gonorrhoeal Allergic Reaction, Demonstrating the Papulo-erythe- matous Cutaneous Response on the Third Day after the Intra- dermic Injection of One Cubic Millimetre of a Killed Polyvalent Suspension of Gonococci 189 FIGURES FIG. 1. Diagrammatic Representation of Structure of Different Antibodies 25 2. Illustrative of Opsonic Curve, Showing Immunity of Rabbit 39 3. Illustrative of Inoculations and Curve of Opsonic Indices Demon- strating Immunity of Ram 40 4. Capillary Teat Pipette for Removal of Senun from Clotted Speci- men of Blood 74 5. Widal's Test, Positive 78 6. Widal's Test, Negative 78 7. Method of Obtaining Blood from Sheep's Ear 105 8. Showing Method of Intravenous Injection or Immunization of Rabbit 107 9. Demonstrating Method of Obtaining Complement by Bleeding to Death an Anaesthetized Guinea-pig 109 10. Titration of Antigen 113 11. Showing Method of Collecting Blood from Vein of Arm with Keidel's Vacuum Ampoule 114 12. Showing Authors' Method of Obtaining Blood for Complement- fixation Reactions 115 13. Titration of Complement 119 14. Showing Arrangement of Tubes in Performance of Wassermann Reaction on One Unknown Case, with Positive and Negative Controls 126 ziii xiv ILLUSTRATIONS 15. Tuberculous Mastoiditis 177 16. Bilateral Tuberculous Epididymitis 178 17. All-glass Tuberculin Syringe, Graduated into Hundredths of a Cubic Centimetre 179 18. Chronic Pulmonary Tuberculosis 207 19. Tuberculous Coxitis 208 20. Electrical Mechanical Shaker 219 21. Various Forms of Containers for Storage of Bacterins 223 22. Capillary Glass Capsules for Collection of Specimens of Blood. . . 231 23. Showing the Collection of Blood in Sodium Citrate Saline Solution 231 24. Blood after Centrifugation in Decalcifying Medium 231 25. Electric Centrifuge 231 26. Various Ingredients Necessary for the Determination of the Opsonic Index 231 27. Washing the Culture of the Given Bacterium from the Culture Medium 232 28. Opsonizing Capillary Pipettes 233 29. Opsonizer or Thermostat 233 30. Illustrating the Construction of Kuhnhardt's Spreader 233 31. Kuhnhardt's Spreader Property Held for Preparation of a Satis- factory Smear 234 32. Analysis of Curve of Opsonic Indices 236 33. Case of Gonorrhoea! Arthritis of Knee 238 34. Pneumonia 239 35. Sho^dng Effect of Variable Concentrations of Bacterial Suspension on Determinations of Opsonic Index 240 36. Case of Gonococcal Arthritis 245 37. Case of Tuberculous Cervical Lymphadenitis 246 38. All-glass Hypodermic Syringe 249 39. Case of Long-standing and Obstinate Pustular Acne Vulgaris 260 40. Furunculosis of Nostril 261 41. Carbuncle of Neck 262 42. Case of Typhoid Fever 263 43. Burns One-third to One-half Body Surface; Multiple Subcutaneous Abscesses 263 44. Recurrent Erysipelas 264 45. Tonsiintis, Peritonsillitis and Toxic Arthritis 265 46. Temperature. Subdiaphragmatic Abscess Drained per Laparotomy 266 47. Pelvic Abscess with Recto-urethro-vaginal Fistulse 267 48. Pyonephrosis 272 49. Reno-lumbar Fistula Following Nephrohthotomy Complicated by Pyonephrosis 273 50. Pyelitis and Cystitis 274 ILLUSTRATIONS xv 51. Cystitis and Toxic Neuritis 275 52. Chronic Prostatitis 277 53. Typhoid Fever Complicated by Epididymitis 278 54. Acute Osteomyelitis of Tibia Followed by Septicaemia 281 55. Corneal Ulcer with Hypopyon 285 56. Cultures both from Ethmoidal Sinuses and Bronchial Expectoration 287 57. Illustrating Typhoid Fever Rates in United States Army 296 58. Antityphoid Inoculation or Immunization 297 59. Site for Deep Intramuscular Injection 330 60. Position of Patient for Intravenous Injection of Salvarsan 332 61. Apparatus Ready for Preparation of Salvarsan or Neosalvarsan . . 333 62. Thomas' Salvarsan and Neosalvarsan Outfit 333 63. Water Still as Used in Authors' Offices 333 64. Illustrating Method of Eliminating Air from Tubing 335 65. Thomas' Salvarsan and Neosalvarsan Burette 335 66. Showing Position of Patient for Spinal Puncture 341 67. Lumbar Puncture with Strauss Needle 341 68. Intraspinal Administration of Serum, Using Syringe 341 APPLIED IMMUNOLOGY INTRODUCTION IMMUNITY AND IMMUNIZATION— NATURAL AND AC- QUIRED IMMUNITY— ACTIVE AND PASSIVE IMMUNIZA- TION—MECHANISM OF THE PRODUCTION OF IMMU- NITY—HISTORY AND DEVELOPMENT OF IMMUNOLOGY Definition of Immunity, — Immunity is the resis- tance manifested by man and various animal species to infectious microorganisms or other foreign proteins. It is influenced by numerous factors, as changed en- vironment, physical condition of the animal, species, idiosyncrasies, virulence of the prevalent microbe, etc. Conversely, the absence of this resistance implies susceptibility. Occasionally, hypersusceptibility to certain proteins is observed and to this state of supersensitiveness Richet has applied the term " ana- phylaxis " (see Chapter IV). Two kinds of immunity are recognized, natural and acquired. Natural Immunity. — The natural or spontaneous resistance of the animal organism to disease is only relative, never absolute. The ability of animals to 1 2- •:*•'•'• '• APPLIED IMMUNOLOGY ward off disease varies with different species and among individuals of the same family. Under nor- mal conditions, an animal may be protected indefi- nitely from infection. Nevertheless, if his vital resis- tance be permitted to fall or he be exposed to a viru- lent infection, his defences may crumble instantly and disease be contracted. On the other hand, the natural immunity of certain species to infection is remarkable ; the negro to yellow fever and most of the lower ani- mals to the venereal diseases. In this connection allusion should be made to local immunity. By this we mean a natural state of certain organs or tissues, prevalent from birth, due to " in- fective tolerance." For example, the mouth and an- terior urethra normally harbor many different patho- genic bacteria without ill effect, owing to life-long local tolerance with resultant immunity. Introduce some of these germs into the synovial membrane of a joint or into the peritoneal cavity and a virulent in- fection results. Again, the intestinal tract tolerates colon bacilli normally in numbers, which, if access be gained to the urinary tract, may precipitate a grave pathological process. Infections common in warm-blooded are rare among cold-blooded animals and vice versa. Verte- brates and invertebrates are not subject to similar in- fections. Field mice are susceptible to glanders. INTRODUCTION 3 while house mice are immune. Tuberculosis is more prevalent among Jersey than Holstein cattle. Birds and reptiles are not necessarily subject to the same diseases that victimize man. Thus the problem of natural immunity presents many interesting phases, but still lacks an absolutely satisfactory solution. Explanations for the existence of natural immu- nity are founded on the protection afforded: first, by the external and internal surfaces of the body ; second, by inflammatory processes ; third, by natural antibac- terial and antitoxic substances, and fourth, by the natural metabolic activity or vital resistance of the organism. Bacteria are unable to penetrate the unbroken cu- taneous epithelium, but may reach the subcutaneous tis- sues through abrasions although microscopical in size, through sudoriferous and sebaceous ducts and glands although their secretions are mildly antibacterial, and through the hair follicles. In the subcutaneous tissues, bacterial encroachment is further combated by cellular proliferation and extravasated plasma containing serum, fibrinogen, and leucocytes. Bacteria entering the nasal and oral passages encounter in the mucus and saliva both physical and chemical barriers. The gastric, biliary and pancreatic juices exert antibac- terial and neutralizing functions. Inflammation, a manifestation of tissue injury, is 4 APPLIED IMMUNOLOGY a process designed to resist infection, if that be its cause, and proves successful for mechanical and im- munological reasons, if the reactive forces of the in- dividual be capable. The invading bacteria develop a condition of positive chemotaxis and leucocytes swarm to the battle-field. Already the bacteria have stimulated the tissue cells to the production of specific substances or antibodies and many bacteria are killed by lysis (see Chapter XI) ; the remainder, by virtue of sensitization with their specific antibodies (opsonins of Chapter XX), are ingested and destroyed by the phagocytes (see Chapter XVII). While the mortal conflict between bacteria and bacteriolysins and phag- ocytes is being waged, inflammatory exudate and proliferation of fi:xed tissue cells occur and raise bar- riers to the further extension of the morbid process. Coincidently, the antibodies are formed in excess and impregnate the blood-serum, establishing the phenom- enon of immunity. There is little evidence pointing to the presence of natural antitoxic substances in animals, although they have been claimed to occur to a limited extent in horses and more abundantly in children and adults. Natu- ral antibacterial substances are more extensively de- monstrable in the tissue fluids and blood-serum. Buchner has given the name " alexins " to these nor- mal bacteriolytic substances. They appear to be INTRODUCTION 5 identical with opsonins and may be increased by active immunization. Acquired Immunity, — Acquired immunity is that condition of protection against disease, resulting from recovery from infection or arising by virtue of arti- ficial inoculation. It may be produced in two ways, namely, by active or passive immunization. Active immunization signifies the process by which the bodily cells of an animal are sti7nulated by a toocin or foreign body {antigen) to the production of cer- tain other bodies {antibodies) specific against the given foreign substance (see Chapter II). Thus the animal is actively concerned in the elaboration of its own antibodies, hence the process is termed active. Untreated disease terminates in either one of three ways — death, recovery, or chronicity. If the viru- lence of the infection is great and the dose large or overwhelming, the animal succumbs, especially if his vital resistance be slight. If the infection is relatively avirulent, even though the dose be large, recovery al- though protracted may take place provided the ani- mal's resistance is great. If the degree of virulence of the infection be merely the average, but the vital resis- tance of the animal be only mediocre, its cellular ac- tivity or infective dose may prove inadequate for the normal generation of sufficient specific antibodies to insure immunity, and chronic invalidism results. Thus 6 APPLIED IMMUNOLOGY in patients suffering from chronic infections, particu- larly, and also in certain acute conditions, the number of specific antibodies may be materially increased and convalescence shortened or recovery insured by artifi- cial inoculation employing homologous bacteria. This result may be achieved by injecting the bacteria with a hypodermic syringe directly into the tissues, or by carefully regulated and graduated auto-inoculation, by manipulations, as massage, hyperemia, etc., of the infected area. The discovery of the feasibility of pro- ducing auto-inoculation in patients afflicted with a localized infection was one of great magnitude and vital consideration in the study and correct interpreta- tion of infection and immunity, particularly in connec- tion with bacterin therapy. By its utilization difficult diagnoses have been established, successful treatment conducted in selected cases and in constitutional dis- eases, clinical interpretations correctly deduced, and proper treatment applied (see Chapter XXI) . It is evident, therefore, that when an individual recovers from an infectious disease he enjoys for an indefinite time immunity against repeated attacks due to the same infective organism. It can also be readily under- stood that immunity against particular diseases can be conferred by artificial inoculation, using the specific bacteria, viruses, etc. (prophylactic inoculation or vaccination). INTRODUCTION 7 The methods by which immunity may be acquired through active immunization are as follows, enu- merated in their order of efficiency: 1. Inoculation with virulent living bacteria, typi- fied by the subcutaneous injection of spirilla of Asiatic cholera. 2. Inoculation with attenuated or relatively aviru- lent microorganisms. This method is exemplified by vaccination against smallpox and antirabic inocula- tion. 3. Inoculation with dead bacteria. This embraces the prevalent practice of bacterin as prophylactic (vaccine) therapy, notable examples of which include bacterial inoculations against typhoid fever, plague, tuberculosis, furunculosis, carbunculosis, etc. A higher immunity may be produced by supplementing the dead bacterins with inoculations of attenuated and finally living virulent bacteria. 4. Inoculation with the excreted bacterial prod- ucts. The injection of the horse with the tetanus toxin for the production of antitoxin is the familiar example of this method. 5. Inoculation with the disintegrated products of dead bacteria (autolysates). Little of value has at- tended this procedure and for practical purposes it may be disregarded. 8 APPLIED IMMUNOLOGY Passive immunization signifies the process by which immunity is acquired when artificial antisera are injected into the animal body (see Chapter V) . Thus the inoculated animal plays no part in the production of the antibodies or antitoxin which he receives, and the process is termed passive. Hence the animal is in- jected with the specific cellular products (antitoxin) of another animal previously actively immunized, and by a process of simple neutralization the toxins in the diseased animal are destroyed and immunity con- ferred. In passive immunization the antibodies bear a close chemical combination to the cells. The acquired immunity of passive immunization is of vastly shorter duration than that resulting from active immunization. Mechanism of the Production of Immunity There is a group of foreign chemical substances, conveniently styled antigens, to which the animal body reacts in a definite manner. This group must be dif- ferentiated from and not confounded with the poison group, as the common poisons are not antigens. The group embraces the foreign proteins, including the bacterial proteins, also certain complex and more or less unknown bodies, as bacterial toxins, parenteral proteolytic ferments, enzymes and other animal and vegetable toxic substances. Following the subcutane- ous, intra-abdominal or intravenous injection of an INTRODUCTION 9 appropriate quantity of such an antigen, after a vari- able interval, during which a leucopaenia may exist, leucocytosis supervenes. After an incubation period of a few days to several weeks, it is found that the body fluids, particularly the blood-serum, react in a novel manner, that is, they possess the property of neutralizing the antigen. Although these are known biological facts, explanations as to when and how these antibodies are formed and when and how they unite with their specific antigens are pure speculation. Nevertheless, immunologists generally have accepted the ingenious so-called side-chain theory of Ehrlich, which conception, although regarded as too visionary by some, has sufficed in a remarkable manner as a working basis for almost all the great problems and discoveries in serology (see Chapter III). On the other hand, JNIetchnikofF's doctrine of phagocytosis (Chapter XVII), not in its original simplicity, plays a not unimportant role in the mechanism particularly of active immunization in view of the researches of the opsonic school. The efficiency of a polynuclear leuco- cytosis in pneumonia has long been recognized as of momentous prognostic value and undoubtedly is inti- mately concerned in the production of immunity. The bactericidal and cytolytic substances normally present in the blood-serum and body fluids of some animals, and capable of marked increase by antigenic 10 APPLIED IMMUNOLOGY inoculations, are extremely important considerations in immunology ( see Chapter XI ) . Other notable antibodies conspicuous in serologi- cal work are agglutinins (Chapter IX), important in certain bacterial agglutination tests, and precipitins ( Chapter X ) , of value in the identification of bloods in forensic medicine. The relationship of these spe- cific antibodies to the immunity of the body is in doubt. The production of anaphylaxis, hypersuscepti- bility or supersensitiveness to all proteins save gelatin, and its association with immunization contributes a most interesting and important subject and is fully discussed in Chapter IV. History axd Development of Immunology Historically, the first reference to any attempt at protection against disease by the utilization of biologi- cal products was made by Mithradates, who, it is al- leged, took small quantities daily of certain poisons in order to render himself immune. Similarly, the cus- tom of hunters of certain wild tribes to inoculate them- selves systematically with snake venom to safeguard the effects of snake bites is well known. Immunization, however, was not placed on a truly practical basis until the eighteenth century, although the practice had existed in the East a long time pre- viously. Lady JNIontagu, the wife of the British Am- INTRODUCTION 11 bassador to Constantinople, permitted her son to be inoculated against smallpox with the matter from variola pustules and subsequently, in 1721, introduced the method in England. Despite the brilliant results that followed, the practice encountered bitter opposi- tion, owing to the fact that although it protected the inoculated it did not prevent conveyance of the disease in a virulent form to the uninoculated, and was finally prohibited by law. Sixty years later, the attention of Jenner was di- rected to a peculiar disease of the udders of cows, from which the hands of milkmen became infected, render- ing them immune to smallpox. Jenner investigated the subject for a number of years, and in 1796 in- oculated a boy with " cow-pox," after which inocula- tion with smallpox showed him to be immune. Two years later, in 1798, Jenner published his classical re- port, which was soon followed by systematic and uni- versal vaccination against the world's greatest scourge. Although the causative organism in the virus of small- pox has not been discovered, even to this day, Jenner firmly established the doctrine that it is possible to con- fer immunity against an infectious disease by the em- ployment of a modified materies morhi. Almost a century passed before any further not- able advance occurred in immunology. Indeed it re- quired the stimulus of the era of bacteriology to pro- n APPLIED IMMUNOLOGY mote research along this line. Schwann showed the relationship between decomposition of organic bodies and microorganisms. This was supplemented by Pasteur's work on fermentation and the yeast fungus. In 1863 Davaine pointed out that certain bacilli de- scribed in the blood by him thirteen years before were the causative factors in anthrax. Thirteen years later, Koch succeeded in growing these bacilli on an artificial medium in pure culture, and anthrax was reproduced in animals. The next problem was to attenuate the bacteria, that is, preserve their identity and life, at the same time reducing their virulence, so that inoculation would not result fatally. In 1880, Pasteur succeeded in preparing a " vaccine " from attenuated anthrax bacilli and inoculated sheep, thereby rendering them immune. In 1885, after much animal experimentation, Pasteur inoculated the first human subject against rabies. The etiological microorganism of the virus of this disease is still undetermined and the procedure of antirabic inoculation has undergone no material change. Thus far the belief prevailed that immunity oc- curred only as the result of recovery from disease. Salmon and Smith at this time demonstrated that immunization could be produced by the products of INTRODUCTION IS bacterial growth. Supplementing the work of Traube and Gscheidlen, in 1887, Buchner showed the specific- ity of the bactericidal properties of blood-serum. In 1888, Roux and Yersin, and Kitasato dis- covered the toxins respectively of diphtheria and tetanus. In 1890, Behring discovered antitoxin in the serum of animals immunized against the toxin of diphtheria, thus furnishing the first antitoxic serum. In the same year, with the collaboration of Kitasato, immunity to tetanus was conferred on mice from the serum of rabbits inoculated with tetanus toxin. In the following year with Wernicke, he immunized other animals with antidiphtheric serum. Behring, there- fore, discovered the whole principle underlying serum therapy in its relationship to modern therapeusis and serological studies, and became thereby its honored founder. The following year Ehrlich demonstrated anti- bodies in the serum of animals inoculated with vege- table poisons, as ricin, abrin and crotin, and three years later Calmette claimed similar results with snake venom. Although it was recognized, even before Behring' s discovery, that the serum of animals inoculated with certain bacteria possessed a specific anti-infectious effect or protection, it was about this time, 1892, that Metchnikoff called attention to a particular substance, 14 APPLIED IMMUNOLOGY " stimulin," in antibacterial sera, capable of stimulat- ing leucocytes to increased ingestion and destruction of bacteria. JNIetchnikoiF claimed that immunity de- pended upon this property of phagocytosis. It was at this time that Buchner described " alexins," and Bordet " sensibilisators " in sera and their role in the process of immunization. Numerous foreign albuminous substances were utilized for the production of antibodies and conceptions respecting the nature of immunity definitely distinguished two types, active and passive. JMoreover, it was realized that eccotoocic bacteria were adaptable chiefly to pas- sive, and endotoxic bacteria to active, immunization. In 1895, Pfeiffer demonstrated his phenomenon of bacteriolysis, thus dealing a severe blow to INIetchni- koiF's doctrine. While the discussion was still warm over PfeifFer's discovery, in the next year Gruber and Durham directed attention to the diagnostical value of " agglutination " of bacteria in their specific antiserum and Widal described the serodiagnosis of typhoid fever. The following year, Kraus described other antibodies, more or less closely related to bacteriolysins and agglutinins, which he styled " precipitins." The year 1896 is notable in immunological annals as marking the advent of antityphoid inoculation by Wright. Previously, Ferran and Haffkine had em- ployed attenuated living cultures, prophylactically. INTRODUCTION 15 against cholera. Wright's work, however, marked a new epoch, since he demonstrated that antibodies may be produced by inoculation with dead bacteria. The following year, Haffkine immunized himself against the plague by inoculation with a sterilized culture of B, pestis. Up to this time, bacterial inoculations on the human had been employed solely in a prophylactic capacity. Wright enjoys the distinction of being the first to realize that any bacterium responsible for local disease and capable of isolation in pure culture may be employed in the form of a bacterial suspension or bacterin to cure the disease it causes. Seven years later, Wright and Douglas, taking ad- vantage of Leishman's studies on comparative phag- ocytosis, showed that phagoc}i;osis does not occur save in the presence of serum, thus claiming a specific sensi- tization of bacteria by certain substances in the blood- serum to which the name " opsonins " was given. An ingenious and clever laboratory method was devised whereby the measure of the ratio of phagocytability could be determined and this was styled the " opsonic index." The work of Wright attracted universal at- tention and popularized active immunization by bac- terin therapy to an unprecedented and world-wide extent. Synchronously with Wright's studies, the discov- eries and advances in serology were truly remarkable. 16 APPLIED IMMUNOLOGY In 1898, Belfanti and Carbone discovered that the serum of horses immunized with the blood of rabbits was very toxic for these animals. The analogy be- tween these cytotoxins or cytolysins and the specific antibodies formed after bacterial inoculations was im- mediately realized. Experiments by Bordet, Ehrlich and iMorgenroth demonstrated the presence of haemol- ysins in the serum of animals inoculated with red blood-cells, and the interesting and important phe- nomenon of haemolysis or solution of erythrocytes was demonstrated. Almost all tissue cells were employed in research work for the production of their specific antibodies, and to these various heterogeneous elements the name " antigens " was applied. Thus, leucotoxins, spermo- toxins, trichotoxins, syncytiolysins, hepatolysins, ne- phrolysins and neurotoxins were produced and their influence as causative factors in the pathology of cer- tain diseases, as hepatitis, nephritis, etc., became a much mooted subject in scientific research. In 1900, Uhlenhuth appreciated the formation of specific cyto- toxins for carcinoma and sarcoma in patients, and em- ployed such sera therapeutically. The year 1902 is memorable in serology as mark- ing the discovery of the " complement-fixation reac- tion " by Bordet and Gengou. They demonstrated that no haemolysis can occur if the thermolabile ele- INTRODUCTION 17 ment of haemolysin be absorbed or fixed in the interac- tion between antigen and antibody. This discovery proved to be of the greatest importance and practical value and underlies many modern serological clinical tests. Wassermann, in 1906, the year following the dis- covery of the Trepone7na pallidum, put the comple- ment-fixation or deviation reaction of Bordet and Gengou to a practical and successful test for the diag- nosis of syphilis, and the so-called " Wassermann re- action " was the result. Subsequently the principles employed in Wassermann' s technic have been applied to many other infectious diseases, notably gonorrhoea. In 1909, Ascoli, employing Traube's stalagmome- ter, annomiced the " meiostagmin reaction," and de- monstrated the feasibility of differentiating between fluids containing mixtui'es of antigens and antibodies by measui*ement of the drops contained in given vol- umes. The method has been utilized in the diagnosis of typhoid fever, tuberculosis, malignant tumors, foot- and-mouth disease, and for the detection of many lipoidal substances. Finally, reference should be made to certain para- doxical reactions that have been observed in immu- nology during recent years. Instead of the immunity which commonly follows the injection of a foreign protein substance into an animal, occasionally, al- 18 APPLIED IMMUNOLOGY though immune bodies have been demonstrated to be present, the inoculation of heterogeneous substances, without apparent reason, produces a singular effect, in consequence of which the previously treated organ- ism reacts differently from the normal. To this re- action von Pirquet has given the name " allergy." Allergy may be expressed by either a refractory state of the organism, namely, immunity, or by supersensi- tiveness, that is, anaphylaxis. Examples of anaphy- lactic reactions are recognized in " Arthus' phenome- non " in rabbits, " Theobald Smith's phenomenon " in guinea-pigs and "serum disease " in man. Less im- portant, although more practical illustrations are com- monly seen in the cutaneous and ophthalmic tuber- culin reactions of tuberculous individuals, the mallein reaction in glanders, the luetin reaction in syphilis, the intradermic reaction in typhoidal subjects, the sero- diagnosis of cancer, etc. In this connection, the hypothesis of Friedberger that a number of pathological processes are due to the occurrence of albumen anaphylaxis, and the coincident poisons of infections are referable to the influence of the concurrent anaphylotoxin, must receive serious consideration. II ANTIGENS AND ANTIBODIES TOXINS AND ANTITOXINS— AGGLUTININS— PRECIPITINS— LYSINS—ISOCYTOLYSINS— OPSONINS— ANTIFERMENTS— AUTO-ANTIBODIES— ALLERGY AND ANAPHYLAXIS We will now discuss briefly the various defensive properties of the fluids and tissues of the body in re- sisting the invasion of disease-producing substances. The latter are usually termed antigens, while the spe- cific substances produced by the body-cells to neu- tralize and thus render the antigens innocuous are termed antibodies. By the term antigen, therefore, is meant an organic foreign substance, usually protein in nature, the product of animal or vegetable cells, which on being introduced into the body has the prop- erty of stimulating the production by the body-cells of a specific substance or antibody which unites chemi-^jjn^^,,^^^^^^^ cally with it and thus neutralizes it. Antigens may be of various types. They may consist of the soluble products of bacterial growth, of substances bound up in the bodies of the bacteria themselves, of protein and lipoid extracts of animal tissues, etc. These different forms of antigens produce corresponding types of antibodies which differ in their methods of attacking the antigens. Antibodies thus specifically produced, 19 20 APPLIED IMMUNOLOGY as distinguished from those naturally existing in the normal animal, are called immune bodies, and the animal is said to be immune to the particular antigen in question. Various Forms of Antibodies (a) Toxins and Antitoxins, — Toxins are the solu- ble products of bacterial and plant growth, also the secretion of certain reptiles, and in the case of some bacteria are the means through which the deleterious effects of the bacteria are brought about. Antitoxins are specific substances produced in the blood-serum whereby the action of bacterial toxins is antagonized. {h) Agglutinins. — JVIany forms of bacteria, when introduced into the body, stimulate the body-cells to produce specific substances called agglutinins, which have the property of causing the bacteria to lose their motility and to mass together in clumps. This phe- nomenon is of great practical importance in the diag- nosis of certain bacterial infections. For example, the blood- serum of a patient affected with typhoid fever contains agglutinins, so that when it is brought in contact with typhoid bacilli agglutination of the latter will take place. {c) Precipitins, — Precipitins are closely allied to agglutinins, and are antibodies which bring about the precipitation of soluble foreign proteins. They also ANTIGENS AND ANTIBODIES 21 have considerable diagnostic importance, to which at- tention will be called later. (d) Lysins, — Lysins are among the most impor- tant of all antibodies, both from the stand-point of protection against disease and also from the fact that many diagnostic tests are based upon their action. Lysins are antibodies that bring about the solution of cells, and the term cytolysin may therefore be applied to them all. Lysins which bring about the solution of bacteria are termed bacimwlysins ; those which cause solution of red blood-cells are known as hcEinolysins, Thus, the terms cytolysis, bacteriolysis and hcemolysis should be readily understood. Lysins are formed as the result of introduction of bacteria or of cells of an animal of an alien species into the body. Thus, the red blood-cells of the sheep, when introduced into the body of the rabbit, produce antibodies or h^emolysins in the rabbit's blood-serum, which when brought sub- sequently in contact with sheep's corpuscles under cer- tain conditions will dissolve the latter. {e) Isocytolysins. — It has been found that lysins may not only be produced against the cells of an ani- mal of a foreign species (heterolysins) but also that the introduction into the body of cells of an animal of the same species will cause the formation of specific cytolytic antibodies (isocytolysins). (/) Opsonins, — Opsonins are substances present 22 APPLIED IMMUNOLOGY in the blood-serum which prepare bacteria for phag- ocytosis or absorption by the leucocytes. Normal opsonins are present in ever}^ seruni. Opsonins which are called forth by the introduction of particular bac- teria are called immune opsonins or bacteriotropins. They are more resistant to heat (thermostable) than normal opsonins. {g) Antiferments. — These are antibodies which resist the action of the different ferments. Owing to their presence, self-digestion by the various cells of the body is probably prevented. Thus for the fer- ments, pepsin, trypsin, rennin, lipase, etc., we have the corresponding antipepsin, antitrypsin, antirennin, antilipase, etc. {h) Aiito-antihodies. — It has further been shown that an animal can be made to form antibodies against its own cells when these are introduced parenterally. Allergy To the altered condition of an animal into whose tissues has been introduced an antigen or foreign cell product, von Pirquet has given the term allergy. Under allergic phenomena are therefore included all subsequent reactions on the part of the body as a re- sult of the parenteral introduction of foreign protein materials, i.e., introduction by other channels than through the gastro-intestinal tract. An animal thus ANTIGENS AND ANTIBODIES 23 treated is said to be " sensitized," i.e., its cells have pro- duced an excess of antibodies against the particular foreign material introduced. Anaphylaxis Anaphylaxis is the term given by Richet to a state of hypersusceptibility or supersensitiveness to the ac- tion of a foreign protein, after the animal has been first " sensitized " by an injection of the protein in question. The phenomena of anaphylaxis will be discussed in greater detail in Chapter IV. Ill EHRLICH'S SIDE-CHAIN THEORY Our present view of the mechanism of the de- fensive processes of the body against disease is based upon the side-chain theory of Ehrlich. This theory- explains the interaction of the various antigens and antibodies and is almost completely supported by ex- perimental evidence. It is most important to realize that the interaction between antigens and antibodies is of a chemical nature, i.e., the antibody does not destroy the antigen but forms a chemical combination with it. Ehrlich's theory conceives of each body-cell as con- sisting of a central molecular complex or nucleus upon which its life depends and a number of processes or " side chains " capable of combining with foodstuffs for the nutrition of the cell and with foreign substances which might prove injurious to it. These side chains or processes are termed receptors. Each receptor has a special affinity for a certain kind of foodstuff or toxin. Certain receptors are common to all cells, while others are foimd only in special cells. It is also conceivable that some receptors may not be normally present but are formed only by the stimulation of cer- tain forms of toxins. The toxic molecule which unites with the cell receptor consists of two groups, a hapto- 24 ■o 3 5 3 O 3J ai to .3 EHRLICH'S SIDE-CHAIN THEORY 25 phore group, which binds it to the cell receptor, and a toxophore group, which actually bears its toxic prop- erties. When the receptor combines with the toxin molecule, the cell throws it off into the circulation, and similar receptors are formed to take its place. These, however, are formed in excess, and the cell throws them off also. These free receptors then imite with corresponding toxin molecules in the circulation. There is a receptor for each particular form of toxin molecule. Thus the diphtheria toxin combines only with the specific receptor provided for it, and will not unite with those intended for tetanus toxin. Ehrlich's theory, which at first only covered the simple union of toxin with antitoxin, was extended also to explain the action of more complex antibodies, and may be said to cover completely all forms of antigen-antibody ac- tion. There are three recognized types or orders of receptors, which are conveniently illustrated by the accompanying diagram (Fig. 1). Receptors of the first order possess only a single haptophore or com- bining group, by which they unite with the hapto- phore group of the corresponding antigens. To this order belong antitoxins and antiferments. Receptors of the second order, in addition to the haptophore group, possess a second or zymophore group, by means of which the anchored antigen can be subjected to further change. Under this head belong agglu- 26 APPLIED IMMUNOLOGY tinins and precipitins. Receptors of the third order possess two combining groups, and are termed ambo- ceptors. The first of these anchors the antigen to the cell and is known as the cytophilic group, while the second combines with the complement of the blood- serum and is known as the complementophilic group. Thus, for the action of receptors of the third order, an outside substance, the complement of the blood-serum, is necessary. Among these are cytolysins (bacterioly- sins, h^emolysins, opsonins, etc.). It is mmecessary to go into the experimental evidence that has been brought forward in support of Ehrlich's theoiy of im- munity, but numerous investigators have fully con- firmed his views by experiments. It is well known that poisons of known composi- tion, such as alkaloids, glucosides, etc., when intro- duced into the body do not produce antibodies, thus differing essentially from true antigens. The true antigens are closely related to foodstuffs and conse- quently have an intimate chemical relation with the body-cell, which provides special receptors for them. The poisons, on the other hand, not having this spe- cial chemical affinity, are not closely bound to the cell- substance, but become physically stored up. Many of these substances can be recovered from the body by extraction, which could not occur if chemical union had taken place. IV ANAPHYLAXIS OR HYPERSUSCEPTIBILITY Ix connection with the administration of antisera for therapeutic purposes, the possibihty of the occur- rence of the phenomenon known as anaphylactic shock must be borne in mind. The phenomenon of protein sensitization was rec- ognized by Vaughan, von Behring and others for many years before the name anaphylaaJis, under which it is now generally known, was given by Richet in 1911. The term indicates absence of protection, as opposed to prophylaxis. Anaphylaxis consists in a series of apparently deleterious effects produced by a second injection of specific protein material into an animal that has been previously *' sensitized " by a first injection of the same material. For instance, if an animal receive one injection of a foreign protein material, say of antitoxic serum, no deleterious effects are usually observed, but if this injection be repeated after several days, even in a very small dose, the second injection may be followed immediately by a marked reaction, such as convulsions and respiratory difficulty, in some cases with a fatal result. The ana- phylactic phenomena usually follow when the second injection is given at least twelve days after the first. 27 28 APPLIED IMMUNOLOGY One of the earliest investigators to devote special at- tention to this subject was Richet. He injected an animal with a dose of protein poison, and after com- plete recovery from the symptoms produced, by inject- ing a very much smaller dose which in a non-sensitized animal would have no deleterious effects, was able to cause death in a few hours. This hypersusceptibility in the sensitized animal Richet ascribed to the forma- tion of a special antibody by the first injection, which on the second injection causes the splitting off of a highly toxic substance from the toxin injected, giving rise to the symptoms. Richet's conclusions were based on the assumption that the phenomena only occurred when toxic substances were injected. Arthus in 1903 showed that hypersusceptibility occurred when non- toxic protein material was used, such as normal horse serum, milk, etc. (Arthus' phenomenon). He also showed that the symptoms could only be produced by means of the same substance that w^as originally in- jected; e.g. J that an animal first sensitized with horse serum would not be affected with a subsequent in- jection of milk or other protein material, but only with horse serum. The studies of von Pirquet showed that a certain interval must elapse after the first in- jection of serum before a second injection will pro- duce s}Tnptoms, and he concluded that this interval represented a period of incubation necessary for anti- ANAPHYLAXIS OR HYPERSUSCEPTIBILITY 29 body formation. He applied this theory to the period of incubation and symptoms of infectious diseases, all of which may be regarded as anaphylactic phenomena. In short, according to von Pirquet, the causative agents of disease do not show their deleterious effects until antibodies are formed which split them up, set- ting free toxic substances. Theobald Smith observed that guinea-pigs, which had been injected with toxin- antitoxin mixture, were hypersensitive to subsequent injections with normal horse serum and rapidly suc- cumbed to small doses of the latter. The most attrac- tive and rational explanation of the phenomena fol- lowing protein sensitization or anaphylaxis was first brought forward by Vaughan in 1907 {Jour. Infec- tions Diseases, 1907, iv, 476). Vaughan's views may be briefly stated as follows: Every protein molecule consists of a poisonous group and a non-poisonous or specific group. The latter group, when the molecule is introduced into the body, induces the development in the body of a specific proteolytic ferment, which has the power of splitting up similar protein mole- cules, setting free the poisonous or non-specific group. The poison thus set free produces the phenomena of anaphylaxis. Upon the first injection of a serum, therefore, the specific group of the molecule causes the formation in the body of a specific proteolytic ferment. The animal is now said to be *' sensitized." If a second 30 APPLIED IMMUNOLOGY dose of the same serum then be given, its molecule is split up by the proteolytic ferment, and the toxic con- stituent is set free, giving rise to anaphylaxis. There is no setting free of the poisonous elements of the pro- tein molecule immediately after the first injection of serum, because the specific proteolytic ferment de- velops gradually in response to the stimulation of the specific group of the molecule. Hence the toxic ele- ment is only liberated in small amounts as the ferment is formed. But by the time a second injection of serum is administered, a considerable amount of the proteolytic ferment is stored up, and is ready to split up the molecules of the second injection and set free the toxin which gives rise to symptoms of anaphylaxis. The anaphylactic poison affects chiefly the nervous system, and particularly the respiratory centres. The reaction manifests itself in respiratory difiiculty and convulsions, which may result fatally, and is particu- larly liable to occur in asthmatic persons. The injec- tion of antitoxic horse serum has been known to pro- duce anaphylactic phenomena in persons in whom at- tacks of asthma are induced by contact with horses. We do not yet know the nature of the anaphylactic poison. It is now recognized that not only serum, but all forms of toxic and non-toxic protein material, in- cluding milk, extracts of normal and pathologic ani- ANAPHYLAXIS OR HYPERSUSCEPTIBILITY 31 mal tissue, and bacteria, may give rise to anaphylactic phenomena. Anti-anaphylaxis. — If an animal receive a second injection of foreign protein material before the twelfth day after the first injection, a condition of resistance or decreased susceptibility may be set up (anti-ana- phylaxis), and the symptoms of anaphylaxis will not occur. This resistance may be kept up by repeated injections at short intervals. This fact is of impor- tance in the repetition of antitoxic serum for therapeu- tic purposes. The mechanism of anti-anaphylaxis is as yet unknown. Passive Anapliylaccis, — Anaphylaxis may be brought about passively as well as actively. Thus, if one animal be sensitized by an injection of foreign protein and sufficient time be allowed to elapse for the formation of the specific protein-splitting ferments, introduction of its serum into a second animal will sensitize the second animal to the protein in question. Anaphylaxis will then follow immediately after the injection of the second animal with the protein. The anaphylaxis reaction is made use of in the diagnosis of several diseases. The various tuberculin reactions of von Pirquet, WolfF-Eisner, Calmette, ]\Ioro and others depend on this hypersusceptibility. Tuberculous persons being " sensitized " by the tuber- culous process, the application of tuberculin in these 32 APPLIED IMMUNOLOGY cases will give rise to the various reactions. A similar reaction of diagnostic value occurs in gonococcic in- fection, a rise of temperature and other anaphylactic symptoms following the injection of gonococcus bac- terin in persons suffering from this infection. Noguchi's luetin reaction in syphilis also is an anaphy- lactic manifestation. Many of the phenomena of disease may be ex- plained on the basis of anaphylactic or allergic phe- nomena. As pointed out by von Pirquet, the incuba- tion period of an infectious disease is the time required by the body to form specific antibodies to the infecting organism. The onset of symptoms marks the setting free of the toxin, when the infecting organism is split up by the specific antibody. Studies in anaphylaxis have also furnished a very plausible explanation for certain individual idiosyncrasies. It is probable that hay fever and allied conditions are manifestations of anaphylaxis. The persons susceptible to these idio- sjmcrasies are probably sensitized by the pollen of plants and other substances, so that they manifest symptoms w^hen placed in contact with small quanti- ties of this proteid. In the same way may be explained idiosyncrasies to various articles of food, such as straw- berries, pork, lobster, exposure to exhalation from horses, etc. Certain precautions may be taken to avoid ana- ANAPHYLAXIS OR HYPERSUSCEPTIBILITY 3S phylactic shock in the administration of serum for therapeutic purposes. Rosenau and Anderson point out that anaphylaxis is to be feared in all persons who have shown a tendency to astlima, or who have received previous injections of serum at least twelve days be- fore. In these persons it is well to make a preliminary injection of a small trial dose of 0.1 to 0.2 c.c. of serum, and if no symptoms appear within two hours, the full dose may be given. Serum Sickness. — The symptoms, grouped under the term " serimi sickness," occasionally seen after the injection of antitoxic serum in diphtheria and other diseases, are for the most part manifestations of ana- phylaxis. These symptoms usually consist in fever, urticarial eruptions, lymphatic nodal enlargements, and kidney irritation. Usually these symptoms are transitory, but fatal cases have been reported. In order for these phenomena to follow a single injection, as occasionally happens, a large dose of serum must be employed; but the appearance of symptoms may occur after a very small second injection. The mild cases of serum sickness require no special treatment. With the refinement of preparation and concentration in small bulk of antisera, untoward symptoms following in- jection have become comparatively rare. ANTISERA PREPARATION OF ANTISERA— INDICATIONS FOR THERA- PEUTIC USE OF ANTISERA Therapeutic antisera for protective and cui^ative passive immunization fall into two general classes: (a) antitoxic sera, and (b) antibacterial sera, accord- ing to the mode of action of the bacteria against which they are directed. Antitoxic sera are sera which are applied to the group of bacteria whose deleterious action depends upon the toxins separated from them in the process of growth. The best examples of this class of bacteria are the bacillus of diphtheria and the bacillus of tetanus. Antibacterial sera are employed particularly in in- fections by those bacteria whose toxins are contained within the bacterial protoplasm (endotoxins). They act by destroying the bacteria themselves. Generally speaking, passive immunization has not been so suc- cessful in dealing with infections by bacteria belong- ing to this group, as with those whose action depends on separated or extracellular toxins. 34 ANTISERA. 35 Preparation of Antitoxic Sera When bacteria whose action depends upon toxins separated from them in the process of growth enter the body, they, as a rule, remain at the point of en- trance, from which the toxins spread through the cir- culation. In response to the stimulation of the toxins, the body produces antitoxins in the blood-serum, by which their deleterious action is neutralized. The anti- toxins at the same time render harmless to some extent the living bacteria at the point of entrance. This proc- ess is termed active immunization. Antitoxin production is artificially induced by ac- tive immunization of suitable animals by subcutaneous, intraperitoneal or intravenous injections of the toxins of certain bacteria. The antitoxin thus formed not only protects the animal inoculated, but can also be removed from the original animal and used as a cura- tive and protective agent against the same infection in other animals, by means of injections. The process in the second animal is known as passive immunization. So we distinguish active from passive immunization by the fact that in the former the animal produces its own antibodies to combat an infection, while in the latter the antibodies have been produced by another animal ( see Chapter I ) . As an example of the preparation of antitoxic sera, the method of artificial production of antidiphtheritic serum (diphtheria antitoxin) will be described. 36 . APPLIED IMMUNOLOGY First of all it is necessary to obtain a quantity of the toxin, separated from the bacteria. The diph- theria bacilli are grown in bouillon, and the fluid con- taining the toxin freed from bacteria by passage through a Berkefeld filter. The toxin strength of the fluid must then be measured. The unit of toxin strength is the smallest quantity necessary to kill a guinea-pig weighing 250 grammes in 5 days or less. This quantity of toxin is called the minimal lethal dose. It should be noted that not all strains of diphtheria bacilli form toxin suitable for the production of anti- toxin. For the formation of antitoxic serum, the horse has been found to be the most suitable animal, but individual horses difl*er in the quantity and quality of antitoxin they are able to produce. No definite rules can be laid down for the active immunization of the horse against diphtheria toxin. But in a general way, small doses of the toxin are first injected, and gradually increased, until a very large quantity of the toxin can be injected at one time. In regulating the amount of toxin to be injected, one is guided by the effects of the previous dose, which are not shown im- mediately as a rule, but appear after a few days. From time to time, small quantities of blood are re- moved from the jugular vein of the horse, and tested for antitoxic strength. When a sufficiently powerful antitoxic content has been demonstrated in the horse's ANTISERA 37 blood-serum, as described below, a large amount of blood is drawn off, and prepared for therapeutic pur- poses. The same horse can be used over and over affain for the formation of antitoxin, if allowed a sufficient period for recuperation" after each bleeding. The formation of a sufficiently potent antitoxic serum usually requires from six weeks to two months. The strength of antitoxin is measured in units. The antitoxic unit is that quantity of horse's serum which will render harmless the injection of 100 mini- mal fatal doses of toxin. It is now ascertained what is the smallest quantity of toxin which when mixed and injected with 1 antitoxin unit will kill a guinea-pig of 250 grammes in 4 or 5 days. This quantity of toxin is then mixed with diif erent dilutions of the horse's serimi and injected subcutaneously into several guinea-pigs weighing 250 grammes each. Supposing that 1/1000 c.c. of the antitoxin neutralizes the dose of toxin so that the guinea-pig injected with this quan- tity remains alive, then 1 c.c. of the horse's serum is said to contain 1000 units of antitoxin. The strength of the antitoxic serum having been ascertained in this manner, it is placed in suitable quantities in syringes for therapeutic use. All these procedures are of course carried out under strict aseptic conditions. Antidiphtheric serum, like other antisera, loses its potency after variable lengths of time. 38 APPLIED IMMUNOLOGY Antitetanic and other antitoxic sera are produced and tested in a similar manner, the horse being the ani- mal usually immunized for their production. Preparation of Antibacterial Sera The bacteria belonging to this group do not act by means of extracellular toxins. Their deleterious ef- fects depend upon toxins set free from the bacterial protoplasm when the organisms undergo disintegra- tion (endotoxins). These toxins cannot be separated from the bacteria themselves in vitro, except to a very slight extent. At any rate it is doubtful if the sub- stances which call forth the production of antibodies can be thus separated. Antibacterial sera are there- fore produced by injecting the bacteria themselves into the body of the animal that is to be actively im- munized. These are injected, usually at first inac- tivated, but also at times in an attenuated or even in their living state (Fig. 2), in gradually increasing doses until an antibacterial serum of suitable strength is obtained, when the animal is bled, and the serum preserved in ampoules containing convenient doses for therapeutic injection. The antibodies contained in antibacterial sera are not of the comparatively simple nature of those found in antitoxic sera, but belong to the second and third orders of Ehrlich. The action of antibacterial sera depends upon several factors, in- ANTISERA. 39 eluding opsonins, bacteriolysins, and to some extent agglutinins and precipitins ( see Chapters IX, X, XI and XX). The standardization of dosage of antibacterial sera is therefore more difficult and uncertain than that of iMMWiiTY CwvE OF Rabbit Inoculated with Living Tubercle Bacilli or Low Virulence O* Opsonic Ijidcii. X' tNocuLATion''«sis. FEBKUARY MARCH 5 ^ 10 If ir e^ a u so \. l- ^^ T ^v 2 !s t I zk * '-J ^^ t ^y " it- 7 ^ ^ J ^ 7 ^^ - IZ 5) I - t 2 ^: ^ 7 a ^^ I t - y^ --^ ~ y ^^2t 7 t y * x ^ ^ u-|==4i^ — -_i — ""■" ::: — :"":":" Fig. 2. — Illustrative of opsonic curve, showing immunity of rabbit inoculated with liv- ing tubercle bacilli of low virulence. © = opsonic indices ; X = inoculations. antitoxic sera, but the potency of an antibacterial serum can to some extent be gauged by determination of the opsonic index from time to time during the im- munization of the animal (Fig. 3) , and also by testing the agglutinating power of the serum, though the lat- ter method is of only slight value as an indication of 40 APPLIED IMMUNOLOGY therapeutic strength. The dosage of antibacterial sera is chiefly determined by the therapeutic effects ob- tained. In general, antibacterial sera are not of so much value as antitoxic sera, though in the case of a Chart Showing Inoculations and © - Opsonic Inde teSEPTXMBER^ 7 ?5^\°' Curve or Opsonic Indices kusTRATiNa Immunity or Ram ■X. X« iNOCtlLATlOM OF STAPHYtOCOCCUS AUREUS. B^En^ NOVEMBER^ ^ ^'I'^^^S^^s'^^ «°^ 4r -■*■ '" i S i p ov i -i\ ^ t • * * « s = . = = 7 .V il ^ 1 -t ■ "• _ 25 b . . . , . . . = . V . fj^ L t- § §s sggjLoL Sfe £•6 g g § o o' 1^ i|i^ ii § ''^ o § o ^ S § s § I 13 1 ' ^i ? S? § ^ ^f i 2_ ^' evj— o fo g:J CD ~ "i i ^ g ^ J. : f-1 ;; J_ S - _L^ : :: ^ -^ " i - i - - ^ ^ - t -,- 4 7 i 3-:S >, t ~ - ;i : - oi~ 51." >- c1 C^l ^^^ - fif(^ - - - ..'rr " — : . Fig. 3. — Illustrative of inoculations and curve of opsonic indices demonstrating immu- nity of ram. © = opsonic indices; X = inoculations of Staphylococcus aureus. few organisms good results have been obtained. Active immunization offers a greater field of usefulness than passive immunization in infections of this type. Indications for the Therapeutic Use of Antisera In the case of infections with bacteria which act through extracellular toxins, antitoxic sera have a ANTISERA 41 high therapeutic value both for protective and cura- tive purposes. By their use in persons liable to be ex- posed to an infection, protection for a limited period of time may be conferred. This protection by passive immunization is not so durable as by active immuniza- tion, though the time necessary to acquire it is much less, a fact of considerable importance in some cases. It has been estimated, experimentally and clinically, that the duration of passive immunity persists from three to six weeks, while active immunization may endure for a year or two. Moreover, studies in pas- sive immunization have sho^vn that the duration of immunity from homologous antisera lasts three to four times as long as that from heterologous immune sera. Antitoxic serum in some infections has a high curative value in persons already suffering from the disease, and indeed in certain infections is the most important means of treatment. In the case of other diseases, the value of the antitoxic serum as a curative agent is problematical. Antibacterial sera, in infections where they are of any value at all, are indicated especially to overcome the immediate infection, owing to their rapid effects, while more lasting protection in this class of infections is conferred by active immunization which pursues a slower course. The indications for active and passive immunization in specific cases will be taken up later. VI ANTITOXIC SERA Antitoxic sera are of recognized value particu- larly in diphtheria and tetanus, and are employed as routine therapeutic agents in these diseases. Antitoxic sera have also been prepared and used from time to time in other infections with varying success. Examples of these are antigonococcic serum, antityphoid serum, antituberculosis serum, antidysen- teric serum, anticholera serum, antibotulism serum. In addition, antitoxic sera have been employed in hay fever ( antiphy totoxic sera), and against snake ven- oms (antizootoxic sera). Antidiphtheeitic Serum (Diphtheria Antitoxin) With antidiphtheritic serum are obtained the most brilliant results in the domain of serum therapy. Sta- tistics from all sources attest its value. The serum is employed both for protective and curative pm'poses. The method of preparation of antidiphtheritic serum has already been given in Chapter V. Protective Use of Diphtheria Antitoxin, — The average duration of immunity conferred by a protec- tive dose of diphtheria antitoxin is three weeks. This varies with the severity of the epidemic present. In 42 ANTITOXIC SERA 43 ordinary epidemics a suitable protective dose is 500 units, injected subcutaneously. Site and Method of Injection, — The usual sites of injection are under the skin of the abdomen, the thigh, or between the shoulder blades. The skin is thoroughly cleansed with alcohol, lifted w4th the thumb and forefinger, and the needle of the syringe quickly introduced beneath it. The serum is allowed to enter slowly, the needle quickly withdra^^n, the pmicture site compressed and stroked with a pledget of cotton soaked in alcohol, and the skin gently massaged. It is usually unnecessary to apply any dressing at the site of puncture of the skin. Infec- tion rarely follows if asepsis has been observed. A protective dose of antitoxin should be given to all persons coming in close contact with a case of diph- theria, especially to children. Some hospitals require that all children admitted receive a prophylactic injection. Von Behring's Method of Prophylaocis, — Recently von Behring has brought forward a new method of prophylaxis against diphtheria, by combined active and passive immunization. He injects a mixture con- sisting of strong diphtheria toxin and antitoxin in such proportions that the toxin is slightly in excess of the antitoxin. Experimental evidence proves that more lasting immunity is conferred by this means than by 44 APPLIED IMMUNOLOGY the injection of antitoxin alone. Statistics showing the increased incidence of cases of diphtheria in large cities demonstrate the necessity for improvement in methods of prevention, and it is hoped that this method will fulfil its promise. Few statistics are available as yet upon which to base conclusions. Curative Treatment of Diphtheria, — Antitoxin should be used as early in the disease as possible, for with every day of delay there is a great increase in the mortality rate. The death-rate is practically nil in patients who receive injections on the first day of the disease. The average dose in the ordinary case when seen on the first day is 5000 miits, regardless of the age of the patient unless extremely young. In a severe case the dose should be 10,000 or 15,000 units. In cases seen later the dose should be proportionately larger, sometimes as much as 100,000 units being given. A single large dose of the serum is preferable to re- peated smaller doses. Beneficial results should be seen from the injection in about eight hours, charac- terized by a subsidence of the throat symptoms and fall of the temperature and pulse-rate. Otherwise the injection may be repeated in larger dose. While the administration of antitoxic serum is by far the most important point in the treatment of diphtheria, other measures, such as rest in bed, liquid diet, sprays. ANTITOXIC SERA 45 and cardiac and renal therapeusis, must not be omitted. Diphtheria Carriers, — After disappearance of the clinical symptoms of diphtheria it is found that many cases still harbor the bacilli in the nose and throat for variable lengths of time, thus becoming sources where- by the disease is spread to others. Moreover, many persons harbor the organisms who have never had an attack of the disease. Schiotz, of Copenhagen, in 1909, after noticing that persons with staphylococcic infections of the nose and throat seldom contracted diphtheria, began to use, with good results, a spray made from a culture of Staphylococcus aureus in per- sons infected with diphtheria bacilli. Reports by many observers show almost uniformly good results from this treatment. Lorenz and Ravenel {Jour, A. M. A,, August 31, 1912, p. 690) recommend a fresh suspension of Staphylococcus pyogenes aureus in nor- mal saline solution or a bouillon culture twelve hours old. This is used as a spray for the fauces, pharynx and nose, after the local signs of diphtheria have dis- appeared, while the cultures for the bacilli are still positive. The spray is repeated at four-hour inter- vals on two succeeding days, and continued there- after until the cultures become negative to diphtheria bacilli. Untoward results are rarely seen. The period of quarantine after diphtheria is greatly shortened by 46 APPLIED IMMUNOLOGY this method when compared with cases treated by anti- septic sprays alone, and many cases of " carriers " that have resisted all other forms of treatment have responded rapidly to it. Antitetanic Serum Tetanus, like diphtheria, is a disease, manifesta- tions of which depend upon a toxin separated from the tetanus bacilli in the process of growth. When a wound becomes infected with tetanus bacilli, the lat- ter remain at the point of entrance and give off the toxin which is disseminated along the nerve sheaths mitil it reaches the spinal cord. Here the toxin com- bines with the cells of the central nervous system, and gives rise to the typical s}Tnptoms of the disease. This affinity of tetanus toxin for nerve tissue has an im- portant bearing in the treatment of tetanus. The method of artificially producing antitetanic serimi is similar to that of producing antidiphtheric serum. Tetanus toxin is formed by growing tetanus bacilli anaerobically in bouillon for several dsLjs, the bacilli being then removed by passing the fluid through a Berkefeld filter. The filtrate is now injected into a horse, at first in small amounts (0.5 c.c.) mixed with a quantity of antitetanic serum. The doses are then gradually increased, the antitoxin being omitted after the first few injections. There are several methods of ANTITOXIC SERA 47 standardization of tetanus antitoxin. The unit for the United States is the smallest amount of immunized horse's serum that will protect a guinea-pig weighing 350 grammes against 1000 times the fatal dose of tetanus toxin. The serum having been standardized, it is stored in syringes convenient for therapeutic use. As in the case of diphtheria antitoxin, antitetanic serum loses its potency after a variable length of time. Therapeutic Use of Antitetanic Serum. — ^The therapeutic efficiency of antitetanic serum depends chiefly on whether it is able to reach and neutralize the toxin before the latter becomes fixed by the cells of the central nervous system. Failure of tetanus antitoxin to have therapeutic effects is due in most cases to faulty administration. Ashhurst and John in a recent article {Am, Jour, Med, Sc, June and July, 1913) present what is undoubtedly the most able and com- prehensive view of the whole subject of the treatment of tetanus. Their recommendations will be followed here to a great extent. Antitetanic serum is used both as a preventive and a curative measure. For prophylactic use, in the case of all wounds in which the development of tetanus is feared, at least 1500 units of the antitoxin should be injected as early as possible into the muscles or if possible into the nerves in the immediate neighborhood of the wound. The longer the interval that is allowed 48 APPLIED IMMUNOLOGY to elapse between receipt of the wound and injection of the serum, the larger the dose of antitoxin required to be efficient. Owing to the fact that the antitoxin is all eliminated from the system in eight or ten days after the injection, and that dormant tetanus spores in the wound may develop after that time, it is ad- visable to repeat the injection during the second week, and if possible again during the third week. In the prophylaxis of tetanus, however, removal of any tetanus bacilH that may be present by thorough open- ing and disinfection of the woimd is of greater im- portance than administration of antitoxin. Antitetanic Serum as a Curative Measure, — The efficiency of tetanus antitoxin as a curative agent de- pends on three factors; (1) the site of the injection, (2) thefrequency of the injection, (3) the quantity of antitoxin injected. (1) The Site of the Injection, — Of all methods, the subcutaneous inoculation of tetanus antitoxin is least efficient, because, as Ashhurst and John point out, " by this method only a homoeopathic dose ulti- mately reaches the motor nerves through which the toxin is being carried to the spinal cord, while by far the greater part of the antitoxin is distributed to the viscera, where it can be of no possible use. Adminis- tered in this way, overwhelming amounts are required to produce any effect." Clinical and experimental ANTITOXIC SERA 49 evidence points overwhelmingly to intraspinal (sub- dural) and intraneural injections as the best methods of administering tetanus antitoxin, for by these means the tissues affected by the toxin are reached directly. Intravenous injection ranks next, as by this means all the spinal nerves are reached at once, though the anti- toxin reaches them much diluted. (2) Frequency of Injection, — The frequent fail- ure of tetanus antitoxin to effect a cure is due in part to the fact that it is not given often enough. The anti- toxin can be given subcutaneously every three hours, intravenously once or twice in the twenty-four hours, intraspinally and intraneurally every day if necessary. (3) Quantity of Antitoxin Injected, — The sheet- anchor in treatment is to get the maximum quantity of antitoxin indicated into the patient's body as soon as possible. One of the authors (B. A. T.), in the successful treatment of a case of tetanus, reported in Monthly Cyclopcedia and Medical Bulletin, June, 1911, administered 213,740 units of antitetanic serum. Of this enormous and unprecedented quantity, 15,340 units were injected intraspinally, the remainder subcutaneously. The largest inoculation at any one time was 35,400 units and the maximum quantity in twenty-four hours was 97,940 units. Subsequently, Ashhurst and John state that subcutaneously, in the usual acute type of case, at least 100,000 units 50 APPLIED IMMUNOLOGY are required in the first twenty-four hours; intra- venously, probably 15,000 to 25,000 units should be administered at first; intraspinally, from 3000 to 10,- 000 units should be given, according to the severity of the case. The writers mentioned recommend for in- traneural injections as great amounts as the nerves will absorb^ ranging from 750 to 1500 units. For intraspinal injection the same technic is employed as for lumbar puncture and spinal angesthesia. For intraneural administration the motor nerve trunk supplying the region of the wound is exposed as near to the spinal cord as practicable, and the antitoxin slowly injected directly into the sheath. We do not regard it as out of place here to quote in full Ashhurst and John's recommendations for the rational treatment of a case of tetanus: The patient will be placed in quiet, with competent nursing facilities. As soon as possible after coming under observation, whether this be in the small hours of the night or at bright noon-tide, the motor nerves lead- ing from the wounded part will be exposed, as near to the cord as practica- ble, and as much antitoxin as each will contain will be injected toward the spinal cord. An intraspinal injection of at least 3000 units will then be made according to the usual technic for spinal anaesthesia. If it is possible to prick the cord with the needle, so much the better. Next the wound of entrance of the infection will be widely opened, all foreign bodies, sloughs, etc., will be removed by forceps, scissors, or scalpel; the wound will be irrigated with hot peroxide of hydrogen, swabbed out with 3 per cent, alcoholic solution of iodine, and loosely filled with gauze soaked in the same solution, and injection of antitoxin will be made (1500 to 3000 units) deeply into the muscular tissues around the wound. Con- tinuous proctoclysis, as used in cases of peritonitis, will be given; and by mouth or in the rectal fluid will be administered effective doses of chloral ANTITOXIC SERA 51 and bromides, at appropriate intervals.^ Feeding will be enforced, by the nasal tube passed under chloroform anaesthesia, if necessary. During the course of the first day a moderate amount of antitoxin will be admin- istered intravenously; probably 10,000 units will suffice. The intraneural and intraspinal injections of antitoxin will be re- peated daily, under chloroform anaesthesia, until marked decrease in spasticity occurs. Every twelve hours, or less often, a moderate amount of antitoxin will be injected intravenously, or even subcutaneously, so as to neutralize the circulating toxins; but the main reliance will be placed on intraneural and intraspinal injections. The administration of spinal depressants will be continued as long as they are indicated; a comatose state or muscular relaxation naturally are contra-indications. The wound will be dressed daily, as above described, until a healthy granulating sur- face is obtained. The administration of cardiac, pulmonary and renal stimulants to meet the particular conditions, is invariably a matter of necessity. The application of the treatment as above outlined and commenced within twelve hours of the onset of symptoms, should reduce the mortality of tetanus to less than 20 per cent. Antigonococcic Serum. — Attempts have been made by Torrey and others to produce an antitoxic serum by injecting a toxin separated by filtration from cultures of gonococci. But it has been found that while this substance is toxic for laboratory animals, it does not produce antibodies that render the animal in- ^ The authors feel from their experience that complete reliance should not be placed on these drugs, and recommend intraspinal injections of chemically pure magnesium sulphate in 25 per cent, solution to allay mus- cular spasm and convulsive seizures, in quantities of 2 to 5 c.c, the maxi- mum being 1 c.c. for each 25 pounds of body weight. Should signs of respiratory failure supervene, chloretone administered by mouth or by rectum, preferably the latter in one drachm doses, may be substituted for or alternated with the injections of magnesium sulphate. 52 APPLIED IMMUNOLOGY jected immune to the toxin. Efforts to produce immune sera by injections of gonococci themselves have been more successful, and will be discussed under antibacterial sera (Chapter VII). Antituberculosis Serum. — IMariagliano has pro- duced a serum by immunizing horses with toxins made from the filtrate of cultm-es of tubercle bacilli. His favorable results have not been generally confirmed, and antiserum is little used in tuberculosis. Antidysenteric Serum, — The dysentery bacilli of the Shiga type form an extracellular toxin and from this it is possible to prepare a true antitoxic serum. The toxin, together with antidysenteric serum, is in- jected into the horse in gradually increasing doses. Killed cultures of the bacilli are also injected to give the serum antibacterial properties. The standard strength of the antiserum is such that 0.5 c.c. will pro- tect a 1500-gramme rabbit against three times the smallest fatal dose of toxin (Schorer). In Japan by the use of this serum, the mortality of dysentery of the Shiga type has been reduced from 28-37 per cent, to 8-12 per cent. In mild cases one dose of 10 c.c. of serum is injected, in severer cases two injections of 10 c.c. are made six to ten hours apart, never more than 20 c.c. in one day. Dysentery caused by bacilli of the Shiga type is rare in the United States, being largely confined to tropical and semitropical coun- ANTITOXIC SERA 53 tries. The form of bacillary dysentery met with in the United States is usually due to the mannite- fer- menting or Flexner type of organism, which will be dealt with in the chapter on antibacterial sera. Antibotulism Serum. — Meat poisoning is due in many cases to a toxin produced by the Bacillus botu- linus. This organism is anaerobic, and depends for its action upon an extracellular toxin. This toxin can be obtained artificially from bouillon cultures of the or- ganism. The Institute for Infectious Diseases in Berlin has produced an antitoxin by immunization of animals, but data are still insufficient to pass judg- ment upon its curative properties. Antiphytotoocic Serum, — Many forms of hay fever are manifestations of anaphylaxis or hypersensibility to the pollen of certain plants, such as golden-rod, rag- weed, honeysuckle, chrysanthemum, etc. Dunbar has produced an antitoxin, known as " pollantin," by in- jecting horses with extracts from various pollens. The antitoxin is put up in liquid form for inoculations, in powder form for local application. In certain cases the antitoxin gives protection and relief, but as a rule only for a limited time. Its use is occasionally attended by severe symptoms of anaphylaxis, so that caution must be observed in beginning treatment with it. Antivenin, — Antivenin is an antitoxic serum pre- pared to counteract the effects of snake poison. The 54 APPLIED IMMUNOLOGY nature and mode of action of snake venoms have been studied extensively, especially by Calmette and Flex- ner and Noguchi. Their researches have shown that poisonous snakes can be classified into two broad groups, according to the effects produced by the toxins of their venom. To one group belong the cobras, whose venom is principally in the nature of a neurotoxin, acting especially on the respiratory centre in the medulla, while in the other group, containing the vipers and rattlesnakes, the venom contains a hemorrhagin, and causes extravasation of blood in various regions of the body. In addition, all snake venoms possess hsemolytic properties, found most markedly in cobra venom. Against the neurotoxin of cobra venom, Calmette has successfully produced an antitoxin (antivenin) by injecting horses with the venom. This is only of therapeutic value, however, against cobra bites, and is useless as an antidote for rattlesnake bites. Attempts have been made, but unsuccessfully so far, to produce a reliable antihemor- rhagic serum for the treatment of rattlesnake bites. The antivenin of Calmette may be injected in doses of 10 to 20 e.c. To be of any value it naturally must be used as soon as possible after the bite is re- ceived, and is probably useless after 3 or 4 hours. The serum has, therefore, only limited application as a therapeutic measure. VII ANTIBACTERIAL SERA It has been experimentally possible to produce antibacterial sera for a much larger number of organ- isms than antitoxic sera, though in the case of none of them have such brilliant therapeutic results been ob- tained as in the case of diphtheria and tetanus anti- toxin. Yet in infections by a few organisms, notably the staphylococcus, streptococcus, pneumococcus, gon- ococcus, meningococcus, typhoid bacillus, colon bacil- lus, dysentery bacillus, cholera vibrio, plague bacillus and anthrax bacillus, antibacterial sera of consider- able therapeutic value have been produced. Antistaj^hylococcic Serum, — Various attempts have been made on horses and other animals to pro- duce a potent antistaphylococcic sermn, but they have almost invariably resulted unsuccessfully, although Doyen and Paltchikowsky assert that they have suc- ceeded partially. Schorer states that the value of the serum is inconsiderable, and its injection in the treat- ment of staphylococcus infections is seldom or never warranted. Such noted authorities as Ehrlich, Bordet and Citron omit even to mention, in their works on immunity, the existence of antistaphylococcic serum. The failure, or only partial success, in producing 55 56 APPLIED IMMUNOLOGY potent antistaphylococcic serum has possibly been due to utilization of avirulent or univalent cultures of the organisms. One of the most important factors con- ducive to the successful production of this serum is the employment of staphylococcus cultures from many sources. With this essential in mind, one of us (Thomas) isolated eighteen strains of Micrococcus aureus in pure culture from many sources, as fur- uncles, carbuncles, abscesses of the scalp, thoracic empyema, axillary abscess, and septicaemia. From these strains 24-hour cultures were grown and from the mixed growth a suspension in physiologic salt solution was prepared containing 32,400,000,000 cocci to the cubic centimetre. This was then heated for one hour over a water-bath at 60° C. The animal selected for the purpose of immunization was a full-grown ram weighing 165 pounds. The degree of immunity of the animal was governed by determination of the opsonic index for the polyvalent staphylococcic sus- pension (see Fig. 3). At the first injection the ram received intraperitoneally 972,000,000,000 dead sta- phylococci. Thereafter at weekly intervals either grad- ually increasing doses, or slightly smaller doses heated for a shorter time, and finally unheated organisms, were injected. After twelve inoculations, the index was found to be 2.6, and the animal was bled to death from the carotid artery. The serum was hermetically ANTIBACTERIAL SERA 57 sealed in glass ampoules containing 1 and 2 c.c. each. The appended chart (Fig. 3) shows the inoculations and curve of opsonic indices illustrating the immunity of the ram. From therapeutic employment of this antistaphy- lococcic serum in many cases of carbuncles and fur- uncles, in doses ranging from 1 to 6 c.c, the following conclusions were drawn. 1. The antistaphylococcic serum as herein pre- pared and described possessed unquestionable thera- peutic efficiency in a series of conditions, both general and local, due to infections by the Micrococcus aureus, 2. Biologic therapy by a potent polyvalent anti- staphylococcic serum is more effective in the presence of a staphylococcic bacterisemia than is the correspond- ing autogenous bacterin. 3. By virtue of the more immediate and decisive effects of the antiserum, it deserves first choice over the bacterin in the treatment of furunculosis and car- bunculosis; on the other hand, a more intensive and lasting immunity can be conferred on the individual by supplementing the serum with two or three inocula- tions of the autogenous bacterin. 4. It is to be regretted that no attempt was made to standardize this antiserum with respect to standard units, since it must be conceded that the therapeutic failure or inefficiency of many serums is referable to 58 APPLIED IMMUNOLOGY the deficiency of the immune-body content of that particular serum, or in other words, to an improper or incomplete immunization of the animal utilized for the production of the antiserum. Antistreptococcic Serum, — Various antistrepto- coccic sera have been employed for some time with more or less success. They are nearly all made by in- jecting horses with increasing doses of killed strepto- cocci recovered from different lesions, thus securing polyvalent antisera. Standardization of the strength of these sera is difficult, the initial dose being more or less empirical, and succeeding doses being guided by clinical effects observed. The serum is indicated in all streptococcic infec- tions, particularly in septicaemia, where rapid effects are desirable, and where bacterial vaccines are not suit- able. The dose varies from 10 to 100 c.c, to be repeated in accordance with the symptomatic indications. Antipneumococcic Serum, — Passive immunization in pneumococcus infections has been attempted by means of antibacterial serum obtained from horses previously injected with different strains of pneumo- cocci. This serum depends for its action chiefly upon an increased production of bactericidal and opsonizing substances. In pneumonia the results of its use by most observers have been disappointing, the dose of ANTIBACTERIAL SERA 59 serum ranging from 10 to 20 c.c. in this disease. Cole finds the results of treatment of pneumonia by anti- pneumococcic serum to be encouraging in certain cases, when used in large doses (100 c.c. or more). He states that this action is probably in part due to antitoxic substances. In other pneumococcus infec- tions, especially serpiginous ulcer of the cornea, bene- fit from its use has been seen. Antigonococcic Serum. — Torrey and Rogers have produced an antibacterial serum that is of considerable therapeutic value in certain gonococcus infections. This serum is made by injecting strong, full-gro^vn rams with 24-hour polyvalent cultures of virulent gonococci. Nine or ten in j ections are usually required to produce a serum of high potency, the bacterial sus- pension being heated for half an hour at 65° C. be- fore the first two or three injections, after which the unheated gonococci are used. When sufficiently im- munized the animal is bled from the carotid artery, the serum separated, and stored in sterile ampoules. Standardization of this serum in units is difficult, but some idea of its strength may be gained by testing its agglutinating power. The dose of the antigonococcic serum varies from 2 to 6 c.c, injected under the skin, and repeated every few days. The serum is only to be used as an adjunct to other methods of treatment. It is of little or no value in acute and chronic urethritis. eo APPLIED IMMUNOLOGY prostatitis, and conjunctivitis, but has beneficial ef- fects in cases of gonorrhoeal arthritis, endocarditis, peritonitis, and septicaemia. Antimeningococcic Serum, — Kolle and Wasser- mann, Jochman, and others have succeeded in immu- nizing animals by injections of the Diplococcus intra- cellularis of Weichselbaum, the antisera thus obtained proving of considerable value in meningitis due to this organism. The serum of Flexner and Jobling, how- ever, both on account of its potency and also the method of its administration, has superseded all others. Flexner and Jobling prepare their serum by inject- ing into horses, first, gradually increasing doses of heated meningococci, followed by injections of un- heated organisms, and finally injections of an autoly- sate or extract of the meningococci which contains the endotoxin. The serum thus produced acts both by opsonizing and bacteriolytic properties. Accurate standardization of the serum is not possible, the dose depending largely upon clinical indications. Flexner and Jobling administer the serum by subdural injec- tion, a lumbar puncture being first done and some of the spinal fluid removed. The quantity of serum to be injected ranges from 30 to 60 c.c. In severe cases the injection must be repeated in 12 or 24 hours and there- after as long as meningococci are found in the spinal fluid. By means of this senmi, the mortality of epi- ANTIBACTERIAL SERA 61 demic cerebrospinal meningitis due to the Diplococcus intracellularis has been reduced from 70 per cent, to about 30 per cent. The antimeningococcic serum may also be used as a preventive measure in persons exposed to the disease. Antityphoid Serum. — Antibacterial sera have been prepared by immunizing horses by injections of mixed strains of killed and live typhoid bacilli. The serum thus produced has been injected into persons suffering from typhoid fever, in daily doses of 10 c.c. or more. So far the results have been disappointing either in shortening the disease or lessening the severity of the symptoms. For protective purposes, active immu- nization by injection of killed cultures of typhoid bacilli has proved more successful. Anticolonic Serum. — Hans ]Much finds that nor- mal blood-serum has a bactericidal action upon certain strains of colon bacilli, while on other strains blood- plasma has this action but not blood-serum. He has employed normal blood-plasma and serum locally in cystitis caused by infection with the colon bacillus with some success. Much has also been able to produce a powerful specific antiserum by injecting animals with several strains of colon bacilli, which have bacteriolytic properties against all strains of colon bacilli. He has used this also with benefit as a local application in colon bacillus pyelitis and cystitis. 62 APPLIED IMMUNOLOGY Antidy sent eric Serum, — We saw that an antitoxic serum has been successfully produced and used in in- fections with the Shiga type of dysentery bacillus. The form of dysentery more common in the United States is due to the mannite-fermenting or Flexner type of organism. An antibacterial serum has been obtained for treatment of infection with this organism, but used with indifferent success. Anticholera Serum, — Attempts have been made to obtain both antitoxic and antibacterial sera for use in cholera. Kolle and Wassermann, MetchnikofF and Roux tried to get a soluble toxin from cultures of the vibrio, and from that to make an antitoxin. But it is probable that they only obtained the intracellular toxin from disintegration of the organisms. The sera produced by inoculation of animals with these endo- toxins or with the bacteria themselves, have been of little value in the treatment of cholera. More favor- able results have followed active immunization with cultures of the organisms. Antiplague Serum. — Two types of antiplague serum have been employed. The first is made by in- oculation of horses with plague bacilli, and the other by inoculation with nucleo-proteids produced from cultures. Extensive trials of these sera in India show little benefit from their use in the treatment of plague, though life may be slightly prolonged by them. ANTIBACTERIAL SERA 63 Anti-anthrcur Serum, — Of the many antisera that have been produced for the treatment of anthrax, that of Sclavo is the best. This is prepared by inoculation of asses simultaneously with cultures of the Bacillus anthracis and antiserum, a very powerful serum being thus obtained. Sclavo advises a dose of 30 to 40 c.c. divided into three or four parts, injected into the ab- dominal wall in different regions. This dose may be repeated in 24 hours if necessary. In grave cases, in- travenous injection of the serum is recommended. Statistics tend to show that use of the serum reduces the mortality of anthrax from about 26 per cent, to 6 per cent. Antimelitensis Serum, — A curative serum for the treatment of Malta or Mediterranean fever was first prepared by Wright by immunizing goats with cul- tures of M, meliteims. Horses were later employed for immunization with more or less encouraging re- sults. However, the method of passive immunization in this disease has been overshadowed in recent years by active immunization. VIII MISCELLANEOUS SERA AND EXTRACTS ANTIRABIC SERUM— ANTILEPROSY SERUM— ANTITYPHOID EXTRACT OF JEZ— LEUCOCYTIC EXTRACT— ANTIFER- MENT — ANTICARCINOM ATOUS EXTRACTS — PYOCYA- NASE— ANTITHYROID SERUM AND EXTRACTS— SPANG- LER'S CROTALIN— PHYLACOGENS There are several extracts and sera that have been introduced as methods of treatment in various diseases, but which require httle more than brief mention, either because they have not yet been given thorough trial, or because results with them have not been sufficiently striking as to establish them as routine measures. Antirabic Serum. — The success of active immu- nization in the prevention of hydrophobia has led several workers to attempt to obtain a sermn for pas- sive immunization in this disease. Such immunity has been experimentally produced by means of serum from dogs that have been actively immunized by in- jections of rabies virus. The practical value of the serum, however, is not yet sufficiently established. The recent announcement of the isolation and arti- ficial culture of the rabies organism by Noguchi leads to the hope of success along this line. Antileprosy Serum. — As yet, it has not been pos- sible to cultivate the leprosy bacillus on artificial 64 MISCELLANEOUS SERA AND EXTRACTS 65 media, a fact that has retarded considerably any re- search on immunization in leprosy. Passive immu- nization has been attempted by Carrasquilla, Herman and Abraham, and others, by injecting the blood of lepers or the juice from nodular lesions into the horse, and employing the serum from the animal thus in- jected in the treatment of the disease. Slight im- provement has been reported in a few cases. Antityphoid Ecctract of Jez. — Jez claimed anti- toxic properties from an extract prepared from the bone-marrow, spleen, and lymph-nodes of an animal immunized to typhoid bacilH. This has been found to have but little value in the treatment of typhoid fever. Leiicocytic Extract, — Attempts have been made to increase the phagocytic powers by preparing and injecting extracts made from leucocji;es, especially in pneumonia. Both human and lower animal ex- tracts have been employed. Manoukhine uses leuco- cytes from the patient's owti blood, removing them from 7 c.c. of the blood by centrifuging. He kills the leucocytes by freezing, and then suspends them in 1 c.c. of salt solution, which he injects. He claims to have obtained beneficial results in pneumonia by this method. Antiferment. — The antiferment treatment of in- fections was introduced by A. Miiller in 1907. In the destruction of the polymorphonuclear leucocj^tes dur- 66 APPLIED IMMUNOLOGY ing suppuration there is liberated from them a pro- teolytic ferment known as proteolysin which has the power of dissolving the albumin of the tissues and thus breaking down the limiting wall of the abscess and allowing it to extend. Theoretically, therefore, much good would be accomplished if this tissue de- struction could be inhibited. Normal blood-sermn con- tains antiferments which neutralize the proteolysis of the leucocytes, but this under most conditions of in- flammation is unable in sufficient quantity to reach the tissue that is being broken down. JVItiller there- fore reconmiended the direct introduction of the anti- ferment in large amount by injecting normal serum into the inflamed area, or by packing abscess cavities, after evacuation of the pus, with gauze saturated with the serum. The patient's own blood-serum or ascitic or hydrocele fluid is recommended for the purpose. Where it is impractical to obtain these, normal horse serum can be substituted. While this form of treat- ment has some theoretical justification, practically it has not met with any notable success. The injection of acute abscesses is not unattended with danger of anaphylaxis. Irrigation of abscess cavities or pack- ing with gauze saturated with the fluid may in some cases be a useful adjunct to the ordinary surgical measures. MISCELLANEOUS SERA AND EXTRACTS 67 Anticarcinomatous Extracts. — Extracts have been prepared from various normal and abnormal animal tissues for use in the treatment of cancer. While cer- tain of these gave great promise at first and ap- parently brought about cures, none of them so far have stood the test of time. Again, certain substances that have given successful results in the cure of the arti- ficially implanted malignant tumors in lower animals, have not had the same effects in corresponding disease in man. Among non-specific products that have been tried for this purpose may be mentioned extracts of thymus and thyroid glands, spleen, and pancreas. Coca and Gilman prepared emulsions from human carcinomata and injected them into patients suffering from growths of similar histology. This method was hailed at first as the long-looked- for specific non-surgi- cal treatment for cancer, but has not lived up to ex- pectations. The injection was supposed to stimulate the formation in the patient's blood of substances capa- ble of dissolving the tumor tissue. Recognizing that malignant tumors are analogous in histological and physiological characteristics to embryonic tissues, Fichera {Lancet, Oct., 1911) announced the results of his experiments with autolysates of fetal tissues in patients suffering from carcinoma. Of 36 patients, in 18 the results were inconclusive, in 8 there was no 68 APPLIED IMMUNOLOGY marked benefit, in 5 there was distinct benefit, while 5 were apparently cured. The autolysates were made by mincing fetal and embryonic tissues, placing them in normal salt solution and incubating for two months. The clear fluid was then injected subcutaneously or directly into the neoplasm. Recently, Babcock {In- ternational Clinics^ Vol. II, 23rd Series, 1913) has reported a trial of fetal autolysates prepared by Fichera's method in 21 cases of recurrent or inoper- able cancer, and concludes that this method is of no distinct value in the treatment of malignant disease in man. It would seem, therefore, that Fichera's treat- ment is doomed to the same failure that has so far attended all other attempts at cure of carcinoma by organo- or serum therapy. Pyocyanase, — Pyocyanase is a ferment isolated from cultures of B, pyocyaneus, said to have high di- gestive powers for proteids. It has been employed in the treatment of diphtheria, the protein digestive action facilitating the removal of the membrane. Pyo- cyanase has also been used in gonorrhoeal urethritis, in follicular tonsillitis and as a spray for disinfection of the nose. Antithyroid Serum and E attracts, — Working upon the established theory that the symptoms of ex- ophthalmic goitre were due to a hypersecretion of the MISCELLANEOUS SERA AND EXTRACTS 69 thyroid gland, investigators have attempted to formu- late a method of treatment upon rational lines of or- ganotherapy. In 1899 Otto Long employed the milk of thyroidectomized goats, believing that this pos- sessed some property antagonistic to the gland secre- tion. Under the name of rodagen, a powder was pre- pared from the milk of thyroidectomized goats, 50 per cent, of which was composed of the active constituent of the milk and 50 per cent, sugar of milk. The dose of this is 1 to 3 drachms a day. Considerable improve- ment was noted following its use in some cases, but no disappearance of the exophthalmos, the goitre, or the tachycardia. Others have employed antithyroid serum from the blood of thyroidless sheep in doses of 1 to 5 c.c. daily. This had no conspicuous effect. Thyroi- dectin, a powder consisting of the desiccated blood of thyroidectomized animals, has also been used, it is claimed, with marked benefit in some cases of exoph- thalmic goitre. The dose is one or two 5-grain cap- sules three times a day. None of these preparations can be said to have fulfilled expectations. Crotalin in Epilepsy, — The apparent improve- ment in an epileptic after being bitten by a rattlesnake led Spangler to treat a number of epileptics by injec- tions of increasing doses of rattlesnake poison or cro- talin. Spangler reported a certain number of favora- 70 APPLIED IMMUNOLOGY ble results, which, however, have not been confirmed by others. The irrational basis for this treatment and the dangerous nature of the poison are such as to make its use unjustifiable. Among others, Yawger (Penna. Med. Jour,, Sept., 1914, p. 964) , after a trial of crotalin in six cases of idiopathic epilepsy, gives the results of his experience as follows : " Two patients were uninfluenced; two were worse during the treat- ment; one, early in the course, developed such in- tolerant toxic symptoms that further experimentation was im justified, and the last patient died two and a half months after treatment. While we did not feel that death resulted from the use of crotalin, the pa- tient's disease certainly was not benefited by the treatment." Phylacogens. — Within the last year or two con- siderable exploitation has been given to certain bac- terial products for the treatment of infections of all kinds, under the name of phylacogens. It is claimed by the manufacturers that these " modified bacterial derivatives '* are superior to bacterial vaccines as ordi- narily prepared. Phylacogens are sterile aqueous solutions of bacterial derivatives prepared by growing the bacteria in artificial culture media, killing them by heat, and then removing their soluble products by filtration. In preparing special phylacogens for the MISCELLANEOUS SERA AND EXTRACTS 71 treatment of various diseases, the mixed products of several varieties of bacteria are employed, whether these varieties of bacteria are found in the particular infection or not. Without going into further detail, we are of the opinion that the therapeutic use of phylacogens is based upon erroneous conceptions of infection and im- munity, is unscientific, empirical, and unjustified. Moreover, there is evidence that their administration is not unattended with risk to the patient. IX AGGLUTININS THE WIDAL PHENOMENON AND OTHER AGGLUTINATION REACTIONS Agglutinins are antibodies belonging to the second order of Ehrlich (see Chapter III). They are formed in the blood-serum as a result of infection with specific bacteria. Agglutinins have the power of causing the specific bacteria, in response to which they have been called forth, to lose their motility and to be drawn together in clumps. Agglutinins are probably not very important factors in immunity, but are of great value for diagnostic pm'poses, as their ac- tion can be studied in vitro. Thus blood-serum in various dilutions, when brought in contact with a cul- ture of the particular organism that has infected the patient, will after a time cause loss of motility and clumping of the bacteria in the culture. The principal infections in which agglutination tests have proved most serviceable in diagnosis are those caused by ty- phoid, paratyphoid, dysentery, and colon bacilli. These tests are of great value, sometimes in differen- tiating closely related organisms. Agglutinins be- long to what are known as " group-reaction " anti- bodies, that is to say, they act upon closely allied 72 AGGLUTININS 7S members of the same group of bacteria, such as B, typhosus, and B, paratyphosus A and B, though not with the same intensity. Members of this group can be differentiated by using different dilutions of the agglutinating serum, the specific organism involved being agglutinated by the greatest dilution of serum. Agglutination tests are occasionally employed in the diagnosis of glanders, cholera, Malta fever, and in staphylococcus and streptococcus infections. The Widal Reaction The agglutination test for the diagnosis of typhoid fever is known generally as the Widal reaction, be- cause this investigator, though by no means the dis- coverer of the method, first used it extensively in clinical work. There are two methods of carrying out the test: microscopically and macroscopically. Of these, the microscopic method is that more generally employed. A. MICROSCOPIC METHOD For the performance of the test we require blood- serum from the suspected patient, and a recent culture of typhoid bacilli. The description of the test may therefore be divided into three portions, viz.: {a) collection and dilution of the patient's blood-serum; (b) prepara- 74 APPLIED IMMUNOLOGY tion of the culture of typhoid bacilH ; ( c ) teclmic of the test. (a) Cpllection and Dilution of the Patient's Serum. — There are several methods of collecting blood from the patient, which will be given in the order preferred. (1) By means of Wright's capsule: A piece of %-inch glass tubing is drawn out to a fine capillary stem at each end, leaving a portion with the original calibre about 2 inches in length. One of the capillary ends of the tube should be bent (see Fig. 22, B), The Fig. 4. — Capillary teat pipette for removal of serum from clotted specimen of blood. patient's blood is drawn into the curved end of the cap- sule by capillar}^ attraction from a puncture of the finger or lobe of the ear. When the tube is almost full, the ends can be sealed in a small flame. After the blood has clotted and the serum separated, a file mark is made at a convenient level on the tube, which is then broken, and the clear serum drawn off by means of a capillary pipette (Fig. 4). Any desired dilution of the serum can now readily be made by taking one drop of serum from the pipette, and the required number of drops of distilled water with the same pipette. The dilutions usually employed in performing the Widal AGGLUTININS 75 reaction are 1 in 40 and 1 in 80. The dilutions are conveniently made in small test-tubes or watch-glasses. For the 1 in 40 dilution, one drop of serum is placed in the test-tube or watch-glass by means of the pipette, and 19 drops of distilled water are then added with the same pipette. This makes a 1 in 20 dilution of the serum, a platinum loopful of which added to a loopful of bouillon typhoid culture gives the desired 1 in 40 dilution. Similarly for the 1 in 80 dilution, one drop of serum and 39 drops of water are mixed in a test- tube, making a dilution of 1 in 40. A loopful of this together with a loopful of typhoid culture gives the 1 in 80 dilution. This is the most reliable method of preparing the patient's serum, as it is free from red cells, and the dilution is accurate. (2) By means of a leucocyte counting pipette: The blood is di-awn up into the leucocyte counting pipette of the Thoma-Zeiss apparatus, as far as the 0.5 mark, just as for a leucoc}i;ic count. Instead of the acetic acid used in the latter procedure, however, the tube is filled up to the 11 mark with distilled water, thus immediately giving a dilution of 1 in 20. This is the most rapid method of preparing the patient's blood, and works very satisfactorily in hospital prac- tice, where the test is usually carried out immediately lafter collection of the blood. The red cells interfere very little, if any, with the reaction. 76 APPLIED IMMUNOLOGY (3) Employment of dried blood: Two or three drops of patient's blood are collected upon a clean glass slide or non-absorbent paper and allowed to dry there. In making the dilution, enough water is added to the dried blood to replace that lost in drying, and the clot well broken up in it by means of a platinum needle. The dilution can then be made by drops from a capillary pipette as described above. This method is useful in cases where the necessary apparatus for either of the other two methods is not at hand, and where the blood has to be sent for examination a dis- tance. While the matter of dilution by this means is largely guesswork, reliable results can be obtained from it, though if possible it is better to use one of the other methods above described. {b) Preparation of the Culture of Typhoid Bacilli. — A stock agar culture of typhoid bacilli should be kept at hand, and should be renewed by transfer- ring to a fresh agar tube every three or four months. For use in the test a bouillon culture is made from this. The bouillon culture should be 24 to 48 hours old, grown at room temperature. In such a culture the typhoid bacilli are found to be present in sufficient numbers, of large size, and very motile. A satisfac- tory bouillon culture can be kept constantly on hand by inoculating a fresh tube with the preceding one every 48 hours. If the bacilli die out or prove other- AGGLUTININS 77 wise unsatisfactory, a bouillon tube can be inoculated from the original agar culture. Before using a cul- ture for the Widal test, the organisms should always be examined for motility. (c) Technic of the Widal Reaction. — The micro- scopic test is usually carried out with a depressed hanging-drop slide. The edge of the depression is ringed with vaseline. Upon a clean cover-slip are placed one platinum loopful of the bouillon culture of typhoid bacilli, and one loopful of previously diluted 1 in 20 patient's serum, giving a dilution of 1 in 40. The slide is pressed down upon the cover-glass, and quickly inverted so that the hanging-drop remains in the centre of the depression. A similar slide is pre- pared with a dilution of 1 in 80, and a third slide with culture only, to servx as a control for the motility of the typhoid bacilli. Each slide is marked with the dilution and time of beginning the test by means of a grease pencil.^ Each slide in turn is now placed under the microscope, the high-power dry objective (% in.) being used. The typhoid bacilli will be seen to move in all directions through the field. In from % to 1 hour in the case of a positive reaction, all the typhoid bacilli * We have found that plain glass slides can be used with equal satis- faction, as reconunended by Stitt. A ring of vaseline is made on the centre of the slide, which is then gently allowed to rest on the cover-glass without any other pressure than its own weight. Sufficient space is thus preserved to allow of motility of the organisms. 78 APPLIED IMMUNOLOGY in dilution 1 in 40 and probably also in 1 in 80 will have lost their motility, and most of them will be collected together in clumps throughout the field (Fig, 5) . In the control slide, containing no serum, the organisms at the end of this time \W11 still retain their motility, and there will be no clumping ( Fig. 6 ) . In case of a negative reaction, the bacteria in all the slides remain motile. If the slide in which a positive reaction Fig. 5. — Widal's test, positive (aggluti- Fig. 6. — Widal'e test, negative, nation or clumping). is taking place be observed from time to time, at first a few of the bacteria will be seen to lose their motility, and one will become attached to another, giving a V- or Y-shaped appearance. These groups will gradu- ally be added to by other bacilli until clmnps are formed and all motility is lost. Various grades of in- tensity of reaction are seen, ranging from formation of a few clumps and many organisms retaining their AGGLUTININS 79 motility to complete clumping and loss of motility of all. The degree of clumping should be reported as a partial reaction, which, although suggestive, is not necessarily specific for typhoid fever. Various grades of intensity are sometimes seen in the different dilu- tions. A positive reaction with 1 in 40 dilution may practically always be regarded as specific for typhoid fever, though occasionally blood of patients infected with B, paratyphosus A or B may give a positive re- action in this dilution with typhoid bacilli. A positive result with 1 in 80 dilution renders the diagnosis doubly sure. B. MACROSCOPIC METHOD OF PERFORMING THE WIDAL REACTION For this method small test-tubes, such as are em- ployed in the Wassermann reaction, are used. Serum in different dilutions is placed in the tubes by means of small pipettes, and quantities of bouillon culture of typhoid bacilli are then added. A control tube con- sisting of the culture alone is also used. At the end of 12 to 24 hours at 37° C, in the case of a positive re- action, the bacilli become clumped and fall as a sedi- ment to the bottom of the tube, leaving a clear fluid above. In the case of a negative reaction and in the control tube, the fluid remains uniformly cloudy. The reaction can be studied best by means of a reading glass. In this method killed cultures of the typhoid 80 APPLIED IMMUNOLOGY bacilli may be employed, and have the advantage of keeping in good condition for some time. With ordi- nary laboratory facilities, however, the microscopic method is preferable. Occurrence and Significance of the Widal Reaction The Widal reaction is usually not seen in typhoid fever until the second week of the disease, as some time is required for the production of the specific ag- glutinins in the patient's serum, upon which the test depends. The reaction usually persists for several months after recovery from typhoid fever, and per- sons have been known to react positively for years after the infection. Artificial immunization with ty- phoid vaccine also gives rise to a positive reaction which persists for some time. These facts should be remembered in weighing the results of the test per- formed in suspected cases of the disease. The diag- nosis of typhoid fever should not rest upon the agglu- tination reaction alone. Other symptoms must be con- sidered along with the laboratory test. The reaction may fail to appear in a small percentage of cases of undoubted typhoid fever, and may be positive occa- sionally in other conditions, particularly in jaundice. Probably the range of error on either side does not ex- ceed 3 per cent., so that in the Widal reaction we have one of the most important cardinal signs of typhoid fever. AGGLUTININS 81 Other Applications of the Agglutination Phenomenon For the identification of certain species of bacteria, the agglutination reaction is of the greatest value. For this purpose, agglutinins are artificially produced by injection of animals, usually rabbits, with cultui^es of the various organisms whose presence is suspected in given cases. Thus we have conditions reversed from those obtaining in the Widal reaction for typhoid fever, the serum being the known factor, and the or- ganism the unknown. For example, we have isolated an organism that we strongly suspect to be the ty- phoid bacillus. If we find that a culture of this or- ganism is agglutinated by the greatly diluted serum from an animal previously immunized to typhoid bacilli, and is not agglutinated by similar dilutions of sera from animals immunized to other organisms, such as B. jparatyphosus, dy sentence, etc., our suspicion is converted into certainty. This method is of great value, especially in the detection of typhoid bacilli in milk and other fluids, as well as in other procedures. X PRECIPITINS SIGNIFICANCE AND APPLICATION OF PRECIPITINS— TECHNIC OF REACTION— SPECIFIC IDENTIFICATION OF BLOOD AND OTHER PROTEINS When a protein is brought in relation, in vitro, with blood-serum of an animal which has been immu- nized against that particular protein, the protein is thrown out of solution, the fluid becomes cloudy, and a sediment finally settles to the bottom of the tube. This is known as the precipitation phenomenon, and is due to the presence in the blood-serum of substances known as precipitins. Precipitins are antibodies formed in the blood in response to injection of unorganized pro- tein material. They are antibodies of the second order of Ehrlich, and are analogous to agglutinins, which act upon organized proteins or bacteria. The precipitation phenomenon is made use of in the identification of various proteins. Each species of ani- mal has a specific protein, which can be detected by the precipitation reaction. Practically, the test has been made use of chiefly in ( I ) detection of adulteration of meat products such as sausages, etc. ; and ( II ) in the medicolegal identification of blood. (I) Detection of Adulteration of Meat Products This test can be applied to determine whether sausages represented as being made purely of beef or 82 PRECIPITINS 83 pork, contain horse meat. Preparation for the test consists in the immunization of animals, of which rab- bits are the most suitable, by injections of extracts made from the various meats to be tested for, as horse, beef, pork, etc., each of which has a specific protein. The immune serum, on being brought in contact with an extract of the particular meat whose protein is specific for it, will bring about precipitation. (II) Medicolegal Identification of Blood This test occasionally assumes the utmost impor- tance when the question arises in murder cases as to whether blood stains are human or not. The chemical tests for blood do not distinguish one species from another, while microscopical examination of dried blood is unsatisfactory. Dried blood in very small quantities, however, will respond to the precipitation reaction, and by this means we can differentiate human blood from that of the lower animals. Uhlen- huth, Wassermann, and Schiiltz were the first to make use of the precipitation test in this connection. The Precipitin Reaction The technic of the reaction is divided into two parts. ( a ) Production of the Precipitating Serum, — For this purpose rabbits are employed. Three or four in- 84 APPLIED IMMUNOLOGY travenous injections of human blood-serum are made into the rabbit in quantities of 1-2 c.c. every five or six days. The strength of the antihuman serum thus formed may be tested after three or four injections. This is obtained by piercing a vein in the ear of the rabbit, collecting a few cubic centimetres of blood, al- lowing the clear serum to separate, and titrating it with different dilutions of human serum. The dilu- tions are to be made with normal salt solution. The antiserum may be regarded as of sufficient strength when 0.1 c.c will produce distinct cloudiness when mixed with 1 c.c. of human serum in 1-1000 dilution after incubation for five minutes at 37° C. The anti- serum, moreover, must give no cloudiness with plain salt solution, nor with the serum of any other animal in 1-200 dilution after half an hour at 37° C. The fol- lowing table illustrates the titration of a suitable anti- human serum : Antihuman rabbit serum Human serum 1 c.c. Controls Result after incuba- tion for y^ hour at 37° C. 0.1 0.1 0.1 0.1 0.1 0.1 0.1 1-10 l-IOO 1-1000 1-5000 1-10000 1 c.c. normal salt solution 1 c.c. normal guinea-pig ser- um (1-200) Marked turbidity- Marked turbidity- Marked turbidity- Turbidity Slight turbidity- Clear Clear PRECIPITINS 85 (b) Identification of Blood. — An extract of the blood clot is made with normal salt solution. This is then filtered until absolutely clear, and diluted until we have approximately a 1-1000 solution of the serum. The latter point can be determined by boiling a por- tion of the solution, as a 1-1000 dilution will then show a slight haziness, while greater dilutions remain clear. One-tenth c.c. of the antihuman rabbit's serum is then mixed with 1 c.c. of the suspected serum thus pre- pared, and incubated for half an hour at 37° C. In another tube 0.1 c.c. of normal rabbit serum is placed with 1 c.c. of the suspected serum. In a third tube is placed 0.1 c.c. of antihuman rabbit serum and 1 c.c. of normal salt solution. The results in the case of a positive reaction are shown in the following table : Antihuman rabbit serum Suspected blood Normal salt solution Normal rabbit serum Result. S?" C. After 5 minutes After half hour 1 2 3 c.c. 0.1 0.1 1 (1-1000) i" (i-ioob) c.c. 1 0^1 Turbidity Negative Negative Marked turbidity Negative Negative It has not been found possible to employ this test for differentiation of the blood of various races of human beings, such as negro from Caucasian, etc. The same test can be used for the blood of various lower animals by preparing suitable antisera. Closely related species, however, such as hens and pigeons, are not differentiated by it. XI LYSINS BACTERIOLYSINS AND CYTOLYSINS (HEMOLYSINS) AiviONG specific antibodies of the third order of Ehrlich we find hacteriolysins, which are substances acquired by the blood-serum of an animal that is im- munized to specific bacteria. Bacteriolysins have the property of dissolving the particular bacteria for which they are specific. Analogous substances are likewise formed against body-cells known as cytoly- sins. Cytolysins that act upon red blood-corpuscles are called hcemolysins. In order for the phenomenon of bacteriolysis to take place, in addition to the specific bacteriolysin (also known as amboceptor) , the pres- ence of a second or non-specific substance is necessary. This substance is known as a complement, and is pres- ent in varying amount in the blood-serum of all ani- mals whether immunized or not. The same comple- ment, being non-specific, may take part in any bac- teriolytic or cytolytic reaction. Amboceptors are said to be thermostable, Le., they are not destroyed by heat- ing to 56° C, while complement is thermolahile , and is destroyed at a temperature of 5Q° C. The non- specific element or complement can therefore be re- 86 LYSINS 87 moved from a serum by heating the latter to this tem- perature, rendering the serum inactive although it con- tains the specific element or amboceptor. The serum can be made active again by adding more complement in the form of fresh unheated normal serum. This discovery that two elements, one specific and the other non-specific, are necessary for bacteriolysis, was made by Bordet. Pfeiffer's Phenomenon. — Our knowledge of bac- teriolysins is dependent largely upon the following experiment carried out by PfeifFer. He injected a suspension of cholera spirilla into the peritoneal cavity of guinea-pigs that had been previously immunized to cholera spirilla, and also into non-immunized guinea- pigs. In the case of the non-immunized animals the bacteria increased in number until they destroyed the animals. In the case of the previously immunized guinea-pigs, examination of the peritoneal fluid from time to time showed that the bacteria quickly died, be- came clumped together in granules, became broken up, and finally were dissolved. Practically, this phenome- non may be made use of in the identification both of bacteria and of specific immune sera. It is especially of value in the identification of cholera spirilla. For this purpose, guinea-pigs previously immunized to chol- era are injected intraperitoneally with the suspected organisms, and the peritoneal fluid examined from 88 APPLIED IMMUNOLOGY time to time for the occurrence of bacteriolysis. Pas- sively immunized animals may also be employed for the observation of Pf eiff er's phenomenon. That is, the reaction will occur on injection of a mixture of cholera immune serum and a culture of cholera spirilla into the guinea-pig. This phenomenon can also be observed in vitro, by mixing immune serum and bacteria in a test- tube, and incubating at 37° C. for some time. What has been said of bacteriolysins regarding the manner in which they operate, applies likewise to cytolysins and haemolysins. Thus, for example, if a specific immune serum, which has been prepared by injecting the blood-corpuscles of a sheep into the peri- toneal cavity of a rabbit, is mixed with the corpuscles of the sheep, and incubated at 37° C, the sheep's cor- puscles will after a short time be dissolved (haemoly- sis). If the rabbit's serum be previously heated to 56° C. for half an hour, haemolysis will not take place because the complement or non-specific factor in the reaction will have been destroyed. The immune serum, though it still retains its specific factor or hsemolysin (amboceptor), has been inactivated. The serum can be re-activated by the addition of fresh normal serum of any animal, thus supplying complement, after which the hsemolytic action will take place as before. In the reaction by antibodies belonging to the third order of Ehrlich, whether they be bacteriolytic, LYSINS 89 cytolytic, or hasmolytic, the substances acted upon (bacteria, blood-corpuscles, etc.) are known as anti- gens; the specific antibody (bacteriolysin, cytolysin, hsemolysin) is spoken of as amboceptor; while the non- specific factor in the reaction is known as complement. Various diagnostic tests of the greatest practical importance have this reaction as a basis, and will re- ceive consideration in the following chapter. XII FIXATION OF COMPLEMENT PRINCIPLES OF THE REACTION— BORDET-GENGOU PHE- NOMENON—WASSERMANN-NEISSER-BRUCK MODIFICA- TION—TECHNIC OF THE WASSERMANN REACTION IN THE DIAGNOSIS OF SYPHILIS— MODIFICATIONS OF THE WASSERMANN REACTION— HECHT-WEINBERG MODIFI- CATION—CLINICAL APPLICATION OF THE WASSER- MANN REACTION— EFFECTS OF TREATMENT ON THE WASSERMANN REACTION In the foregoing chapter we have seen that bac- teriolysis, cytolysis, and hsemolysis take place by means of a specific substance known as amboceptor, and a non-specific substance called complement. This imion of antigen, amboceptor, and complement is spoken of as fixation or ahsoi'ption of complement. The exact nature of the reaction is not known. In order that the complement-fixation reaction be under- stood in its application to specific diagnosis, it is neces- sary to explain first in some detail the process of hgemolysis. When red blood-corpuscles remain in sus- pension in a fluid, the fluid has an opaque red color. If haemolysis occurs, the haemoglobin leaves the cor- puscles, the fluid becomes transparent, and has a deep red color. If no haemolysis occurs, the corpuscles gradually fall to the bottom of the tube, leaving clear, colorless fluid above. Haemolysis can occur in a non- 90 FIXATION OF COMPLEMENT 91 specific manner, i.e,, without amboceptor and comple- ment, through several agencies. Thus, if the cor- puscles be placed in plain water, haemolysis will occur, and likewise through the addition of various substances, such as snake venom, tetanus toxin, etc. Specific haemolysis differs from non-specific in that it will take place in an isotonic fluid such as physiologic salt solution. In haemolysis there is no actual solution of the cell. The process consists rather in a disturbance of the os- motic equilibrium between the cell contents and the surrounding medium. The haemolytic amboceptor combines with the stroma of the cell, increases the permeability of the latter, setting free the haemoglobin into the surrounding medium. Bacteriolysis is de- pendent upon a similar mechanism, and differs from haemolysis in that it is practically invisible to the naked eye. The blood of most animals usually contains a cer- tain amount of natural haemolysin for the red cor- puscles of other animals. Thus, human blood-serum possesses a considerable natural haemolytic power for sheep's corpuscles. This normal haemolytic power can be greatly increased artificially by injection of the blood-corpuscles of one animal into another, so that very high dilutions of the second animal's serum will haemolyze the corpuscles of the first, in the presence of 92 APPLIED IMMUNOLOGY suitable amounts of complement. In experimental and diagnostic work, the rabbit is the animal usually employed for the production of specific hemolytic serum. By three or four intraperitoneal or intraven- ous injections of suitable quantities of washed sheep's corpuscles into the rabbit at intervals of 3 or 4 days, the rabbit's serum will become so highly immunized that it will, in 1-1000 or greater dilution, hsemolyze an equal quantity of 5 per cent, suspension of sheep's corpuscles in the presence of a sufficient amount of complement. It has been found that complement is present in varying amounts in the blood-serum of dif- ferent animals, but that the serum of the guinea-pig is most constant in this respect. So in order to insure the presence of a definite amount of complement, the immune hsemolytic serum (rabbit) is heated at 56° C. to destroy the complement present, and a definite amount of fresh guinea-pig serum afterwards added. The technic of the preparation and titration of specific hsemolytic serum will be given under the description of the Wassermann reaction. The Bordet-Gengou Phenomenon Bordet and Gengou were the first to employ the complement-fixation reaction in the diagnosis of spe- cific infections. Since the phenomena of bacteriolysis and of haemolysis each depend upon the presence and FIXATION OF COMPLEMENT 93 fixation of a common non-specific substance termed complement, these observers conceived the idea of combining the two phenomena as a means of diagnosis. The components of the first-mentioned reaction, con- sisting of the bacterial suspension or antigen, the spe- cific antibacterial serum or amboceptor, and comple- vient, together form the bacterial system; the second part is known as the hcemolytic system, consisting of blood-corpuscles (antigen), specific hsemolytic serum (amboceptor) , and complement. Bordet and Gengou took a suspension of cholera spirilla as antigen and mixed it with a certain quantity of cholera-immune serum made by injecting a rabbit with cholera spirilla, together with a definite amount of fresh guinea-pig serum as complement. This mixture was incubated at 37 "" C. for one hour. At the end of this time it was pre- sumed that the interaction between antigen and ambo- ceptor had taken place with fixation or absorption of complement. But since the reaction was not percepti- ble to the eye, a further procedure became necessary to show the result. For this purpose, a suspension of washed rabbit's blood-corpuscles and a quantity of hsemolytic serum for the rabbit's corpuscles were added, and the whole incubated for another hour. At the end of this time, it was found that no haemolysis of the rabbit's corpuscles had taken place, because the complement had been fixed during the first incubation 94 APPLIED IMMUNOLOGY by the bacterial system, none being left free to act in connection with the hsemolytic system. In a second experiment, exactly the same compon- ents were used, except that the cholera-immune serum was replaced by normal serum. Hence, complement was not fixed by the bacterial system, but remained free to become a part of the hsemolytic system, and haemolysis occurred. The first or positive and the second or negative re- actions may be graphically illustrated thus : 1. Positive Result Suspension of cholera spirilla Haemolytic serum No ^ v^ haemolysis X _^^,,^ Complement Cholera-inamune serum '^''''''^ Red corpuscles 2. Negative Suspension of cholera spirilla Haemolytic serum Complement ^ Haemolysis ^^ T Normal serum Red corpuscles The specific complement-fixation reaction may be applied to the diagnosis of several diseases. By test- ing an unknown serum with a known bacterial anti- gen, it is thus possible to determine whether or not the individual furnishing the serum is infected by the par- ticular organism from which the antigen is made. For some time after Bordet and Gengou's announcement, the reaction had chiefly a scientific rather than a prac- tical interest. The first great advance toward render- ing the method of practical importance was the dis- FIXATION OF COMPLEMENT 95 covery by Wassermann and Bruck that extracts fur- nished by lysis of the bacteria will sei^'e as antigens for the complement -fixation reaction equally as well as, if not better than, suspensions of the bacteria them- selves. This fact was applied by the workers men- tioned in the diagnosis of typhoid fever, tuberculosis, meningitis, etc. The Wassermann Reaction A further step was now taken by Wassermann, Neisser, and Bruck. They recognized what an enor- mous value the reaction would have if it could be ap- plied to syphilis, the causative organism of which had not been artificially cultivated up to that time (1906) . So these observers conceived the idea of employing as antigen, extracts of organs kno\\Ti to be rich in tre- ponemata (spirochastae). From this the now uni- versally used and important " Wassermann reaction " had its origin. In their first experiments Wassermann, Neisser, and Bruck made water}^ extracts from syphi- litic fetal liver, in which Treponemata pallida are very abundant. Using these extracts they obtained fixa- tion of complement with the sermn of syphilitic in- dividuals, and from their results concluded that the re- action was specific, just as that obtained in the case of typhoid bacillus extract and typhoid serum. But it was then found that alcoholic extracts of syphilitic tis- 96 APPLIED IMMUNOLOGY sues would give the reaction in syphilis with equally good results. Since it is the lipoid material in tissue that is dissolved by alcohol, it followed that this lipoid material must contain the substance that is antigenic for syphilitic serum. It was then found by Marie, Levaditi, and Land- steiner that complement-fixation with syphilitic serum occurred when alcoholic extracts of normal tissues, such as liver, human heart, or guinea-pig heart, were used as antigen, and that the results of reactions with these extracts were as reliable as those where extracts of syphilitic tissues were used. These observations showed that the complement-fixation reaction in syph- ilis is not entirely a specific antigen-antibody reaction as in the case of those occurring between bacterial anti- gens and antibodies, but this fact in no way lessened the practical value of the test in the diagnosis of syphilis. It has still further been shown that extracts pre- pared from lecithin, cholesterin, etc., can be used as antigens and some workers restrict themselves to these artificial products. The various forms of antigen in use at the present time account to a great extent for the divergence of results in some cases when serum from the same patient is examined by different workers. This has a tendency to throw doubt on the value of the reaction in the minds of skeptical clini- FIXATION OF COMPLEMENT 97 cians. A great deal of work is being done upon the antigen question, and it is hoped that in the near fu- ture some definite standard will be adopted and uni- versally employed. We believe, in the light of pres- ent knowledge, that in the Wassermann reaction there is probably a specific factor as well as a non-specific, certainly that the organisms of syphilis cause an over- production of the special lipoid antigenic substance in the tissues, and that the most reliable results will be obtained by using an alcoholic extract of syphilitic tissue, in which this substance is found in greatest abundance. We adhere to this view as the result of the use of syphilitic liver-extract in several thousand tests, in which our results have differed in no material way from those of reliable workers. We recently reported (J. A. M. A,, Jan. 31, 1914) the results of a trial of some cholesterinized ex- tracts of guinea-pig heart and human heart. These extracts are made by adding cholesterin to the ordi- nary alcoholic extract of normal heart in the propor- tion of 0.4 per cent. The cholesterinized guinea-pig heart-extract was used by us along with our antigen of alcoholic extract of syphilitic liver in the routine ex- amination of 100 cases, the cholesterinized human heai-t-extract being employed in thirty-three cases. Careful preliminary titration of the antigens was carried out before doing the tests, and at no time was 7 98 APPLIED IMMUNOLOGY more than one-third of the anticomplementary dose used. The comparative results of tests with syphilitic liver-extract and cholesterinized guinea-pig heart-ex- tract may be divided into four groups as follows : 1. In twenty-two cases the results were positive with both antigens, with little or no diiFerence in de- gree of reaction. 2. In eight cases the syphilitic liver-extract gave a weakly positive reaction, while the cholesterinized ex- tract gave a stronger reaction. 3. In thirty-six cases both antigens gave a negative result. 4. In thirty-four cases the syphilitic liver-extract gave a negative reaction, while the cholesterinized heart-extract gave a weakly positive or medium posi- tive result. This is naturally the most important group from the clinical point of view. Nine of these thirty-four cases were treated cases of syphilis ex- hibiting no symptoms, showing that it is more difficult to render the serum negative to the cholesterinized heart-extract by anti-syphilitic treatment. The re- maining twenty-five of this group were cases sub- mitted for diagnosis or which had no signs or history of syphilis. When one antigen reacted more strongly than the other, it was always the cholesterinized heart- extract. In no case did the syphilitic liver-extract give a stronger reaction than the cholesterinized extract. FIXATION OF COMPLEMENT 99 In the comparative tests with the cholesterinized extract of human heart, of thirty-three cases there were six in the first group, that is, equally positive with the two extracts; four in the second group, in which the cholesterinized human heart-extract gave a distinctly stronger reaction than the syphilitic liver antigen; sixteen in the third group, in which both antigens gave negative results; seven in the fourth group, which were negative with the syphilitic anti- gen but positive with the cholesterinized heart-extract. In the first series, therefore, the results with the two antigens disagreed in 42 out of 100 cases, and in the second series in 11 out of 33 cases. We see from these results that the cholesterinized extracts appar- ently give a more " delicate " reaction than the ex- tract of syphilitic liver, but we also find that many weakly positive results are obtained by them in non- syphilitic cases. This fact alone is quite sufficient, in our opinion, to offset any advantage in delicacy of the reaction obtained with the use of cholesterinized ex- tracts. We feel that just as high a percentage of positive results is obtained in known or clinically ap- parent syphilitics with the syphilitic liver-extract as is obtained with the cholesterinized antigens, and the former do not give positive results in non-syphilitic cases. The success in detecting mild degrees of syphi- litic infection is doubtless due in part to the use of one 100 APPLIED IMMUNOLOGY properly standardized unit of complement and hsemo- lytic amboceptor, as suggested to us by Laird, instead of double or triple units as employed by many workers. We must conclude that with cholesterinized antigens, varying degrees of inhibition of hsemolysis may be obtained in serums from many conditions other than syphilis, and in normal persons. While ex- perimental investigations are to be highly commended, we therefore regard the employment of these artificial antigens for routine clinical use at the present stage of our knowledge, instead of being an advance in sero- logic technic, rather as a distinct step backward. Schamberg, Kolmer, and others report that they obtained positive Wassermann reactions, using the cholesterinized antigens in over 28 per cent, of twenty- two cases of psoriasis, in a great many of which syphi- lis could almost certainly be excluded, thus providing evidence that weak reactions with these antigens do not necessarily mean syphilis, and that a diagnosis of syphilis cannot be based on weak and medium inhibi- tions when they are employed. We hold that weakly positive reactions with syphilitic liver-extract mean nothing but syphilis. Even though it were true that the cholesterinized antigens give a more " delicate " reaction and may furnish positive results in cases of syphilis that are negative to the syphilitic liver-ex- tract, it is a very much less serious error to overlook an FIXATION OF COMPLEMENT 101 occasional case of syphilis than to saddle a diagnosis of the disease with all it entails on a patient who does not have the disease. Considerable harm is being done at present by the use of unreliable non-specific or artificial extracts, in two ways: 1. The marked discrepancies between the results of the Wassermann test and the clinical findings in many cases are causing skeptical clinicians to lose con- fidence in the value of the reaction, and thus they are being deprived of an important diagnostic and thera- peutic aid, 2. A great many unfortunate persons are being treated for syphilis who have not and never had syphi- lis, as the result of weakly positive and doubtful re- ports of workers using these antigens. Since Noguchi, in 1911, first cultivated the Tre- ponema pallidum in a pure state, much work has been done by Noguchi, Kolmer, and others with antigens made from the treponemata themselves. The results have been disappointing for diagnosis of pri- mary and early secondary syphilis, as only a relatively small proportion of these cases gives positive reactions when the spirochsetal antigens are employed. But the positive reactions obtained in late secondary and ter- tiary cases do prove that there is a specific fixation of complement occurring when lipoidal substances are 102 APPLIED IMMUNOLOGY brought in relation with syphihtic serum, but as yet we are in the dark in regard to the true nature of the phenomenon. Several factors other than the antigen are impor- tant in obtaining reliable results with the Wassermann reaction; the most essential of which is the titration and use of minimal amounts of complement. Technic of the Wassermann Reaction The following is a list of the apparatus, animals, etc., required, and has been made as complete as possible : Electric centrifuge. Several 15 c.c. graduated centri- fuge tubes. Several 1 c.c. pipettes, graduated in tenths of a c.c. Several 1.2 c.c. pipettes, graduated in hundredths of a c.c. Two 10 c.c. pipettes, graduated in tenths of a c.c. Capillary pipettes, prepared by drawing out 3/8 in. glass tubing. Rubber nipples for capillary pipettes. 100 1 c.c. glass ampoules. 100 small test-tubes, about 5 c.c. capacity. Several flasks, capacity 500 c.c. Two flasks, 100 c.c. capacity. Two graduated 100 c.c. cylinders. Several Petri dishes. Normal salt solution (0.85 per cent.). Sodium citrate solution (1 per cent, in normal salt solution). Wire racks for holding small test- tubes. Platinum loop with glass handle. Scissors, scalpel, dissecting forceps. 2 c.c. all glass hypodermic syringe with small needle. Wax pencil. 95 per cent, alcohol, ether, xylol, glycerin. Water-bath. Centigrade thermometer. Incubator. Bunsen burner. Refrigerator. Sheep, rabbits, guinea-pigs. All glass-ware and solutions mentioned above must be sterilized before using. FIXATION OF COMPLEIVIENT 103 HEMOLYTIC System We employ the anti-sheep hasmolytic system, i.e., washed sheep's red blood-corpuscles and anti-sheep hemolytic rabbit serum. As the hsemolytic ambocep- tor requires some time in preparation, its production will be described first. This anti-sheep heemolytic serum — which hereafter for the sake of brevity will be termed amboceptor — is prepared by injecting quantities of washed sheep's red blood-corpuscles into a rabbit until in the presence of a suitable amount of complement the rabbit's serum in high dilution will dissolve corpuscles from the sheep when properly in- cubated in vitro. Collection of Sheep's Blood. — The collection of blood and preparation of suspension or red corpuscles as described below is the same for the test itself as for injection into the rabbit. A sheep may be kept on the premises, or the blood may be obtained from a nearby slaughter-house. The latter if available saves a good deal of time. The sheep's blood can be drawn from the internal jugular vein by plunging a needle of large lumen into the vein and allowing the blood to fall into a flask containing pieces of glass or wire. The blood is defibrinated and thus prevented from clotting by shaking the flask immediately after collection of the blood. For the reaction, a 5 per cent, suspension of sheep's corpuscles is employed. Whole blood consists 104 APPLIED IMMUNOLOGY approximately of one-half serum and one-half cor- puscles. To make up a 5 per cent, suspension there- fore, 1 c.c. of the defibrinated blood is placed in a graduated centrifuge tube by means of a 1 c.c. pipette, and the contents of the tube brought up to 10 c.c. by the addition of normal salt solution. This tube then will contain 0.5 c.c. or 5 per cent, of sheep's corpuscles. Several tubes can be prepared in the same way, to furnish sufficient suspension of corpuscles for the test. A convenient method used by us for obtaining blood from the sheep is as follows : In several gradu- ated centrifuge tubes are placed 9 c.c. of 1 per cent, sodium citrate solution in normal saline. The sheep's ear is washed off with alcohol, and one of the large veins severed with a scalpel (Fig. 7) . One c.c. of the blood is now allowed to drop into each of the tubes, the sodium citrate preventing it from clotting. This gives a 5 per cent, suspension of sheep's corpuscles. It is necessary that all serum be removed from the sus- pension, in order to avoid anaphylactic symptoms if for injection into the rabbit, and to remove all complement if the corpuscles are to be used in the test itself. This is accomplished by centrifuging the tubes for 5 or 6 minutes in an electric centrifuge, at the end of which time the corpuscles wiU be found at the bottom. The volume of the corpuscles will vary ac- cording to the length of time used in centrifuging, the FIXATION OF COMPLEMENT 105 longer the time the more closely will they be packed. From this it is easily seen that the method used of tak- ing a definite amount of whole blood in the beginning and from that calculating the dilution required is more accurate than drawing out a quantity of the centri- fuged corpuscles and making the dilution from them, Fig. 7. — Method of obtaining blood from sheep's ear. which is the method commonly employed. The clear fluid above the corpuscles is poured off, and replaced with normal salt solution, the tubes shaken, and again centrifuged. This washing and centrifuging is re- peated three or four times, in order to remove all serum. If the corpuscle suspension is to be used in the test itself, the fluid in each tube is finally brought 106 APPLIED IMMUNOLOGY up to its original volume with normal salt solution, making a 5 per cent, suspension. Practically, a larger amount of suspension can be prepared in the same number of centrifuge tubes, by originally placing say 3 c.c. of whole blood in each, washing and centrifug- ing three or four times, and after the final washing bringing the volume up to 30 c.c. For injection into the rabbit, a 10 per cent, suspension of the corpuscles is more suitable than a 5 per cent, suspension, so after the final washing the fluid in a tube containing origi- nally 1 c.c. of whole blood or 0.5 c.c. of corpuscles is brought up to a total volume of only 5 c.c. Immunization of the Rabbit to Sheep's Corpuscles, — The suspension of sheep's corpuscles prepared as described i^ove may be injected into the rabbit either intraperitoneally or intravenously. Since much larger amounts of suspension and a longer time for immu- nization are required by the former method, we have discarded it entirely in favor of the intravenous method. The latter is just as simple, and if proper precautions against infection and injection of air be taken, is quite safe. For the first injection about 1 c.c. of the 10 per cent, corpuscle suspension is drawn up into an all-glass hypodermic syringe of 2 c.c. capacity and fitted with the usual fine needle; the smaller the needle the better. The marginal vein of the rabbit's ear is made prominent by wiping it with FIXATION OF COMPLEMENT 107 xylol, and the suspension slowly injected into it through the skin in the direction of the circulation (Fig. 8). Usually, to obtain a sufficiently powerful h^emolytic amboceptor, three injections at intervals of 3 or 4 days are required. The second injection should Fig. 8. — Showing method of intravenous injection or immunization of rabbit with 10 per cent, suspension of sheep's blood-corpuaclea for the production of haemolytio amboceptor. consist of 1.5 c.c. and the third of 2 c.c. of the sus- pension. Three or 4 days after the third injection, about 2 c.c. of the rabbit's blood is drawn from the ear by puncturing a vein, and the serum tested for its hsemolytic power against sheep's corpuscles. Thus in 108 APPLIED IMMUNOLOGY ten days, or two weeks at the most, we are able to pre- pare a powerful hgemolytic amboceptor, which re- quires four or five weeks by the intraperitoneal method. Titration of Hcemolytic Amboceptor, — For use in the Wassermann test we aim to produce a hsemolytic amboceptor of such strength that 1 c.c. of not less than a 1-2000 dilution of rabbit's serum will hasmolyze 1 c.e. of a 5 per cent, suspension of sheep's corpuscles in the presence of 0.1 c.c. of fresh guinea-pig serum used as complement. Usually, by the intravenous method described, the rabbit can be immunized so that its serum in 1-3000 dilution will fulfil these re- quirements. To determine the strength of the hsemo- lytic serum, three or four days after the third injection of the rabbit, about 2 c.c. of blood are drawn into a small test-tube from a vein of the rabbit's ear. When the blood has clotted and the clear serum separated, the latter is drawn oiF and the natural complement in it destroyed by heating the serum in a water-bath for half an hour at 56° C. This deprivation of comple- ment is known as inactivation. The complement used in titrating the hsemolytic amboceptor and in the Wassermann test itself is furnished by serum of the guinea-pig, as it has been found that this animal has an almost constant amount of complement present in its blood. To obtain complement, a full-grown guinea- /^ --, -R Fig. 9. — Demonstrating method of obtaining complement by bleeding to death an anaesthetized guinea-pig. FIXATION OF COMPLEMENT 109 pig is etherized by placing its head in a glass contain- ing gauze soaked with ether, and before death occurs, the blood-vessels of the neck are severed and the blood collected in a Petri dish (Fig. 9) . More blood will be obtained if drawn before respiratory and cardiac movements cease, and from 10 to 15 c.c. should be col- lected in this manner. The blood is allowed to coagu- late, and the clear serum to separate, which it does in about two hours. The serum is then drawn off with a capillary pipette and diluted in the proportion of one part in ten with 0.85 per cent, salt solution, i.e., to 1 part of serum 9 parts of salt solution are added. The complemental property of serum is soon lost, and the guinea-pig serum should therefore be used on the day it is obtained, or not later than the following day, if kept on ice over night. The different reagents having been prepared as above described, we are now ready for the titration. The rabbit's serum, after having been inactivated, is made up in the following dilutions: 1-500, 1-1000, 1-1500, 1-2000, 1-3000, 1-4000, and 1-5000, in nor- mal salt solution. In a series of test-tubes 1 c.c. of each of these dilutions is placed, together with 1 c.c. of a 5 per cent, suspension of sheep's corpuscles and 0.1 c.c. of the 1-10 dilution of guinea-pig complement, with sufficient salt solution to bring the total volume up to 4 c.c. After their contents have been thoroughly 110 APPLIED IMMUNOLOGY mixed by inversion, the tubes are placed in the incu- bator for one hour at 37° C After this time, if the amboceptor is sufficiently powerful for practical use, the corpuscles in the tubes containing 1-500, 1-1000, 1-1500, and 1-2000 dilutions of rabbit's serum should be completely hasmolyzed. Those in the tubes con- taining higher dilutions generally show partial hsemol- ysis. The following table illustrates the titration of haemolytic amboceptor: Amboceptor dilution 1-500 1-1000 1-1500 1-2000 1-3000 1 1-4000 1 1-5000 Comple- ment 0.1 0.1 0.1 0.1 0.1 0.1 0.1 5 per cent, suspen- sion of sheep's cor- puscles p ex. o' 1 13 1 t-* 1 1 P 1 CO 1 o 1 P Result Complete hsemolysis Complete liEemolysis Complete haemolysis Complete hsemolysis Almost complete hsemolysis Partial hsemolysis Partial hsemolysis If the 1-2000 tube does not show complete haemol- ysis, the rabbit must be given another injection of sheep's corpuscles in order to raise the hsemolytic power of the serum. If the serum is found to be of sufficient hsemolytic strength, the rabbit should be etherized and bled to death from the carotid artery ' without delay, as the serum in the living animal will lose its hsemolytic power after a time. The rabbit's blood is collected in Petri dishes, and set aside on ice imtil the clear serum separates. This is drawn oif FIXATION OF COMPLE^IENT 111 with a capillary pipette, inactivated by heating for half an hour at 56° C, and stored for future use in 1 c.c. glass ampoules. Amboceptor, when collected in this way under aseptic conditions, will retain its ha^mo- lytic power for several months. We have found that the serum is less likely to deteriorate or become con- taminated when mixed with an equal quantity of glycerin, as suggested by Much. The glycerin in no way interferes with the Wassermann test. We also prefer to store the glycerinized serum in short lengths of glass tubing with sealed ends, as suggested to us by Laird, because a larger amount of air must be en- closed in the ampoules, with consequent greater risk of contamination. In the Wassermann test itself we em- ploy 1 c.c. of a 1-1000 dilution of hemolytic ambo- ceptor that gives a titre of at least 1-2000. We avoid having to open a fresh tube of amboceptor and thus wasting a great deal, by making a 1-100 dilution from one tube, placing it in sl sterile flask, and making the 1-1000 dilution from this for several days. Syphilitic System Antigen. — For reasons given above, we prefer to use as antigen an alcoholic extract made from the liver of a syphilitic foetus that has been found on micro- scopic examination to be rich in treponemata. The method usually employed in preparing such an anti- 112 APPLIED IMMUNOLOGY gen is as follows : One part of the tissue is minced and mixed with four parts of 96 per cent, alcohol. The antigenic substances are then extracted by shaking the mixture in a machine for 24 hours, when the fluid is filtered or allowed to stand until the sediment falls to the bottom of the vessel. The clear fluid is then pipetted off* and placed in dark bottles, to be used as required. This extract must be tested for antigenic power against a number of known syphilitic and known non-syphilitic sera, and must give positive re- sults with the former and negative with the latter in order to be fit for use. Furthermore, the most suit- able dose must be ascertained. When present in ex- cessive amount, the extract may cause fixation of com- plement alone in the absence of syphilitic serum. The optimal dose of antigen is that which gives complete complement-fixation (or inhibition of hsemolysis) with a syphilitic serum and three times which is re- quired to produce partial fixation without syphilitic serum. Briefly, the optimal dose of antigen should not be greater than one-third the anti-complementary dose. This dose is determined by performing the Wassermann test on a syphilitic serum using several tubes containing the serum and various amounts of an- tigen. Other series of tubes are set up containing 2, 3, and 4 times these amounts of antigen but without syphilitic serum, to determine the anti-complementary FIXATION OF COMPLEMENT lis dose of antigen. This procedure will be understood more clearly after the Wassermann test itself has been described (Fig. 10). In our own work we rely en- tirely upon an imported German extract made as de- scribed above, which we have found to retain its anti- Fig. 10. — Titration of antigen. Front row: Increasing quantities of antigen tested with 0.1 c.c. of known syphilitic serum to ascertain optimal dose. Back row: Determination of anti-complementary dose of antigen. Each tube con- tains 3 times the amount of antigen present in corresponding tube of front row, but no serum. genie power almost indefinitely, and which has very slight anti-complementary action. By its use we avoid the rather tedious preparation and titration of anti- gen, except, of course, that we make a test of anti- complementary power with each new bottle, and use 8 114 APPLIED IMMUNOLOGY kno\vn positive and negative controls every day the test is performed. Collection and Preparation of the Patient's Serum, — The patient's blood to be tested may be drawn from a vein at the bend of the elbow with a hypodermic syringe and immediately transferred to a small test- tube. There are several other methods in use, as by means of Keidel's vacuum ampoule (Fig. 11), cup- FiG. 11. — Showing method of collecting blood from vein of arm with Keidel's vacuum ampoule. Observe the tourniquet of rubber dam secured with the easily de- tached, tucked-under loop. ping, etc. At least 2 c.c. of blood should be obtained. The method employed by us is as follows : The ring- finger is wiped off with alcohol, allowed to dry, and then given a deep puncture at the radial extremity of the distal phalanx about a quarter of an inch below the angle of the nail with a large Hagedom needle. The blood is then squeezed out into a small test-tube by intermittently compressing the radial side of the FIXATION OF COMPLEIMENT 115 patient's finger with the tip of the little finger of the right hand holding the test-tube, the thumb and index- finger of the left hand holding and constricting the circulation on the other three sides of the patient's finger (see Fig. 12) . With a little practice, sufficient Fig. 12. — Showing authors' method of obtaining blood for complement-fixation reactions. Note the little finger of the right hand holding the test-tube, free to exert intermittent pressure over the radial aspect of the patient's punctured finger, held and constricted on three sides by the enveloping thumb and index-finger of the left hand. blood can thus be obtained in a minute or two, and the method does not entail any formidable preparation and obviates the possibility of phlebitis incident to faulty venous puncture. In very young children, suf- ficient blood can quickly be obtained by puncturing 116 APPLIED IMMUNOLOGY the lieeL The tube containing the blood thus collected should be plugged with cotton and if possible be kept in the refrigerator until required for the test. It will keep well for four or five days in this way, but should be tested as early as possible after removal from the patient. If blood is to be carried or sent any distance, a cork or other impervious stopper should be put in the tube, as inversion would cause the serum to be absorbed by a cotton stopper. The blood if kept too long, or in too high temperature, may heemolyze, and is then unfit for use, though slight hsemolysis does not interfere with the test. Consequently if the serum is to be shipped from a distance or stored miusually long, the serum had best be separated from the clot. We now arrive at the procedures to be carried out on the day of the test. They will be given, as far as possible, in the order in which they may be carried out in the shortest possible time. Pi^eparation of Comjylement. — The guinea-pig is first of all bled to death in the manner previously de- scribed, so that its serum will have separated by the time it is required. One guinea-pig as a rule will fur- nish sufficient complement for about 40 cases and their controls. The serum is made into a 1 in 10 dilu- tion with normal saline solution for convenience in handling small quantities. Preparation of the Suspension of Sheep's Cor- FIXATION OF COMPLEMENT 117 pnscles. — Sixty c.c. of a 5 per cent, suspension of sheep's corpuscles will usually be found ample for about twenty cases. This suspension is made as de- scribed under the hsemolytic system. The washed corpuscles will keep on ice without deterioration for about three days, after which spontaneous haemolysis usually begins. Titration of Complement, — In performing the Wassermann test it is essential that we know the smallest amount of complement that is necessary to produce haemolysis in the heemolytic system. There- fore before coming to the test proper we must each day, first of all, find out the smallest amount of guinea- pig serum in the presence of which 1 c.c. of a 1-1000 dilution of amboceptor will heemolyze 1 c.c. of a 5 per cent, suspension of sheep's corpuscles. This amount is known as 1 unit of complement, and may vary somewhat in the serum of different guinea-pigs. The employment of not more than one unit of comple- ment in the reaction is of great importance, since an excess might furnish enough to produce complete haemolysis and thus give a negative result when some of the complement employed has been previously fixed by syphilitic serum and antigen. Again, the use of less than one unit of complement would give a positive reaction even in the absence of syphilitic serum, as haemolysis would then be incomplete. The 118 APPLIED IMMUNOLOGY majority of workers employ excessive doses of comple- ment and double or treble the smallest amount of amboceptor necessary to produce complete hemolysis of the corpuscles. The only possible reason for the use of such excessive doses of the two reagents is to overcome the effect of fixation of a slight amount of complement, non-specifically, by the patient's serum and the antigen alone, and to replace the com- plemental power lost in the incubation. This empiri- cal method is responsible in part for negative results in positive cases. By the method in use by us, we are enabled to accurately allow sufficient complement for the overcoming of any non-specific fixation by serum and antigen alone, and also to make up for the complement lost during incubation, and at the same time use in the test proper not more than one hemolytic unit. Previous to titration of comple- ment with hemolytic amboceptor and corpuscles, the complement is first incubated with one dose of antigen and 0.1 c.c. of a mixture of several non-syphilitic sera. This is done for the reason that a slight amount of complement is fixed under these conditions by the serum and antigen, and we are thus sure of allowing for this slight non-specific fixation in the test itself. Different sera present variations in the amount of complement they are capable of absorbing in this FIXATION OF COMPLEMENT 110 manner, but these variations are usually so slight that for practical purposes they can be ignored, particularly if a pooled serum from several non- syphilitic cases be used. The technic of titration of complement is carried out as follows: Into each of a series of six tubes is placed 0.2, 0.3, 0.4, 0.5, 0.6, Fig. 13. — Titration of complement. With the complement in each tube are placed 0.1 c.c. of non-syphilitic serum and 1 dose of antigen. The tubes contain gradually increasing amounts of complement, ranging from 0.2 c.c. (dilution 1-10) to 0.8 c.c. 0.7 and 0.8 c.c. of 1-10 dilution of guinea-pig serum, together with 0.1 c.c. of pooled non-syphilitic serum previously inactivated by heating at 56° C. for half an hour, and the dose of antigen used in the test (Fig. 13) . Sufficient salt solution is then added to bring the volume up to 2 c.c. The tubes are shaken and incu- 120 APPLIED IMMUNOLOGY bated for one hour at 37° C, after which 1 c.c. of 1-1000 hsemolytic amboceptor and 1 c.c. of 5 per cent, suspension of sheep's corpuscles are added to each tube. After a further incubation of one hour, the de- gree of haemolysis that has taken place in the different tubes is noted. Usually complete hsemolysis has oc- curred in all the tubes containing 0.5 c.c. and more of diluted complement. The 0.5 c.c. tube is therefore said to contain one unit of complement plus the small extra amount allowed for non-specific inhibition due to the presence of patient's serum and antigen. This is the amount of complement to be used in the test proper on that particular day. The table given below will illustrate the titration of complement : Pooled Antigen 1-7 Comple- Ambo- Sheep's ^ O normal serum ment 1-10 O g- ceptor 1-1000 cles 5 percent. C cr P Result o h c.c. c.c. c.c. p c c. c.c. E3 1 0.1 0.2 0.2 H- 1 1 1— ' Slight haemolysis 2 0.1 0.2 0.3 1 1 •-1 Partial hsemolysis 3 0.1 0.2 0.4 ! P 1 en- 1 1 Almost complete d- haemolysis 4 0.1 0.2 0.5 ^ 1 1 ii Complete hemolysis 5 0.1 0.2 0.6 p 1 1 o P Complete haemolysis 6 0.1 0.2 0.7 1 1 Complete haemolysis By making two parallel experiments, it is readily shown that where the complement has been previously subjected to incubation with inactivated normal serum and antigen, in the same doses as are used in the test itself, a larger amount is required for hsemolysis than FIXATION OF COMPLEMENT 121 where complement after being incubated for one hour alone is added to the amboceptor and sheep's cor- puscles. In a second series of tubes prepared in this way the results would be : Comple- Ambocep- Sheep's t3 ment tor corpuscles 2 1-10 1-1000 5 per cent. COST. c.c. c.c. c.c. ^® 1 0.2 1 op 2 0.3 1 3 4 04 0.5 1 1 ?l 5 0.6 1 p ! Result Partial haemolysis Almost complete hsemolysis Coiri'plete hceinolysis Complete haemolysis Complete haemolysis By this means we arrive at the exact amount of guinea-pig serum that represents one unit of comple- ment. This will be of practical value later as seen in quantitative tests. Patient's Serum. — The serimi of the patient's blood will usually separate spontaneously from the clot and can be drawn off with a capillary pipette. If not, the clot can be broken up well with a platinum loop and the serum separated by centrifuging the tube. The clear serum can then be readily drawn off. The patient's serum is heated in a water-bath at 5^"" C. for half an hour to destroy the natural complement present (inactivation) . This can be carried out while the complement is being titrated. At the same time a known syphilitic and a known non-syphilitic ^erum should be inactivated to be used in the test as controls. 122 APPLIED IMMUNOLOGY Performance of the Wassermann Test Proper The performance of the test on one unknown serum with positive and negative controls will be de- scribed, though of course any number of sera can be tested simultaneously, using one set of controls. Six small test-tubes are arranged in two rows, three in each row. Tubes 1 of the front and back rows are for the serum to be tested ; Tubes 2 for the known syphilitic serum; and Tubes 3 for the non-syphilitic serum. The back row tubes serve as controls for those of the front row. Into Tube 1 of the front row are placed: 0.1 c.c. of patient's serum; one unit of complement (usually 0.5 c.c. of a 1-10 dilution previously determined by titration) ; one dose of antigen (usually 0.2 c.c. of a 1-7 dilution). Into Tube 1 of the hack row are placed: 0.1 c.c. of patient's serum ; one unit of complement ; no antigen. Tubes 2 and 3 are dealt with in the same way, ex- cept that 0.1 c.c. of syphilitic serum is used in each of Tubes 2, and 0.1 c.c. of non-syphilitic serum in each of Tubes 3, instead of the unknown serum. The back row tubes furnish controls to show that the serum alone without antigen will not fix complement. The total volume in each tube is now made up to 4 c.c. by the addition of normal salt solution. The contents of the tubes are thoroughly mixed by inversion. The FIXATION OF COMPLEMENT 123 finger can be used as a stopper for the tube while mix- ing, provided that contamination from one tube to another is avoided by using a different finger for each case. The tubes are then incubated for one hour at 37° C. to assist in fixation of complement. At the end of this time, no alteration in the appearance of the con- tents is manifest, though complement-fixation will have taken place in the tubes containing syphilitic serum and antigen. In the negative cases there will be no complement-fixation. The occurrence or non-oc- currence of complement-fixation is now determined by the addition of the hemolytic system and re-incubation of the tubes. To each tube is added 1 c.c. of a 1-1000 dilution of anti-sheep hemolytic amboceptor and 1 c.c. of a 5 per cent, suspension of sheep's corpuscles. The contents of the tubes are again thoroughly mixed, and the tubes incubated for one and a half to two hours. At the end of this time a preliminary reading of the results may be made, but final reading should be de- ferred until next morning after the tubes have re- mained all night in the ice-box.^ Reading of Besults, — In case of a positive result, in Tube 1 of the front row there should be no hsemol- ysis, because complement being fixed by the action of the antigen on the syphilitic serum, no complement re- ^ The time consumed in incubating the tubes may be materially lessened by placing them in a water-bath heated to 37° C. instead of in the air incubator, only one-half the time being required for each incubation in the water-bath. 124 APPLIED IMMUNOLOGY mains to take part in hsemolysis of the sheep's cor- puscles by the amboceptor. The corpuscles will have settled to the bottom of the tube, leaving colorless fluid above. In case of a negative result, complete haemol- ysis will have occurred in this tube, because there being no " reagin " in the serum to fix complement with the antigen, complement remains free to act with the hsemolytic system during the second incubation. Hsemolysis is shown by a disappearance of the cloudi- ness due to the corpuscles and the fluid becoming a transparent red color. In Tube 2 of the jront row, containing known syphilitic serum, we should get inhibition of hsemol- ysis for the same reason that we obtained it in case of a positive result in Tube 1. In Tuhe 3 of the front row, containing non-syphi- litic serum, we should get complete hsemolysis, for the same reason that we obtained it in case of a negative result in Tube 1. In all tubes of the hack row, there should be com- plete hsemolysis, because these tubes contain no anti- gen, and therefore no complement-fixation could have taken place during the first incubation, tlie comple- ment remaining free to combine with the haemolytic system. Occasionally, in the serum to be tested, there are substances capable of fixing part or all of the com- plement in the absence of antigen. In these cases, the CD o iO U Tube 1 Tube RESULT Partial Partial iaemolysis Haemolysis Complete Haemolysis or J ^ediurni- Weakly -f Complete Haemolysis Negative T. .L -Ti ^ .. RESULT Tube 1 Front Patient's serum 3 No Partial Partial Haemolysis Hremolysis Haemolysis Complete Haemolysis row 0.1 cc. c Amboceptor 3 O Complement g 1 cc cr "■ n 11 fl 0.5 cc. o Corpuscles .r\ or \ .r II 1 Antigen serum a Ice 5' 1 1 Tube 2 Back 5\/philitic 5erum Amboceptor p 1 S row Oj cc. Complement 0^ P Ice Corpuscles 1 0.5 cc. Ice u Tube 3 Front Non-Syphilitic 5erum 5" 5" .Complete Haemolysis roNA/ 0.1 cc. 2 Amboceptor P Complement * '+ Ice. 5 11 05 cc o' 3 Corpuscles 5 3 1 „ Antigen Ice. _ H o =3- V 1- 0.2 CO. ~: T^ c r\ N«gatl»e u a .ii..plete Haemolysis 1 Tube 3 BacK Non-Syphilitic Serum Amboceptor Ul "IS fow 0.1 c C. p Ice p 11 ■z. Complement 05 c c. Corpuscles Ice \ FIXATION OF COMPLEMENT 125 mistake of giving a positive result by absence of hemolysis in the front row tube is avoided by finding a like result in the control tube in the back row. In reading the results, varying degrees of hsemolysis may be observed in tests of different sera, ranging from complete haemolysis in negative cases, to absence of haemolysis in strongly positive cases. Even slight de- grees of inhibition of haemolysis are generally to be re- garded as positive, the closeness of reading of border- line cases being dependent on clinical facts and the experience of the worker. The stronger the serum in syphilitic " reagin," the more complement will be fixed, less being left free for haemolysis. In a serum containing smaller amounts of the Wassermann sub- stance, only part of the complement will be fixed, leav- ing the remainder free, and we get partial haemolysis (see Plate II). Quantitative Estimation, — While the degree of haemolysis produced gives an idea of the strength of the reaction up to absorption of one unit of comple- ment, yet it does not make any measure of positive cases in which the serum is capable of absorbing more than one unit of complement. In other words, absence of haemolysis as seen by the usual method means a strongly positive reaction, but indicates no difference in the degree of strength of strongly positive sera; so that a serum may show no signs of weakening under 126 APPLIED IMMUNOLOGY the effects of treatment when tested with only one unit of complement, because it was previously capable of fixing two or more units. When we wish to determine accurately the complement absorption power of a Fig. 14. — Showing arrangement of tubes in performance of Wassermann reaction on one unknown case, with positive and negative controls. P.S.I. Tube containing patient's serum and 1 unit of complement. P.S.2. Tube containing patient's serum and 2 units of complement. S.C. Serum control tubes, without antigen. +1. +2. Tubes containing known syphilitic serum and 1 and 2 units of complement respectively. —1,-2. Tubes containing known non-syphilitic serum and 1 and 2 units of complement respectively. A.C. Antigen control, containing a double dose of antigen. serum, in order to gauge the effects of treatment by subsequent tests, tubes should be prepared in doing the test containing two, three, or more units of comple- ment in addition to the usual one unit (Fig. 14). FIXATION OF COMPLEMENT 127 By this means we can readily see whether the usual amount of the serum used is capable of fixing one, two, three, or more units of complement. It is rarely neces- sary to carry this quantitative determination beyond two units of complement, though some sera will fix five or six units. By means of a series of tests at intervals on a case under treatment, we can observe that the serum gradually loses its power to fix complement, ab- sorbing perhaps two units the first time, then one unit, then allowing hsemolysis of half the corpuscles, then only a few corpuscles being left unhaemolyzed, and finally complete haemolysis showing that the serum has become negative. Some workers use signs to desig- nate the strength of the Wassermann reaction, a weak reaction being indicated by + or 1 plus, meaning that there was only 25 to 50 per cent, inhibition of haemol- ysis, between 50 and 75 per cent, inhibition being indi- cated by + + or 2 plus, between 75 and 100 per cent, inhibition as + + + or 3 plus, and complete inhibition as + + + + or 4 plus. The percentage of haemolysis cannot be thus accurately guessed at by the eye, and moreover this method of notation does not give the strength of the reaction beyond specific absorption of one hasmolytic dose of complement. We prefer to designate reactions up to one unit of fixation as nega- tive, weakly positive, medium 'positive, and strongly positive, giving the number of units of complement ab- 128 APPLIED IMMUNOLOGY sorbed in reactions stronger than this. We believe that this method of designation conveys more informa- tion as a rule to the clinician than the plus signs. We are indebted to Dr. John L. Laird for the principles we follow in quantitative determination (Pa. Med, Jour., 1911-12, XV, 97-102). For those who wish to measure the percentage of haemolysis more exactly, the Duboscq colorimeter may be used according to a method described by one of us (Ivy, Jou7\ Amer. Med. Assn., Aug. 10, 1912, p. 432). We regard this accu- rate measurement as seldom called for, yet the method may be fomid useful to those who are in the habit of guessing the percentage of haemolysis in giving their results. The sera that have been tested can be preserved and used as positive and negative controls in future reactions, provided they are reinactivated by heating before subsequent tests are made. The table (Plate II) illustrates the performance of the test on an unknown serum, with positive and negative controls. Modifications of the Wassermann Reaction Each worker is apt to vary from the original technic in minor details to suit his own convenience in the performance of the Wassermann reaction. These slight modifications are in no way to be regarded as radical changes in the reaction. By the original Was- FIXATION OF COMPLEMENT 129 sermann reaction, as we view the matter, is meant the use of extract of syphiHtic liver as antigen, an anti- sheep h^emolytic system, fresh guinea-pig serum as complement, and inactivation of the patient's serum. Various workers have sought to improve on the origi- nal Wassermann technic by the use of other antigens, as above mentioned; by using anti-ox, anti-pig, anti- human, and other ha^molytic systems; by using fresh instead of inactivated patient's serum, etc. All at- tempts to simplify the technic have resulted in sacri- ficing the delicacy of the test. The modification that is most commonly employed in this country is that of Noguchi, who, realizing the value of the laboratory re- action for syphilis to the medical profession and the training and experience necessary for the performance of the test as laid down by Wassermann, attempted to simplify the technic so as to make it accessible to the general practitioner as a routine office procedure. He proposed the use of an anti-human heemolytic system ; thus making unnecessary the use of sheep's blood. He prepared anti-human ha^molytic amboceptor by inject- ing rabbits with human corpuscles. The antigen, am- boceptor, and complement were put up in dry form by saturating strips of absorbent paper with these re- agents. In the performance of the test, it was only necessary to add the antigen and complement papers to diluted patient's serum, incubate, and then add the 9 130 APPLIED IMMUNOLOGY amboceptor paper and washed corpuscles from the pa- tient. But it was soon found that the complement paper rapidly lost its power and became useless, lead- ing Noguchi to discard it in favor of fresh guinea-pig serum. The antigen and amboceptor papers are still employed to some extent, but accurate titrations of the reagents cannot be carried out when they are used, and misleading results are liable to occur. Reliable workers employing the Noguchi system now use the different reagents in their original fluid form, titrat- ing them accurately, so that the technic as carried out at present has no advantage over the original Wasser- mann in point of simplicity. Those accustomed to the Noguchi modification with careful titration of all the reagents, no doubt obtain as reliable results as can be obtained with the Wassermann technic, and even claim that the results are more delicate. We believe, however, that by proper titration and employment of a minimal amount of complement, there will be little difference in the results obtained by the two methods. As in all laboratory procedures, cases will be en- countered on the border-line between positive and negative which can only be judged by a consideration of the clinical features of the cases, and the experience of the worker. Hecht'Weinherg 3Iodification. — The Hecht- Weinberg test modifies the Wassermann reaction for FIXATION OF COMPLEMENT 131 syphilis by utilizing the natural complement and anti- sheep hiemolysin present in the patient's serum in- stead of adding guinea-pig serum and prepared heemolytic amboceptor. Gradwohl {Jour, Amer, Med. Assn., July 18, 1914) regards this test as of great value as a check upon the Wassermann reaction. In a series of one thousand cases he found the Hecht- Weinberg was positive 15 per cent, more often than the Wassermann. Clinical Application of the Wassermann Reaction In a great many cases, the correct interpretation of the examination of the patient's serum in the labora- tory can only be given when the clinical aspect of the case is considered. The clinician therefore should be familiar w^ith the meaning of a given result from the laboratory so that he can intelligently apply it to the case in question. For instance, many practitioners are disappointed upon the receipt of a negative Wasser- mann report in a case clinically diagnosed as syphilis, because the blood has been collected from the patient when under the influence of specific treatment. A familiarity w4th the following facts will be of value in interpreting the results of the reaction. The question as to whether the Wassermann reac- tion is specific for syphilitic infection alone or whether it is found in other diseases, has been the occasion for 132 APPLIED IMMUNOLOGY much study. Observers from time to time have ob- tained positive results in yaws or framboesia, trypano- somiasis, some cases of leprosy, malarial fever, scarlet fever, and occasionally in other diseases. It is not sur- prising that a positive result should be given in yaws, a disease clinically similar to syphilis in many respects, and which is due to the Spirochceta pertenuis, an or- ganism very difficult to distinguish from the Tre- ponema pallidum. Positive results with the Was- sermann reaction are found more frequently in cases of the tuberous type of leprosy than the anaesthetic form. In malaria the reports are conflicting. Some observers have obtained positive results in a certain percentage of cases, which become negative as soon as the malaria parasites disappear from the blood. Others have had uniformly negative results. Similar experiences have been reported in scarlet fever. After summing up all the data at our disposal, and judging from our own results, we must conclude that except in yaws, trypanosomiasis, and leprosy, a positive Was- sermann reaction means that the patient is the victim of syphilitic infection. The diseases named being practically unknown in temperate climates, should cause no confusion in this part of the world. The positive results obtained in other diseases are either due to the fact that syphilis cannot be absolutely ex- cluded, or to faulty technic. FIXATION OF COMPLEMENT 133 On the other hand, we may obtain a negative Was- sermann reaction in the presence of sypliilitic infection under certain conditions to be mentioned below in detail. Primary Stage of Syphilis, — The Wassermann re- action as a rule does not become positive until at least two weeks after the appearance of the chancre, the average time being from three to four weeks, though cases have been known in which it was positive before the chancre appeared. In our records the earliest positive Wassermann reaction was obtained four days after the appearance of the primary lesion. Oc- casionally it does not become positive until after the secondary symptoms have manifested themselves. The Wassermann reaction has proved that many in- stances of so-called " soft sores " are in reality due to syphilitic infection, or at least a mixed infection exists. The earliest means of making an absolute diagnosis of the primary lesion of syphilis is by finding the Tre- ponema pallidum in the secretion from the sore by dark field illumination or by stained smears. This method fails, however, in a certain percentage of cases, especially when local treatment has been applied; so that, while a negative Wassermann reaction in the sus- pected primary stage of syphilis does not exclude the disease, a positive reaction will frequently serve to establish the diagnosis. 134 APPLIED IMMUNOLOGY Secondary Stage. — The reaction is positive in practically all cases of secondary syphilis, and is there- fore of the greatest value in making the diagnosis of atypical cases. In this stage, too, we usually find the strongest reactions. Tertiary Stage, — In tertiary syphilis a positive Wassermann reaction is obtained in 80 to 90 per cent, of cases. A negative reaction in this stage therefore does not have quite the same significance as in sus- pected secondary syphilis. Medium and weakly posi- tive reactions are more frequent here than in the secondary stage. Latent Syphilis, — By latent stages are meant periods in which all symptoms disappear for a time, either spontaneously or as a result of treatment, but in which, as shown by the Wassermann test, the spe- cific reaction between the spirocheetse and the tissues is still going on, indicating therefore that the disease is not eradicated. It has been found that persons in this stage, although exhibiting no symptoms, yet are capable of infecting others, and themselves are liable to develop later manifestations of the disease. It is estimated that about 50 per cent, of latent syphilitics give a positive Wassermann reaction. It is difficult to estimate this percentage, because we cannot always say whether a given case having a negative Wasser- mann reaction is cured or merely in the latent stage. FIXATION OF COMPLEMENT 135 Craig and Xichols {Jour, Amer, 3Ied, Assn., 1911, Ivii, p. 474) have found that the recent ingestion of varying quantities of alcohol has a marked effect on the Wassermann reaction. These authors state that from 180 to 240 c.c. of whiskey, 90 c.c. of 95 per cent, alcohol, and 700 c.c. of Munich beer are capable of causing a positive reaction to become negative for periods ranging from 24 to 72 hours. They therefore recommend, in any case where the ingestion of large quantities of alcohol in the preceding 24 hours is sus- pected, that the removal of blood from the patient for performance of the test be postponed for 3 or 4 days. The authors have recently observed a case of second- ary syphilis in a chronic alcoholic patient, whose Was- sermann did not become positive until the seventh week of the disease. Previously, in an extensive ex- perience with hundreds of cases of untreated syphilis, the Wassermann reaction always resulted positively by the fifth week following the appearance of the chancre. It is now pretty generally agreed that a positive Wassermann reaction means the presence of living spiroch^tse in the body, the organisms having been found in practically all lesions of every stage of ac- quired and inherited syphilis. The reaction differs from true antigen-antibody reactions in that the latter are apt to persist for some time after the infecting 136 APPLIED IMMUNOLOGY agent has disappeared from the body, while the Was- sermann " reagin " usually disappears very rapidly. In iriherited syphilis the Wassermann reaction is positive in a very high percentage of cases, at least 90 per cent. The reaction tends to show moreover that a great many congenital defects and maladies of young infants, until recently regarded as due to syphilis, are not caused by this disease. The Wassermann reaction has thrown considerable light on the significance of the laws of Colles and Prof eta. Colles' law is that the mother of a syphilitic child may show no signs of syphilis and is immune to the disease. It has been found that in a very high percentage of such mothers the blood gives a positive reaction. These cases proba- bly have syphilis in a modified form, and therefore cannot be reinfected. Prof etas law is that the chil- dren of syphilitic mothers may be apparently healthy, and yet not susceptible to syphilitic infection. A high percentage of these children also give a positive Was- sermann reaction, the explanation being the same as in the case of Colles' law. Parasyphilitic A Sections, — Under this name are grouped certain diseases of the nervous system, par- ticularly paresis and tabes dorsalis. The discovery of the Treponema pallidum in the lesions of paresis and tabes has proved them to be true manifestations of syphilis in the vast majority of cases. The Wasser- FIXATION OF COMPLEMENT 137 mann reaction may be performed on cases of these dis- eases by examination of either the blood-serum or the cerebrospinal fluid. In using the latter for the test inactivation is not necessary, as the fluid contains no complement. The technic is the same as when using blood-serum, except that 0.2 c.c. of the cerebrospinal fluid should be employed. According to most ob- servers, in paresis, examination of the blood-serum shows the Wassermann reaction to be positive in prac- tically all cases, while the cerebrospinal fluid gives a positive result in about 90 per cent, of cases. In tabes both the blood-serum and the cerebro- spinal fluid give a positive Wassermann reaction in 60 to 70 per cent, of cases. In the manifestations of syphilis of the central nervous system, which do not differ materially from syphilitic lesions elsewhere, and are grouped apart from paresis and tabes, the Wassermann reaction with the blood-serum is positive in practically all cases, while it is positive in only a small percentage of cases with the cerebrospinal fluid. In this group are in- cluded gummatous lesions, syphilitic meningitis, etc. From personal experience, we have found in general that the cerebrospinal fluid is more frequently positive or gives a stronger Wassermann reaction than the blood-serum in the majority of cases of syphilis in- volving the central nervous system. 138 APPLIED IMMUNOLOGY The Wassermann reaction has shown that a high percentage of cases of aortic disease and aneurism are syphilitic in origin. Effects of Treatment on the Wassermann Reac- tion,— The Wassermann reaction is markedly in- fluenced by treatment. In a syphilitic undergoing treatment with mercury and particularly salvarsan, the reaction may rapidly be reduced from strongly positive to negative. As a guide or measure of the effects of treatment, therefore, the Wassermann reac- tion plays almost as important a role as it does in diag- nosis. True biological antibodies, such as for instance occur in typhoid fever as determined by the agglutina- tion reaction, may persist in the blood for months and even years after the disease has been eradicated. On the other hand, the evidence of the Wassermann reac- tion shows that the substances taking part in it disap- pear from the patient's body very shortly after the destruction of the syphilitic virus by treatment, or when the virulence of any remaining spirochset^ is much diminished. In the primary stage, if treatment by salvarsan or neosalvarsan be instituted as soon as the diagnosis is made (by dark field illumination), the Wassermann reaction may never become positive and secondary symptoms may never appear. With every day that treatment is delayed, and especially after the Wasser- FIXATION OF COMPLEMENT 139 mann reaction has become positive, a proportionate increase in treatment will be required to eradicate the disease. In the tertiar}- stage it is impossible in some cases to render the reaction negative. Under treatment the symptoms usually disappear before the Wassermann reaction becomes negative, though occasionally the opposite holds true; that is, cases under treatment may present a negative reaction although symptoms are still manifest. The reaction may persist for some time after all symptoms have disappeared, indicating that further treatment is necessary. After being rendered negative by treat- ment, the reaction may after a time again become weakly positive, then gradually stronger, and finally be followed by a reappearance of symptoms. It is not yet possible to say when a negative reaction in a case that has received treatment means that the patient is cured of syphilis. If negative Wassermanns have been obtained at intervals of three or four months over a period of two years, during which the patient has had no treatment and with no recurrence of symp- toms, it is probably justifiable to regard the case as cured. But it will require ten to twenty years' exper- ience with the reaction to be positive that the so-called parasyphilitic diseases or other late manifestations will not develop even after such a series of negative tests. JNIuch closer readings of the reaction should be 140 APPLIED IMMUNOLOGY made in using it as a guide to treatment than as a diagnostic aid. Border-line reactions giving very- slight inhibition of hsemolysis (5-10 per cent.) when performed for diagnosis should be regarded as nega- tive. But such a result in a case that has been known to have had syphilis and to have been under treatment indicates that fm^ther treatment is required. Occasionally in a case of late or treated syphilis giving a negative Wassermann reaction, a positive re- sult may be obtained immediately after a so-called provocative dose of salvarsan, neosalvarsan or mer- cury. It is believed that the treatment in such cases is just sufficient to stimulate the spirochsetse without causing their death. In view of the fact that treatment hy mercury or salvarsan often produces a negative reaction in cases where it otherwise would he positive, the test should not he performed on a patient unless all treatment has heen discontinued for at least three weeks. If this precaution be not observed, negative results will fre- quently be obtained in cases that should be positive, greatly to the surprise and chagrin of the practitioner. In employing the Wassermann reaction in differ- ential diagnosis, it must be remembered that a positive result does not necessarily mean that the lesion in question is syphilitic, but that there is syphilitic infec- tion in the body of the patient. XIII FIXATION OF COMPLEMENT (Continued) GONOCOCCUS COMPLEMENT-FIXATION TEST— SERUM DIAG- NOSIS OF ECHINOCOCCUS DISEASE— COMPLEMENT-FIXA- TION REACTION IN TYPHOID FEVER— COMPLEMENT- FIXATION REACTION IN TUBERCULOSIS— THE COMPLE- MENT-FIXATION REACTION AS APPLIED IN PROTEIN DIFFERENTIATION (NEISSER-SACHS REACTION) The Gonococcus Complement-fixation Test In recognition of the recent admirable work by Schwartz and McNeil on the complement-fixation test in gonococcic infections, the fact must not be over- looked that Miiller and Oppenheim, in 1906, were the first to apply this reaction to a gonorrhoeal affection and consequently are entitled to the distinction of being termed the originators. The present popularity of this test has been the outgrowth of the suggestion made by Schwartz and McNeil — namely, that of the employment of a polyvalent antigen. As a result of their labors, these workers contend, and seem to have proved conclusively, through animal experimentation : (1) "that the different strains of the gonococcus differ markedly one from another — so much so that the antibodies produced in the body by the toxin of one strain will in many instances not bind the comple- ment in the presence of an antigen prepared from another strain. Therefore, if only one strain is used in the preparation of the antigen, a great many nega- 141 142 APPLIED IMMUNOLOGY tive results would be obtained in positive cases; (2) an antigen prepared from many strains fixes the com- plement whenever one of its component strains does so, and consequently the necessity of testing a serum against a number of antigens separately is avoided. It is not to be denied that there probably are other strains of gonococci differing widely from any pres- ent in the polyvalent antigen, so that at times a nega- tive result will be obtained in a positive case." While we recognize the fact that a negative reac- tion may mean nothing, in fact, may be erroneously contradictory, the significance, on the other hand, of a positive reaction has been so great — more specific, in fact, than when the lipotropic antigen, commonly em- ployed in the performance of the Wassermann reac- tion, is utilized — that we have applied the test in a large series of diverse cases with the most gratifying results. Discussion of Technic, — Schwartz's method and the technic which we practiced for a time in compari- son with our own gonococcus complement-fixation test is as follows, so far as the quantities of the ingre- dients participating in the reaction are concerned: (1) patient's serum, 0.02 c.c; (2) salt solution, sufficient to equalize the volume in each tube ; (3) antigen (rou- tinely 0.3 c.c. in one tube and 0.15 c.c. in another tube, of a commercial preparation diluted 1 to 10). These quantities are determined by preliminary standardiza- FIXATION OF COMPLEMENT 143 tion with a fresh known negative and a known positive senun; the positive showing the true antigenic dose, and the negative the highest quantity of antigen which will allow complete hsemolj^sis ; in the actual test this large quantity of antigen is placed in one tube and one-half the quantity in a second tube; (4) comple- ment 0.1 c.c. of a dilution 1 to 10; (5) amboceptor, 0.1 c.c. representing twice the lowest quantity that will completely hsemolyze 0.1 c.c. of the cell suspension with 0.1 c.c. of complement in one hour; (6) sheep's red blood-cells (5 per cent, suspension), 0.1 c.c. In- cubation is made for one-half hour at 37° C. in a water-bath or for one hour in dry heat, before and after the addition of the hsemolytic system. The technic on which we have learned to place the greatest reliance is essentially that utilized by us in the performance of the Wassermann reaction, merely substituting the gonococcus specific antigen for the syphilitic lipotropic antigen, employing always the carefully standardized single complement unit and the routine standardization of antigen and ambocep- tor (see Table of Test Reactions). Antigens, — The necessity of a polyvalent antigen is indisputable, presumably owing to the diversity of the strains of the gonococcus. The only question of importance is, how may this antigen be prepared to the best advantage? Schwartz and McNeil in their latest communication say that the " various strains of 144 APPLIED IMMUNOLOGY gonococci are grown on salt-free veal-agar, neutral in reaction to phenolphthalein : 24-hour-old cultures are washed oiF the agar-slants with distilled water and the resulting suspension is heated for two hours in a water-bath at 56° C. It is then centrifuged and passed through a Berkefeld filter. No salt is added to this antigen until it is desired to use it, when it is made up to 0.9 per cent, strength by adding one part of 9 per cent, saline solution to nine parts of antigen. Follow- ing Schwartz and McNeil's instructions to the letter, we have prepared monovalent, trivalent and hexa- valent antigens and have employed them compara- tively in a large series of cases with the result that the hexavalent preparation gave the highest percentage of positive results and in every way appeared to be the most reliable of the three antigens. Even with the hexavalent antigen, prepared as described above, we have been forced, in comparative studies, to the con- clusion that it has not been so useful or reliable as when prepared in the following manner : Forty-eight- hour-old cultures of the same six strains of gonococci, grown on blood-agar, were washed off in sterile dis- tilled water ; shaken for one hour ; and autolyzed for twenty-four hours in a thermostat at the temperature of 37° C. and heated in a water-bath at 60° C. for one-half hour. Before use, this antigen is diluted one to ten by the addition of 0.85 per cent, salt solution. FIXATION OF COMPLEJVIENT 145 Tests and con- trols for the suspected serum Tests for anti- gen stand- ardization and controls Tests for com- p 1 e m e n t standardi- sation en *■ 00 to ^ o o 00 ^ 05 Cn >^ CO to H- No. test-tube .' O O Q o o o to to jj b *> io 0.05 0.1 0.2 0.3 c.c. 0.2? 0.2? 0.2? 0.2? Antigen (dilution 1: 10) bi j-i p J- p H- h- CO b CO b ►— ^o to CO ►f' bi c.c. 1.3 1.2 1.1 1.0 NaCl solution (0.85 per cent.) P Patient's serum (in- activated) H- H* h- ' o p p p . p Known po.sitive ser- um (inactivated) : : P .° P : : : : « Known negative ser- um (inactivated) p 0.4? 0.4? 0.4? 0.4 0.8 0.4 0.4? 0.4? 04 ? o o o o« Cr» rf>. CO ioP Complement (dilu- tion 1 : 10) Incubation at 37° C. for one hour | | ^ i_i n Haemolytic ambocep- tor (antisheep) (titre=l:2000) (dilution 1:1000) b o o b b b b o b b b b b b oT" ^ ^ ^ ^« Red blood- corpuscles (sheep's 5 per cent, washed suspension) b b b b b b b o b b b 'o 'o 'o br* Incubation at 37° C. for one and one-half hours { a> 1 Complete haemolysis. . Complete haemolysis. . Incomplete haemolysis No haemolysis ....... (Positive reaction haemolysis=l unit) No haemolysis (Positive reaction haemolysi3=2 units) Complete haemolysis. . (Complete negative reaction) Incomplete haemolysis Partial haemolysis .... No haemolysis No hffimolvsis Incomplete haemolysis Incomplete haemolysis Complete haemolysis . . Complete hsemols'sis. . Results (immediately or morning after refrigeration) ill tl 1 be used in test proper. To prove that the antigenic dose is not in itself anticomplementary. To prove that twice the antigenic dose is not in itself anticomplementary. To prove that thrice the antigenic dose is not in itself completely anticom- plementary. To determine quantitatively the degree of complement-fixation. To determine quantitatively the degree of complement-fixation. Shows that there was no immune body present in the patient's serum with the aid of the antigen to fix the To determine quantity of antigen to be used in test proper: To determine quantity of antigen to ^ be used in test proper. To determine quantity of antigen to be used in test proper. To determine quantity of complement to be used in test proper. To determine quantity of complement to be used in test proper. To determine quantity of complement to be used in test proper. To determine quantity of complement to be used in test proper. O f s- 2. f 3 g 1 10 146 APPLIED IMMUNOLOGY Analysis of Cases Treated. — The result of our work has been Httle more than a corroboration of the reports that have emanated from Schwartz and McXeil and those who have confirmed their results. We believe, however, that, by utilizing the teclmic herein described, we have added to the accuracy of the test as applied by them and have thereby improved the findings to the credit of the test and its value in clinical diagnosis. To the increased positive results we at- tribute the accurate standardization of antigen on each occasion and the employment of a standardized single complement unit. Reviewing our experience with the gonococcus complement-fixation test in general, it may be stated that a negative reaction is not decisive against the presence of a gonorrhoeal infection, and this is par- ticularly true during the first six weeks of a primary acute urethritis either anterior or posterior in the ab- sence of any complication, previous to which time we have never obtained a positive reaction; on the other hand, the supervention, even during the acute stage of the disease, of complications such as epididy- mitis, arthritis, prostatitis, etc., is prone to result in the production of a positive reaction. On the con- trary in our experience, a positive reaction has been pathognomonic of a focus of gonococcal infection and has assisted many times in elucidating obscure or FIXATION OF COMPLEMENT 147 doubtful lesions. In fact, it appears that the gono- coccus-fixation test enjoys greater specificity than does the Wassermann reaction, since thus far we have found no alien infection or condition capable of pro- ducing a positive reaction. This much certainly can- not be claimed for the Wassermann reaction. INIore- over, there is no drug, as there is in syphilis, which is capable of causing the reaction to be negative during the existence of the disease. The probable explanation for the greater specificity of the gonococcus comple- ment-fixation test rests in the fact that with gonococcic infections we employ a specific antigen — the gono- coccus— while in the case of syphilis a non-specific or lipotropic antigen is employed. The analysis of our cases further illustrates another interesting feature, namely, the persistence in some cases for a short time of a positive reaction, after an apparent clinical cure. This has occurred so often that we no longer discharge a patient cured or give him a clean bill of health so long as he gives a positive reaction, provided he has not been the recipient of immmiotherapy. Usually a persistent positive reac- tion will become negative in two or three weeks follow- ing clinical cure with or without a continuation of treatment. The only explanation is that it requires an indefinite time for the antibodies, formed during the course of infection, to disappear from the blood. 148 APPLIED IMMUNOLOGY Torrey, in animal experimentation, has found that the antibodies in immunized rabbits begin to disappear after ten days, and that the ehmination is practically complete by the fiftieth day in all cases, disappearing much earlier in many instances. Thus a patient, evi- dencing a positive reaction two months after presumed clinical cure, should be regarded as still harboring a latent gonorrhceal focus. Such experience, adopted either as routine procedure in the management of treatment, or discovered accidentally when gonor- rhceal infection or its symptoms were denied, or dem- onstrated by submitting suspected or positive syphi- litic serum to the gonococcus-fixation test, has been encountered in a large number of cases. Because of the generally acknowledged difficulties, at times, of differentiating the pelvic lesions in women, notably certain of the inflammatory from the cystic and neoplastic conditions, and also the differential diagnosis among gonorrhoeal, tuberculous and pyo- genic infections themselves, the gonorrhoeal-fixation test seems destined to play a role. As in the male, in whom a positive reaction seems never to occur, at least so long as the infection is confined to the anterior urethra, so in the female we have been unable to obtain a positive reaction unless the disease has ascended to the level of the uterus. An interesting, if not important, feature connected FIXATION OF COMPLEMENT 149 with this work is the comparative importance and value of the serological and bacteriological examina- tion of cases of suspected gonorrhoeal infection. It is, to-day, a fact that the judiciary courts of our land re- quire that the presence of the gonococcus be demon- strated, culturally, in order to establish its indisput- able and legal identity. Based on this qualification, there are many cases of gonococcic infection impos- sible to determine, and we do not hesitate to state that, in our judgment, many such cultures are in reality the Micrococcus catarrhalis and not the gonococcus. This applies particularly to such isolation of the diplo- coccus of Neisser from chronic inflammatory proc- esses. JMoreover, it must be generally recognized that the demonstration of a Gram-negative diplococ- cus in smear is often insufficient and faulty evidence on which to base a diagnosis of gonococci. Therefore, it is most fortunate that in the chronic stage of the disease with complications, the complement-fixation test seems to be signally meritorious, while in the acute, subacute and frequently in the chronic forms of the diseases when the gonococcus may be demonstrated bacteriologically, the serological test promises little or nothing. Conclusions, — Detailed and careful analysis of the gonococcus complement-fixation test, performed with 150 APPLIED IMMUNOLOGY the serums of the cases tabulated in our series/ would seem to justify the following assertions: 1. A positive reaction is invariably reliable and al- ways denotes the presence of a focus of gonococcic infection. 2. A negative reaction frequently occurs in the presence of disease, especially in the acute and sub- acute stages when the disease is limited to the urethra, and it is always negative when the disease is confined to the anterior urethra or vagina alone. 3. In no alien non-gonorrhoeal infections of sys- temic disease has a positive reaction been obtained ; the test, therefore, appears to be absolutely specific. 4. A positive reaction has been found to be present in 21.05 per cent, of patients clinically cured. Such patients, therefore, should not be discharged from treatment or observation until a negative reaction has been obtained. 5. Not infrequently, either when suspicious lesions are presented or accidentally, positive reactions will be discovered in patients denying gonorrhoea. 6. In only 9.09 per cent, of cases of acute and sub- acute antero-posterior urethritis has the complement- fixation test resulted positively. The earliest ap- pearance of a positive reaction in a primary attack of ^ Archiv. Int. Med., January, 1914, p. 143. FIXATION OF COMPLEMENT 151 posterior urethritis, without complication, occurred in the sixth week. 7. In a number of cases of chronic recurrent ure- thritis with acute exacerbations, the test was invaria- bly positive ; many of these patients undoubtedly had prostatitis. 8. The reaction resulted positively in one-third of all cases of chronic posterior urethritis; undoubtedly many had a mild or low-grade prostatitis. 9. In 52.08 per cent, of cases of chronic prostatitis a positive reaction was obtainable. 10. Two-thirds of all stricture cases demonstrated a positive test. 11. In epididymitis a positive complement-fixa- tion test was observed in 87.5 per cent, of cases. If, from our series, one case, probably tuberculous, may be eliminated, and a time duration of five weeks can be imposed, the positive result in this form of disease has been 100 per cent. 12. In arthritis, undoubtedly gonorrhoeal in char- acter, positive reactions were obtained in 100 per cent, of cases. 13. In the diagnosis and differential diagnosis of pelvic disease in women, the gonococcus-fixation test is destined, unquestionably, to play an important role. We have been unable to obtain any positive results in uncomplicated urethritis, vulvovaginitis and Bar- 152 APPLIED IMMUNOLOGY tholinitis, and it would appear that the infection must ascend at least to the level of the uterus in order to produce a positive blood response. 14. Inoculations of gonococcus bacterin, antigono- coccic serum, etc., may in themselves by the produc- tion of immune bodies be causes of positive reactions. How long these immunizing effects may endure is un- known, but we have observed patients, treated by im- munotherapy, who one year later demonstrated nega- tive complement-fixation reactions. 15. Although the bacteriological demonstration of the gonococcus culturally is the only absolute method for its identification in chronic inflammatory processes, the method as a routine procedure is impractical and susceptible of many failures and fallacious results, so that the complement-fixation test is not only less la- borious, but is productive of a higher percentage of positive findings. A series of comparative studies using non-specific with the specific antigens in the performance of the gonococcus complement-fixation reaction has been carried out (Thomas, B. A., Ivy, R. H., and Bird- sail, J. C, Surgery, Gynecology, and Obstetrics, 1914). Polyvalent antigens were prepared from various non-gonorrhoeal Gram-negative and positive bacteria, namely, the Micrococcus catarrhalis, the Dip- lococcus meningitidis, the Pneumococcus, the Strepto- FIXATION OF COMPLEMENT 153 COCCUS pyogenes, the Micrococcus albus and aureus, the Colon bacillus, and the Cory neb act erium, pseudo- diphtheria'. From these later studies we have deduced the fol- lowing facts ; ( 1 ) In no case have polyvalent antigens prepared from meningococci, pneumococci, strepto- cocci, staphylococci, colon bacilli, or corynebacteria sufficed to fix complement, thereby not jeopardizing the specificity of the gonococcus antigen. ( 2 ) In ten per cent, of sera examined a weakly positive result was obtained with polyvalent Micrococcus catarrhalis anti- gen ; in these cases the reaction was much more marked with the various gonococcic antigens. Thus it may be inferred that the relation between the gonococcus and the Micrococcus catarrhalis is not positively and abso- lutely defined and it is not unlikely, on the one hand, that a culture of the M, catarrhalis is occasionally in- cluded in a supposedly specific polyvalent gonococcus antigen, while, on the other hand, it is undoubtedly true that a mixed infection often due to the M, catar- rhalis exists in patients suffering from gonorrhoea and its complications. Serum Diagnosis of Echinococcus Disease Echinococcus disease is rare in this country in the human being, and therefore serum diagnosis is seldom called for. Results with the complement-fixation test. 154 APPLIED IMMUNOLOGY however, have shown it to be a reliable method of diag- nosis. The technic of the reaction is the same as that for the Wassermann reaction. The antigen used con- sists of the fluid from hydatid cysts of sheep affected with the disease. Suitable amounts of this fluid when brought in contact with the blood-serum of patients suffering from echinococcus disease will cause comple- ment fixation. Complement-fixation Reaction in Typhoid Fever In reporting the results of the complement-fixa- tion reaction in typhoid fever, Garbat {A?n. Jr. Med, Sc, July, 1914) finds that the serum of practically all typhoid fever patients sooner or later gives a positive complement-fixation reaction. A highly polyvalent antigen properly prepared is absolutely essential in order to obtain a maximum of positive results. A positive complement-fixation test throws great cor- roborative diagnostic weight on the side of a doubtful or positive Widal reaction. Occasionally the test is positive before the Widal or blood culture, but usually not before the end of the second week. It generally persists for about six weeks after recovery. Complement-fixation Reaction in Tuberculosis Emplo^^ing as antigen a simple emulsion of liv- ing tubercle bacilli, ]McIntosh, Fildes, and RadclifFe {Lancet, Aug. 22, 1914) obtained positive comple- FIXATION OF COMPLEMENT 155 ment-fixation in 70 per cent, of 85 pathologically cer- tain cases and in 66 per cent, of clinically certain cases of tuberculosis. In 87 controls, taken from normal and disease conditions, without any selection, all were negative but three, these being two cases of leprosy and one of Addison's disease. From these results the reaction may be regarded as highly specific. The Complement-fixation Reaction as Applied to Protein Differentiation ( Neisser-Sachs Reaction) This method may be applied to supplement the re- sults of the precipitation reaction, and may serve to differentiate proteins where the precipitation reac- tion fails. It is of practical importance in the medico- legal identification of human blood. An anti-sheep hgemolytic system is prepared in the same manner as for the Wassermann reaction. For the identification of human blood, an anti-human serum is made by in- jecting a rabbit with human blood-serum. The anti- serum should be prepared of such a strength that 0.03 or 0.04 c.c. will give complement fixation with 0.00001 c.c. of human serum. The antiserum having been previously prepared, an extract of the suspected blood is made in approximately 1-1000 dilution in the same manner as for the precipitation reaction (see Chapter X). The test is carried out according to the following 156 APPLIED IMMUNOLOGY table, by mixing different quantities of the extract of the suspected blood with 0.1 c.c. of suspected blood and 0.03 c.c. of the antiserum. Extract of blood 0.1 c.c. 0.05 c.c. 0.02 c.c. 0.01 c.c. 0.005 c.c. 0.002 c.c. 0.001 c.c. 0.0001 c.c. Complement Antiserum 0.1 c.c. 0.03 c.c. 0.1 c.c. 0.03 c.c. 0.1 c.c. 0.03 c.c. . 0.1 c.c. 0.03 c.c. 0.1 c.c. 0.03 c.c. 0.1 c.c. 0.03 c.c. 0.1 c.c. 0.03 c.c. 0.1 c.c. 0.03 c.c. The tubes containing the above are placed in the incubator at 37° C. for one hour. Then the doses of hemolytic amboceptor and 5 per cent, sheep's cor- puscles are added and the tubes again incubated for one hour. If 0.01 c.c. of the extract of blood prevents haemolysis, the test can be regarded as positive for human blood. Antisera for blood of various animals can be pre- pared in the same way and the extract of unknown blood tested with them. XIV MISCELLANEOUS BIOCHEMICAL REACTIONS ABDERHALDEN'S BIOLOGICAL TEST FOR PREGNANCY— SERO-ENZYME TEST FOR SYPHILIS— ABDERHALDEN- FAUSER REACTION IN MENTAL DISEASES— MEIOSTAG- MIN REACTION— EPIPHANIN REACTION Abderhalden*s Biological Test for Pregnancy This biological test belongs to a class of newer serum reactions. It has been found by Abderhalden of Halle that foreign proteins entering the blood pro- duce specific protein-splitting enzymes. In preg- nancy, Abderhalden assumes that the placenta gives off a protein which causes the production of a specific enzyme in the blood which is capable of splitting up the placental protein with the formation of peptone and amino-acids. The test consists therefore of incu- bating the serum of a suspected pregnant woman with placental material, and then testing the fluid for the end products of protein digestion. Two methods are employed in the detection of these substances, first the dialysis metJiod, in which the presence of dialyz- able peptones are tested for at the end of the incuba- tion by means of certain color reactions; and second the optical method, in which the end products of pro- tein digestion are detected by means of the polariscope. A brief outline of the technic of the dialysis 157 158 APPLIED IMMUNOLOGY method is as follows: Fresh placenta is cut in small pieces and repeatedly boiled with a little acetic acid until all soluble protein is removed. This is deter- mined by testing with the biuret reaction. The pa- tient's serum is collected by puncturing a vein at the elbow. Five or six c.c. of serum should be obtained, and must be absolutely free from haemoglobin. For dialysis, Schleicher and Schull's diffusion shells are employed. These membranes must be impermeable to the protein of blood-serum and permeable to pep- tone, as ascertained by preliminary tests. The ninhy- drin reaction is used for reading the results. This substance, the full name of which is triketohydrinden- hydrat, gives a blue color with the end products of protein digestion. Into one of the diffusion shells are placed 1 gramme of the boiled placental material and 1.5 to 2 c.c. of patient's serum. The membrane is then placed in a small beaker containing 20 c.c. of water. The fluid in and outside the membrane is then covered with toluol and the whole incubated at 37° C. for 16 to 24} hours. At the end of this time, the water in the beaker is tested for products of proteid digestion by adding to it 0.2 c.c. of a 1 per cent, watery solution of ninhydrin. If the reaction is positive a blue color re- sults. The reaction must be carefully controlled by using at the same time other membranes containing BIOCHEMICAL REACTIONS 159 serum alone and placenta alone. Great care must also be exercised to see that all bacteria are excluded, and that the membranes are not permeable to protein, but will allow peptone to pass through. If the shells are used repeatedly, they must be scrupulously cleansed in order to avoid contamination from previous tests. By carrying out all proper precautions, the great ma- jority of observers have obtained results agreeing with those of Abderhalden, the test proving positive in pa- tients ranging from the early weeks of intra- or extra- uterine pregnancy to a few weeks post partum. Pearce and Williams {Surgery, Gynecology , and Ob- stetrics, April, 1913, p. 411) report a series of 36 cases of pregnancy giving positive results, controlled by negative results in a male and a non-pregnant female. Insufficient work has as yet been done using normal serum and serum of persons suffering with various diseases, to place the test on a conclusive basis. Pearce and Williams (loc, cit.) tested the reaction of serum of pregnant women with various organs, such as kidney, liver, and uterus, instead of placenta, and obtained positive results in some cases. While the test gives promise of becoming of importance in the diagnosis of pregnancy, yet further experiments to eliminate pos- sible sources of error must be carried on before it will attain wide clinical application. In view of the pres- ent possible sources of error in the reaction, and par- 160 APPLIED IMMUNOLOGY ticularly the difficulty of sending serum for a distance that is absolutely haemoglobin- free, the advertisements of commercial laboratories that they are prepared to carry it out as a diagnostic measure are premature. The optical or polariscopic method of reading the reaction, for which a polariscope is necessarj^ gives practically the same results as the dialysis-ninhydrin method, but at present entails so much expense that it is out of the reach of the ordinary laboratory. Abderhalden and others are at present working on similar protein-splitting reactions for the diagnosis of cancer and other diseases, which promise much for the future. Pearce and Williams have worked out a technic which promises to do away with many of the serious difficulties of the test, by which the reaction can be carried on in ordinary test-tubes instead of the dif- fusion shells. The mixture of serum and placenta is coagulated by heat and acetic acid, and the products of protein digestion, if present, are then separated from the coagulated serum proteins by filtration. Great caution must be observed that coagulation is complete and that the filtrate is rendered absolutely clear by a second boiling if necessary. This method does away in a large measure with the difficulties due to haemoglobin-stained sera. We quote the following directions as to the technic from Pearce and Williams* BIOCHEMICAL REACTIONS 161 article: "Measured amounts of serum are placed in several tubes, and to one is added boiled placenta, to others boiled kidney, or heart, or whatever the control may be, and with one tube containing serum alone, and others the tissues named above, all are placed in the thermostat at 37 '^ C. for 24 hours. At the end of this time the contents of each tube are poured into a sepa- rate beaker, diluted with 20 c.c. of water, boiled with the addition of acetic acid, and filtered. To 10 c.c. of each filtrate the ninhydrin test is applied." Jamison and Cole {New Orleans Med, Jour., Ixvi, 3, p. 188) using Pearce and Williams' technic have confirmed its reliability as compared with the diffusion method. Sero-enzyme Test for Syphilis Baeslack {Jour, Amer, Med. Assn., Mar. 28, 1914, p. 1002 and Aug. 15, 1914, p. 599) has applied the principles of Abderhalden's technic to the diag- nosis of syphilis in a considerable number of cases. The tissues made use of as antigen in the reaction are the pearly white gummata resulting from the inocula- tion of the testicles of rabbits with syphilitic tissue. This tissue is prepared in the manner prescribed by Abderhalden for the placental tissue in the pregnancy test, and the remainder of the technic is the same as that of the Abderhalden dialysis method, using nin- hydrin as indicator. Baeslack's results correspond 11 162 APPLIED IMMUNOLOGY closely with those of the Wassermann reaction carried out simultaneously. In early cases of syphilis the sero- enzyme reaction was positive earlier than the Wasser- mann. In cases of tabes and paresis in which the cerebrospinal fluid was tested the reaction was never positive, showing that the enzyme is not present in the cerebrospinal fluid. The sero-enzyme test, moreover, was positive in cases of tabes in which the Wasser- mann reaction of the blood-serum was negative. Abderhauden-Fauser Reaction in Mental Diseases Fauser {Milnch. med, Wochenschr,, Nov. 18, 1913, p. 384) applied the Abderhalden technic to the diagnosis of certain mental diseases, using as antigen tissue from the sex glands. In the case of males he employed testicular tissue, and in females ovarian tis- sue. According to his findings, the serum only of patients with dementia pr^ecox contained protective ferments against these tissues, which caused him to re- gard the reaction as specific for the diagnosis of this disease. Later workers, however, have found that other mental diseases, such as paresis, manic depres- sive insanity and epilepsy, occasionally produced posi- tive reactions. In a recent article, Simon {Jour, A, 31, A,j May 30, 1914, p. 1701) concludes that a posi- tive reaction, while not specific for dementia precox as held by Fauser, is the rule in dementia pr^ecox, while BIOCHEMICAL REACTIONS 163 it is the exception in purely functional psychoses, and that the test promises to become of great value when the technic, which at present is open to many errors, has been perfected. Meiostagmin Reaction Ascoli discovered that when a bacterial extract and the specific antibody produced by it in blood- serum were brought together a lowering of surface tension occurs, as shown by an increase in the nmiiber of drops in the fluid. The number of drops can be conveniently measured by Traube's stalagmometer. As an illustration, it may be found that a certain mix- ture of normal serum and extract of typhoid bacilli shows 50 drops; while a mixture of the same quanti- ties of serum from a typhoid case and the extract of typhoid bacilli will show 52 drops. This phenomenon has been observed in the case of several diseases, in- cluding tuberculosis, anchylostoma, and echinococcus disease. For all these diseases, however, there are simpler clinical tests than the meiostagmin reaction. This reaction promises to be of some importance in the diagnosis of malignant tumors, particularly car- cinoma. In this case the serum of the suspected pa- tient is tested with an extract made from cancer tis- sue. It has also been found that an extract made from beef pancreas answers the purpose as well. An extract of the dried tumor tissue or pancreas is made 164 APPLIED IMMUNOLOGY with methyl alcohol in the proportion of 1 : 4 at 50'' C. for 24 hours. It is then filtered while hot, and again after cooling. The serum is employed in 1 : 20 dilution in normal saline solution. In preliminary titration of the extract, decreas- ing quantities, made up to 1 c.c. with distilled water, are placed in tubes with 9 c.c. of 1 : 20 dilution of nor- mal serum. A control is also prepared with plain water and serum (without extract). These mixtures are incubated at 37° C. for 2 hours, after which the number of drops in each tube is noted by means of the stalagmometer. For the test, the dose of extract is employed which is in the tube that contains 3-5 parts of a drop more than the control tube. The following table will illustrate the titration of the extract : 9 c.c. normal serum (1 9 c.c. normal serum (1 9 c.c. normal serum (1 9 c.c. normal serum (1 9 c.c. normal serum (1 9 c.c. normal serum (1 9 c.c. normal serum (1 9 c.c. normal serum (1 20) 4- 1 c.c. extract 1 20) + 1 c.c. extract 1 20) + 1 c.c. extract 1 20) + 1 c.c. extract 1 20) + 1 c.c. extract 1 20) + 1 c.c. extract 1 20) + 1 c.c. extract 1 50 75 100 125 150 200 300 20) + 1 c.c. aqua dest. (control) Number of drops i^Q [59 + 3 parts of a drop. j-o 59+1 part of a drop. o^ 59 + 1 part of a drop. "d!^ 59 ^ 03 1 58 + 9 parts of a drop. g £ 58 + 8 parts of a drop. ^ g 58 + 8 parts of a drop. .J5 I 58 + 8 parts of a drop. In this case the dose of extract to be used in the test would be 1 c.c. of the 1 : 100 dilution, which shows about 3 parts of a drop more than the control. Before using the extract for diagnostic purposes, this dose, as determined by titration, should be tested for reliability with a number of known carcinomatous BIOCHEMICAL REACTIONS 165 sera and known negative cases. If it proves reliable, it may then be used for diagnosis of unknown sera. Known positive and negative controls should always be used in performing the test, and titration of the extract should frequently be carried out, as its strength is not constant. A reaction is regarded as positive when the number of drops is more than 1% in excess of the control. For the test, a mixture of the suspected serum and the dose of extract is made ; also a mixture of the sus- pected serum and distilled water. The same thing is done with normal serum. These mixtures are then incubated for 2 hours at 37° C. In the case of a posi- tive result the mixture of the patient's serum and tumor or pancreas extract must have an increase of more than 1% drops over the mixture of patient's serum and water, and over the mixture of normal serum and extract. The following table gives an example of a positive result : Number of drops Normal serum (control) 9 c.c. 1 : 20 dil. + 1 c.c. extract 1 : 100 dil 59 Normal serum (control) 9 c.c. 1:20 dil. + 1 c.c. aqua dest. 58 + 4 parts Suspected cancer serum 9 c.c. 1 : 20 dil. + 1 c.c. extract 1 : 100 dil 61 Suspected cancer serum 9 c.c. 1 : 20 dil. -\- 1 c.c. aqua dest. 58 -|- 7 parts Different investigators working with the meio- stagmin reaction give varying reports as to its relia- 166 APPLIED IMMUNOLOGY bility. The general opinion appears to be that while it cannot be regarded as infallible in the diagnosis of carcinoma, yet it has a distinct clinical value when taken along with other tests and symptoms. Epiphanin Reaction The epiphanin reaction of Weichardt is based on practically the same principle as the meiostagmin re- action, viz., an acceleration of diffusion in the fluid when an antigen is brought in relation with the specific antibody. SeifFert has shown that this phenomenon is manifested by a change in reaction to phenolphthalein. This test has been applied especially to the diagnosis of syphilis. For this purpose 0.1 c.c. of a 1 : 10 dilu- tion of the patient's serum is mixed with 0.1 c.c. of alcoholic extract of syphilitic fetal liver, 1 c.c. of deci- normal sulphuric acid and 1 c.c. of an exactly equiva- lent solution of barium hydroxide are slowly added, making a neutral mixture. On the addition of a drop of an alcoholic solution of phenolphthalein, the fluid turns red if the serum is syphilitic, but does not change color if it is non-syphilitic. It is doubtful if this test will become as widely used as the Wassermann reac- tion for the diagnosis of syphilis. XV SPECIFIC BACTERIAL REACTIONS ALLERGIC PHENOMENA— TUBERCULIN TESTS— LUETIN, GONORRHCEAL AND TYPHOID TESTS— SCHICK'S DIPHTHERIA TOXIN SKIN REACTION The interesting subject of hypersusceptibility, anaphylaxis or aWeTgy has been sufficiently reviewed in Chapter TV. It will be recalled that allergy is merely an incident in the process of immunization and signifies simply the reactive changes exhibited by an individual after infection or the injection of some foreign sustance or antigen. Allergic phenomena, therefore, may be characterized as those associated with increased sensitiveness ( anaphylaxis ) , and those with diminished susceptibility (prophylaxis). It is apparent that a number of skin manifestations as erythematous and urticarial eruptions of serum sick- ness, exanthemata of eruptive fevers, " disposition to sudden cuticular inflammation " noted by Jenner in his studies of cow-pox in 1798, tuberculin reactions, etc., are pure allergic phenomena, since they are the exponents of localized foreign proteins and the toxic substances incident to their destruction by the specific antibodies in the blood of the sensitized body. Con- spicuous among the allergic reactions to which practi- cal consideration at this time will be given are the 167 168 APPLIED IMMUNOLOGY tuberculin tests, the luetin test and the diagnosis of typhoid and gonorrhoea! infection. Tuberculins. — Tuberculin has been prepared in many ways and, although each preparation may be used diagnostically as well as therapeutically, it is true that one furnishes better results for diagnostic pur- poses, just as others are superior from the therapeutic standpoint. The available products include Koch's " old " and " new " tuberculins, Denys' " bouillon fil- trate," Spengler's " perlsucht," Dixon's " bacillary extract," and Russian " tuberculinum purum." Old Tuberculin (" O. T.").— The original tuber- culin made and used by Koch has been styled " old " in contradistinction to his later or " new " tuberculins. It is prepared from the five or six weeks' pure culture of B, tuberculosis on five per cent, glycerin bouillon. The culture medium is then evaporated to one-tenth of its volume and filtered through porcelain. The filtrate containing all the soluble secretion products of tubercle bacilli is then diluted with glycerin, form- ing a twenty-five or fifty per cent, solution. The glycerin acts simply as a preservative, not as a germi- cide. Hence the stock solution may become con- taminated if frequently opened, and it should be steril- ized before making dilutions by heating on a water- bath for one hour at 60° C. or by boiling in a test- tube for ten minutes. It is customary when making SPECIFIC BACTERIAL REACTIONS 169 dilutions to use a solution of approximately 0.25 per cent, phenol in normal saline. It is advisable to make up fresh such dilutions about once in two weeks, as the phenol may cause some coagulation of the tuberculin, resulting in precipitation and deterioration. Dilu- tions showing marked sedimentation should be dis- carded. New Tuberculin (" T. JR."). — Realizing that old tuberculin was merely a toxin of tubercle bacilli, and that the immunity produced by it was to the toxin only and not to the bacterium itself, Koch, in 1897, seven years after the announcement of " O. T.," described a new tuberculin. This consisted of the residue (Rilcktand) of tubercle bacilli and for brevity is known as " T. R." This tuberculin is prepared by drying in vacuo a virulent culture of tubercle bacilli. The dried substance is powdered in a mortar and triturated with normal saline. After centrifugation the supernatant cloudy fluid is discarded. The resi- due is again dried, ground, extracted with salt solu- tion and centrif uged. This time the clean supernatant fluid is pipetted off and retained. The process is re- peated until the residue is entirely used up. The clear solutions are united and added to glycerin, forming a twenty per cent, solution. ** T. R." is standardized so that one cubic centimetre represents ten milli- 170 APPLIED IMMUNOLOGY grammes of the original dried whole tubercle bacilli or two milligrammes of the active solid substance. New Tuberculin (" B, E.''), — Appreciating the fact that the injection of dead bacilli, as compared with " T. R.," caused an increase in the agglutination of the blood, Koch advocated a second new tuberculin, a bacillary emulsion {Bacillen Emulsion or B, E,), This is prepared by pulverizing finely a virulent cul- ture of B, tuberculosis, and suspending one part of the powder in one hundred parts each of distilled water and glycerin. It is standardized so that one cubic centimetre contains ^ve milligrammes of dried sub- stance in suspension. It will be borne in mind that the " new tuber- culins " are suspensions, not solutions, hence they must be thoroughly shaken before use. IMoreover, therapeu- tically, reactions are not so noticeable as after the use of " old tuberculin " and the resultant immunity is greater and more durable, both of which have added to their popularity. Denys' Tuberculin {'' B, F"),—ln 1905 Denys recommended the filtrate from bouillon cultures of tubercle bacilli. This tuberculin is commonly known as Bouillon Filtrate or " B. F." It is essentially the same as Koch's old tuberculin, differing only in that no heat is used in its preparation. It contains all the normal soluble products of the B, tuberculosis. SPECIFIC BACTERUL REACTIONS 171 Sjoengler's Perlsucht Tuberculin {'' P. T. O/') .— Perlsucht signifies *' pearl disease " in cattle, that is, true bovine tuberculosis. Spengler believed that a tuberculin prepared from this strain of the tubercle bacillus would prove most efficacious in the treatment of human tuberculosis, and indeed such was the case in his experience. The method of preparation of Spengler's tuberculin is precisely identical with that of Koch's old tuberculin. Dixon's Tuberculin. — This product is a saline ex- tract of living tubercle bacilli minus their fat. Six to eight weeks old cultures from four per cent, glycerin veal broth are removed and collected on hard filter paper. Equal quantities, by weight, of human and bovine types of bacilli are placed between sterile filter paper and dried in a thermostat for twenty-four to forty-eight hours. The dried bacteria are then treated in an excess of ether until all water and glycerin are removed. Further extraction of the fat with fresh ether is done and the same is removed from the bottom of the vessel by a Pasteur pipette. After the bacillary mass has been thoroughly dried and freed from ether, it is ground in an agate mortar and suspended in nor- mal saline in the proportion of parts one to five. The suspension is then shaken for eight to ten hours, after which it is allowed to stand at room temperature for several days. Finally it is filtered until the filtrate is 172 APPLIED IMMUNOLOGY free of bacilli, as determined microscopically, cultur- ally and by animal inoculation. One cubic centimetre of this extract represents one-half gramme of the bacillary mass. One-half per cent, phenol is added as a preservative. Tuherculinum Purum {" T, P"), — According to " New and Non-official Remedies," this Russian tuberculin is " the purified filtered extract of human tubercle bacilli in 50 per cent, glycerin, prepared in the same way as in Koch's old tuberculin, but subse- quently treated with alcohol, ether, chloroform and xylol in order to remove deutero-albumoses." These toxalbumins and glycerin-soluble by-products of the culture medium are held responsible for a certain amount of the toxic reaction noted in using old tuber- culin and their elimination has resulted in this so- called purified tuberculin. Technic of Making Dilutions Many pharmaceutical firms to-day market tuber- culins in serial dilutions most convenient for immediate use. However, if it be desirable or necessary to em- ploy stock preparations, the following technic may be found useful : Pipettes. — (a) One-tenth c.c. pipette, graduated in hundredths, is most economical of stock in making tuberculin dilutions. SPECIFIC BACTERIAL REACTIONS 173 (b) One c.c. pipette, graduated in tenths for larger dilutions. (c) Ten c.c. pipette, graduated in tenths of a c.c. for the highest dilutions. Pipettes are best sterilized by dry heat in specially constructed copper containers, or they may be kept immersed in a jar of two per cent, phenol or alcohol, rinsing in sterile diluting solution before use. Dilutions. — A convenient method of making dilu- tions economically is as follows: Dilution No. 1 : 0.1 c.c. stock tuberculin, pipette (a) + 9.9 c.c. diluting solution = 0.001 c.c. tuberculin. Dilution No. 2: 0.1 c.c. of Dilution No. 1, pipette (b) (1 subdivision) + 9.9 c.c. == 0.1 c.c. = 0.00001 c.c, tuberculin. Dilution No. 3: 0.1 c.c. of Dilution No. 2, pipette (b) (1 subdivision) + 9.9 c.c. = 0.1 c.c. = 0.0000001 c.c. tuberculin. In doses of 0.001 c.c. and over it is advisable to use pipette (b) for measuring the stock tuberculin. The Physiological Action of Tuberculin, — Many theories have been advanced. The most tenable ap- pears to be that of Citron, who explains the presence of antituberculin, demonstrated by Wassermann, Bruck and Ludke, in the bodies of tuberculous sub- jects, on the assumption that after an injection of tuberculin the cells in the immediate vicinity of the 174 APPLIED IMMUNOLOGY tuberculous focus unite with tuberculin by their re- ceptors (Ehrlich), and the cells thus attacked pro- duce receptors in excess of the demand. These over- produced receptors or antibodies are then set free in the serum to unite with other portions of tuberculin. Thus repeated tuberculin inoculations lead to the for- mation of large numbers of free agglutinins, anti- tuberculin and opsonins at the point of local infection as well as many fixed receptors or antibodies. Obviously, it will be seen that the tuberculin reac- tion is dependent upon the presence of specific anti- bodies. If the suspected individual is free from tuber- culosis and none exists, no reaction can be produced even by recourse to very large doses of tuberculin; nor can a reaction be elicited in the advance stages of the disease, because all antibodies have been consumed. On the other hand, in the average case of tuberculous infection, a comparatively small dose of tuberculin will suffice to evoke a reaction. The typical tuberculin re- action is threefold: general, focal and local. The general reaction consists of malaise, headache, in- somnia, bodily aches, nausea, cough, tachycardia, and particularly a rise in temperature of one or more de- grees. These phenomena are probably due to the fact that tuberculin, like any other protein, is split up by complement acting in conjunction with antibody, and the split toxic products formed give rise to the symp- SPECIFIC BACTERIAL REACTIONS 175 toms as they are eliminated. The focal reaction con- sists of the fresh inflammatory changes noted at the tuberculous focus, namely, congestion, pain, tender- ness, swelling, redness, etc. The local reaction com- prises the inflammatory signs observed at the site of the injection. The focal and local reactions are ex- plained by the interaction of combined tuberculin, newly formed antibodies and complement, in attract- ing phagocytes, with direct localizing action, produc- ing thereby an inflammatory reaction. If the local reaction be severe, necrotic tissue may be cast off. Following the above reactions, there is a tendency ex- hibited by the pathological process to heal. Tuberculin as a Diagnostic Agent. — The employ- ment of tuberculin in a diagnostic capacity is very ex- tensive, and rightfully so, because, properly and com- petently utilized, its value at times as an aid in difl*erential diagnosis is inestimable. In the authors' experience the positive or negative information thus yielded has been absolutely dependable. Its proper use, however, entails the greatest caution and dis- crimination as to indications and contra-indications on the part of the patient, size and administration of doses, and, not least of all, the correct brand of tuber- culin. Unanimity of opinion prevails that Koch's old tuberculin, " O. T.," whatever may be its method or form of application, is best for diagnostic purposes. 176 APPLIED IMMUNOLOGY The various methods utilized for the application of tuberculin diagnostically comprise, (1) subcutaneous injection, (2) intradermic injection, (3) cutaneous scarification, (4) percutaneous anointment, and (5) mucous membrane instillation. Method of Subcutaneous Injection, — First prac- ticed by Koch, it supersedes in reliability any other method of tuberculin application. Carelessness may render it the most dangerous method, but properly carried out it is absolutely harmless. It has been variously modified as to size and interval of dosage. Old tuberculin (O. T.) is the preparation of choice. Koch stipulated that doses up to 250 milligrammes could be administered to perfectly normal individuals without reaction, but advised a limit of 10 to 25 milli- grammes in practice. We feel that such doses are en- tirely too large, and, with few exceptions, agree with Roth-Schultz respecting the technic of inoculations. The subcutaneous tuberculin test is best carried out with the patient in bed or at rest. His temperature and pulse should be recorded every two or three hours for two or three days, also all clinical signs and symp- toms must be noted prior to starting the first inocula- tion. The primary injection given is 0.5 milligramme. Should the slightest indication of a reaction, either general, focal, or local (see page 174), supervene, the same sized dose is to be repeated two or three days SPECIFIC BACTERIAL REACTIONS 177 after its subsidence. If no sign of a reaction occurs, the dose may be increased to 1.25 milligrammes on the third day. Again if the typical reaction is not pro- duced and merely suggestive signs appear, the same sized inoculation is repeated, since this may be suffi- cient to provoke a marked reaction after the previous sensitization. In the absence of any reactive phe- TEMP MAV 20 21 22 23 24 25 2b 2T 2"8 29 0 ' "5 « O o - tft? ^ 1 J: ^ 102 -^ -- -- —^ - J _ . I _ i _: : X ^ = _. 101 - - -— -J ---- t t - V- ~\- 100 ::_ -I- -- - _: -I . 5 ► i % \ -h ^ „ : j\ j\ _L_ -♦ i_- ,99 ____^_ 7 \ ::" ^ ^ : : ^ H ^ ^^A Vj t ^-^ it --- ^1 k i --Z-- i - L ,j ---- — 1 — --- - - -' Fig. 15. — Edward G., aged 77. Tuberculous mastoiditis. Note typical diagnostic tuberculin reaction after second injection of tuberculin. Associated with the febrile rise and general reactions of malaise and headache, was a local reaction of increased pain and discharge from the mastoid. nomena after three or four days, an injection of 2.5 milligrammes should be given. If this fails to cause a definite response, a final maximum inoculation of 5 milligrammes is permissible. In children, under fifteen years of age, Baldwin recommends doses of 0.05, 0.2, 0.5 and 1 milligramme. A reaction to be pathogno- monic for any suspected lesion must comprise, in 12 178 APPLIED IMMUNOLOGY addition to a rise in temperature of at least one degree, increased focal inflammatory signs, located in the lungs, bones, joints, epididymis or wherenot (Figs. 15 and 16). Considerable importance is to be at- tached, also, to the inflammatory areola ofttimes sur- rounding the site of injection (local reaction). Ob- FiG. 16. — R. W. S. Bilateral tuberculous epididymitis. Observe the suggestive rise in temperature after the first injection of tuberculin, not to be regarded, however, as pathognomonic, owing to the absence of any focal reaction. The reaction occurring on the thirteenth of the month is typical generally, focally and locally. viously, a focal reaction outweighs in significance both the general and local reactions, and under such cir- cumstances a positive test may be pronounced even though the febrile rise has not amounted to a degree. The sites commonly chosen for the injections are the outer or posterior aspects of the arm, the supra- or interscapular areas, the outer aspects of the thigh or SPECIFIC BACTERIAL REACTIONS 179 leg and the lumbogluteal regions. After cleansing the skin with alcohol, the needle is to be introduced well subcutaneously and care taken not to pierce the muscular sheaths or to enter a vein. The syringe best adapted for making the inoculations is an all-glass type graduated in minims or, better, fractions of a cubic centimetre, owing to its cleanliness and ease of sterilization (Fig. 17). Reliable pharmaceutical houses supply tuberculin " O. T." in serial dilutions ready for immediate use. If the above technic be care- fully followed, and the patient restricted to bed on the ''¥7¥^''T'7"4"'wp[HX^ Fig. 17. — All-glass tuberculin syringe, graduated into hundredths of a cubic centi- metre, particularly useful for both diagnostic and therapeutic inoculations. occurrence of a reaction, until its disappearance, the test is devoid of danger, even when incipient and latent pulmonary tuberculosis is present. The subcutaneous tuberculin test, as stated above, is indicated in all forms of localized tuberculosis, when the diagnosis is in doubt. Among these lesions may be enumerated tuberculous ulcerations of the skin, lym- phadenitis, osteitis, synovitis and arthritis, laryngitis and tracheitis, phlyctsenular conjunctivitis, keratitis, iritis, uveitis, nephritis, prostatitis, seminal vesiculi- tis, epididymitis, peritonitis and pulmonary tuber- culosis. 180 APPLIED IMMUNOLOGY The contra-indications to the use of the subcutane- ous tuberculin test may be stated to be general miliary tuberculosis, acute phthisis, advanced pulmonary tuberculosis, tuberculous meningitis, markedly as- thenic individuals and convalescents from an acute ill- ness, conditions in which any tuberculin application may fail to produce reaction; in patients running a marked irregular temperature, and possibly also in children, the scarification or von Pirquet test takes precedence over the method of subcutaneous injection. Intradermic Injection, — Mantoux has strongly recommended this method for the administration of tuberculin, diagnostically, and to determine, by the severity of the reaction, the sensitiveness of the pa- tient prior to therapeutic inoculations. He claims that it is more sensitive than other tuberculin tests. Indeed it would appear too delicate, as almost all adults show a reaction, although tuberculosis, clinically, is absent. The test may be serviceable in very young children, but is not destined to become popular. Man- toux employs 0.01 milligranmie injected into the skin of the thigh. The reaction is not unlike the local in- flammatory process observed in the subcutaneous method. Scarification or von Pirquet Cutaneous Reaction, —This test, like the subcutaneous method, depends upon the antituberculin in the body fluids of the in- SPECIFIC BACTERIAL REACTIONS 181 fected individual. The reaction is very delicate, thus accounting for the high percentage of positive results. The test has its greatest applicability in children and patients exhibiting a comparatively high fluctuating temperature. Old tuberculin (" O. T."), again, is the preferable preparation in the performance of von Pirquet's test. It is conveniently put up in sealed capillary tubes and marketed by a number of pharma- ceutical firms. The technic is quite similar to vac- cination against smallpox. An area on either the arm or leg, usually the forearm, is cleansed with ether, not alcohol or soap and water, and a drop of " O. T." is placed on the skin ; about two inches distant a second drop of glycerin bouillon, utilized as a control, is placed. With a lance, a needle or special scarifier, im- mersed first in the central drop, then the drop of tuber- culin, the epidermis is lightly scratched, removing only the superficial epithelium and avoiding, if pos- sible, any evidence of blood. The drops should be al- lowed to evaporate to dryness, before applying a dress- ing of sterile gauze, otherwise they may be covered with a vaccine shield. At most a very slight hyper- emia, disappearing in twenty-four to forty-eight hours, due to traumatism, may be noted at the control point. A typical positive reaction at the site of the tuberculin drop is characterized by a hypersemic and inflammatory area from four millimetres to three 182 APPLIED IMMUNOLOGY centimetres in diameter. The intensity of the reaction varies from a hyper^emia with papule formation to many papules on an oedematous and inflamed base, to a markedly indurated zone exuding serum from the scarification site ( Plate III ) . The inflamed area may persist for a week or two. The reaction is not at- tended with general fever or other symptoms. A positive test simply indicates the presence of tuber- culosis; it does not specify, as does the focal reaction in the subcutaneous test, that a suspected joint, cor- nea, epididymis, prostate, etc., is tuberculous. A pa- tient with tuberculous peribronchial lymphadenitis may exhibit a positive reaction, while his particular cause for complaint, a troublesome knee, may be gonorrhoeal. Detre had evolved a modification of the above, known as a differential tuherculin test and therapeutic control. It has for its objects the difl'erentiation of the human and bovine types of infection, the measure- ment of the extent of the disease, whether incipient or advanced, the selection of the best variety of tuber- culin for immunization and a control in therapy superior to the opsonic index. Three varieties of tuber- culin are employed: Koch's " O. T.," " B. F." prepared from bacilli of the human type, and " B. F." as in the von Pirquet method, preferably the flexor surface of the forearm is cleansed with ether and a pledget of PLATK III ^. 1) Von Pirquet's cutaneous tuberculin test (positive reaction). SPECIFIC BACTERIAL REACTIONS 183 cotton. At distances of about three inches, from above downward, drops respectively of commercial " O T.," human " B. F." and bovine " B. F." are expressed on the skin from capillary tubes. In the medium of each drop the underlying skin is scarified, care being taken to cleanse the scarifier in passing from one variety of tuberculin to the other. The inoculated sites should be inspected every twelve to twenty-four hours. If no reaction takes place in three days the test may be pro- nounced negative. Positive reactions differ in no re- spect from those described under the von Pirquet test. Detre points out that a greater reaction at the site of inoculation with the bovine tuberculin means tuber- culosis of that type and indicates the use of tuber- culin of the bovine type in therapy ; also that if greater reactions occur with the Denys filtrates than with *' O. T.," Koch's old tuberculin should be employed in immunization and vice versa. Marked reactions are usually observed in early virulent infections, although not infrequently they occur in the chronic latent form of the disease, including surgical cases. The reaction to filtrate is prone to disappear more in old chronic cases. Routine periodic applications of the test are necessary if it is to be utilized as a control of therapy. Method of Percutaneous Anointment. — Moro de- scribes the use of an ointment compounded of equal parts of tuberculin " O. T." and refined anhydrous 184 APPLIED IMMUNOLOGY lanolin. The test is not so delicate or reliable as either the premier subcutaneous method or even von Pir- quet's scarification, but is utilized by some practi- tioners, especially in febrile patients, because of its ease of application and absolute harmlessness. The ointment is applied as follows : An area of skin about five centimetres in diameter on the abdomen or in the mammary region is cleansed with soap and water, alcohol and finally with sterile water to remove all traces of alcohol. A mass of the ointment, about the size of a pea, is thoroughly rubbed into the prepared area for one-half to two minutes. A gauze dressing, covered with oiled silk or wax paper, is serviceable to protect the patient's clothing. The reaction, occur- ring usually within a few to forty-eight hours, rarely delayed for a week, is characterized by a few to a hundred or more discrete papules from one to five millimetres in diameter, in the case of marked reac- tions surmounting an erythematous base and as- sociated with itching, at other times not. The papules dry up and desquamate in a few days and at the end of two weeks merely a brownish pigmentation re- mains visible. The reaction is not accompanied with fever, pain or undue discomfort. The more intense reactions are observed in scrofulous conditions and bone tuberculosis ; weaker reactions are usually seen in infections of the lungs. SPECIFIC BACTERL\L REACTIONS 185 Method of Mucous Membrane Instillation, — Wolff -Eisner first reported on the use of ten per cent. " Alttuberculin " as a valuable diagnostic test when instilled into the conjunctiva. Almost synchronously, Calmette pointed out the irritation due solely to the contained glycerin in ordinary tuberculins, especially '* O. T.," and recommended its application as an oph- thalmic test only after precipitation and purification with alcohol. Even as such the conjunctival test has not been received in all quarters with much favor, owing to a number of serious ophthalmic complications, which have arisen incident to the use of tuberculin in the eye. Therefore, it is strongly contra-indicated in conjunctivitis, ulcers, blepharitis, iritis, keratitis, tra- choma and all diseases of the internal structures of the eye. Scrofulous persons are predisposed to the formation of phl^^ctenules as a result of the reaction. Tuberculin for this test is obtainable, commercially, in either solution or tablet form. Baldwin advises an initial instillation into the conjunctival cul-de-sac of one minim of a one-half per cent, solution; in the absence of a reaction in forty-eight hours, the same quantity of a one per cent, solution is instilled into the other eye. The reaction usually develops in three to twelve hours, but may be delayed a day or two, and often endures for a week. It is characterized by itch- ing, burning, serofibrinous exudate, congestion and 186 APPLIED IMMUNOLOGY redness of the caruncle, sometimes involving the en- tire palpebral conjunctiva and lids. Vaughan, Jr., states that he has never seen any- ill effects as a result of the application of the test. His technic consists in dissolving a tuberculin tablet in five minims of boiling hot water, producing a one per cent, solution. One drop of this, after cooling, is instilled into the conjunctival sac; if no reaction takes place, at the end of a week a second instillation of one drop of one-half per cent, solution is instilled into the same eye. He believes the zymogen created by the first in- stillation is activated by the second, causing the anti- tuberculin ferment to attack the tuberculin, thereby liberating the toxic cleavage product in large amount, even though the second dose is but one-half of the first. As soon as a reaction becomes distinctive, the conjunctiva is copiously flushed with a solution of boric acid, in order to prevent undue inflammation. This tuberculin test has been unsuccessfully applied to the nasal and vaginal mucosa. JLuetin Cutaneous Reaction in Syphilis, — Noguchi, in 1911, described an allergic cutaneous reaction in syphilis. Following the successful cultivation of the Treponema pallidum {S pirochceta pallida) in ascitic fluid and ascitic fluid agar containing pieces of pla- centa, the agar growth was ground to a paste in a mortar, then diluted with the fluid culture, steriHzed 5-g P o V- z n p OD ,3 Cr cc a- g B IR o P D - , ^ < m p n » O X D ^ ? c_ tn P ,"5 z c o p" IR •-« c n SPECIFIC BACTERIAL REACTIONS 187 at 60° C. for an hour in a water-bath, preserved in 0.5 per cent, phenol and standardized with the dark- field microscope, so that forty to one hundred dead treponemata occupied the average field. To this preparation Noguchi assigned the name luetin, and has recommended it as a valuable diagnostic aid, superseding even the Wassermann reaction in certain forms of syphilis. The technic of the test consists in sterilizing a small area of skin of both arms, using alcoholic sublimate solution. Intradermically, in one arm, with a syringe having a very fine needle, 0.05 of a cubic centimetre of a control suspension is injected; in the opposite arm a similar quantity of luetin. Normal or negative reactions consist of a small erythematous and occasionally a papular formation, not attended with pain or itching sensation, occurring in 24 hours, and disappearing usually in 48 hours and always in 72 hours, even if papular. No indm*ation results, although yellowish pigmentation due to ecchy- mosis is commonly seen. Positive reactions assume three forms : ( 1 ) papu- lar, (2) pustular and (3) torpid, and are character- ized by the formation, a day or two after inoculation, of a large, reddish papule usually five to ten millime- tres in diameter (Plate IV) . A hyperamic zone fre- quently surrounds the papule. The inflammatory process increases and extends for the next three or 188 APPLIED IMMUNOLOGY four days, then slowly recedes, all signs disappearing usually in a week or two. At the height of the reac- tion the papule commonly presents a bluish-red colora- tion and may be associated with vesicles, even under- going pustulation and ulceration with secondary crust formation. In the so-called torpid form, the slight reactive signs entirely disappear in three or four days and will suggest a negative result. However, after ten or more days, the injected site may light up and proceed to pustulation as above described. Systemic reactions, as malaise, loss of appetite, diarrhoea and slight rise of temperature occur rarely. It is not assumed that this test will replace the Wassermann reaction. Certainly, the Wassermann reaction is infinitely superior to the luetin test in the primary, secondary and acute tertiary stages of s^^phi- lis, although in the latent nervous and hereditary forms of the disease Xoguchi adheres to the superior- ity of the skin reaction. He found the test positive in 100 per cent, of manifest tertiary lesions, in 94 per cent, of latent tertiar}^ and in 96 per cent, of heredi- tary syphilis. It would seem that the serological reac- tion is more readily influenced to become negative under treatment than is allergic cutaneous reaction. Cutaneous Eeaction in Gonorrhoea. — In 1908, Irons alluded to the diagnostic value attached to the constitutional disturbances provoked by therapeutic PLATE V X \ r \ tilled polyvalent suspension ol gonococci ejection of one cubic millimetre of SPECIFIC BACTERIAL REACTIONS 189 inoculations of dead gonococci and suggested the use of gonococcic bacterin in this capacity. At the time of this report, one of the authors ( Thomas ) had been utihzing an alternation of subcutaneous injections of tuberculin, gonococcic, streptococcic, pneumococcic and staphylococcic bacterins in the differential diag- nosis of the etiological bacterium in obscure articular affections, with a view to the determination of the correct biological therapeutic agent. Contrary to Irons' apparent greater reliance on the general and focal reaction, Thomas attached more significance to the local reaction at the injectional site, employing much smaller doses, 100,000,000 instead of 500,000,- 000 cocci. Recently the substitution of unheated bouillon filtrate or a filtrate similar to old tuberculin for the bacterin has produced more dependable results. Whether the filtrate or bacterin is used in small or large dosage, subcutaneously or intradermically as has been done by London, or by scarification, the reaction to be looked for simulates precisely that observed in tuberculin tests, namely, local hyperasmic areas with or without papules or crusts, focal inflammatory changes at the affected site, malaise, leucocytosis, rise in tem- perature, etc. The reaction appears in six to twenty- four hours and persists for two to three days, usually unattended by systemic phenomena, unless large doses are administered. No control is necessary, as relatively 190 APPLIED IMMUNOLOGY large inoculations of dead gonococci do not produce the reaction in non-gonorrhoeal subjects. The test finds its greatest field of usefulness in the differential diagnosis of obscure synovial, joint, periosteal and gjTisecological affections ; also promises to be of value in the control of candidates for matrimony and as a guide to the insufficiency of treatment. Cutaneous Reaction of Typhoid Immunity, — Gay and Force very recently have announced a decisive skin reaction in 95 per cent, of persons who have had typhoid fever. Furthermore, they found that nine out of fifteen individuals inoculated from four and three-fourths years to eight months previously, in ac- cordance with the method of the United States Army, gave positive reactions, and that of twenty-five per- sons inoculated within eight months, none showed a negative response. They conclude that the test may serve as an index of the duration of typhoid immunity following protective inoculation, thereby governing the time for re-inoculation. The preparation used for the test consists of ty- phoid cultures grown for five days in five per cent, glycerin-broth. This is evaporated to one-tenth of its original volume over acetone, simulating the prepara- tion of Koch's old tuberculin. The test is carried out precisely as is von Pirquet's tuberculin method, pro- ducing reactions of identical character (see page 180) • SPECIFIC BACTERL\L REACTIONS 191 Schick's Diphtheria Toxin Skin Reaction This test, which promises to be of great practical importance, has recently been studied in this country by Veeder (Am. Jour, Dis, Child,, 1914, viii, 154- 162). The technic is similar to that of the Noguchi luetin reaction, and consists of the intracutaneous in- jection of an amount of diphtheria toxin equal to 1-50 of the lethal dose for a 250-gramme guinea-pig. This is so diluted that the amount of toxin is contained in 0.1 c.c. of fluid. A positive reaction is characterized by an area of redness and swelling within 24 or 48 hours, and indicates the absence of antitoxin in the blood. A negative result, on the other hand, is evidence that the individual is well supplied with diphtheria anti- toxin. By actual test it has been found that in the case of a negative result the individual tested possesses at least 0.031 unit of antitoxin per cubic centimetre of blood, which is considered sufficient to protect him from an ordinary attack of diphtheria. The practical importance of the test lies in its use as a preliminary indicator of the necessity of pro- tective injections of antitoxin in persons exposed to diphtheria. It is argued that those giving negative reactions already possess in the blood sufficient anti- toxin for protective purposes, and therefore require no prophylactic injection. Those giving positive reac- tions, on the other hand, possess insufficient antitoxin, 192 APPLIED IMMUNOLOGY and require protective inoculations. Veeder's studies show that in children from birth to 15 years of age, which includes the period of greatest liability to diph- theritic infection, approximately 50 per cent, give negative reactions. If the test proves its reliability, this will mean a saving of one-half of the amount of antitoxin to be used prophylactically, and also a great lessening in the number of cases of temporary dis- ability that sometimes follows antitoxin injections. Kolmer and JNIoshage {Am. Jour. Dis. Child., March, 1915, p. 189) , after extended studies with this reaction, reach the followang conclusions: 1. The toxin skin reaction is a valuable and reliable method for detecting susceptibility to diphtheria. 2. Persons reacting negatively to this test usually contain at least ^Q unit of diphtheria antitoxin per cubic centimetre of serum, and this amount of antitoxin is probably sufficient to protect against infection. 3. Persons reacting weakly or strongly positive usually contain less than %o of a unit of antitoxin per cubic centimetre of serum or none at all. They may be regarded as susceptible to diphtheria and in the event of ex- posure to infection should be passively immunized with antitoxin injection. 4. About 40 to 50 per cent, of children ranging from 1 to 15 years of age react positively to the toxin test; this means that the preliminary use of the toxin test will eliminate the necessity of administering prophy- lactic doses of antitoxin to about 50 per cent, of children. 5. The toxin reaction indicates that the immunity conferred by an injection of antitoxin begins to disappear after ten days and has gen- erally passed away entirely after four weeks. 6. The increased susceptibility of persons with scarlet fever to diphtheria is shown by the toxin reaction; even after the injection of antitoxin about 10 per cent, are susceptible within ten days. 7. According to the toxin reaction the immunity conferred by an attack of diphtheria is usually of short duration or entirely absent. 8. The most practical application of the toxin reaction consists in applying the test as a preliminary measure to all persons who have been exposed to diphtheria and immunizing only those who react positively. XVI TUBERCULIN THERAPY PROPHYLAXIS— THERAPEUTIC ADMINISTRATION OF TU- BERCULIN—AVAILABLE PREPARATIONS— MODES OF ADMINISTRATION AND DOSAGE— CONTROL OF TUBER- CULIN TREATMENT— LIMITATIONS AND CONTRA-INDI- CATIONS— INDICATIONS AND RESULTS Prophylawis. — Although the promises of Koch, Behring, Maraghano, Arloing, Friedmann and others have never measured up to their greatest expectation, hope still exists in the breasts of investigators that the future holds a specific for inoculation against tubercu- losis. Indeed, there appear to be good reasons, both experimentally and clinically, why this should be- come a realization. Auto-immunization in human beings is probably of common occurrence. This is attested to by the prevalence of tuberculosis in child- hood, and the fact that only 25 per cent, of patients succumb to the disease, while in the remaining 75 per cent, almost all exhibit evidence of healed tuberculous foci. Thus it is logical, irrespective of the assertion that tolerance to tuberculin probably never means the production of true immunity in tuberculosis, that we should reinforce our defensive forces, prophylactically as well as therapeutically. Indeed, Hamburger, in view of the frequent contraction of tuberculosis in early childhood, advises immunization in infancy and asserts that the proper administration of tuberculin at 13 193 194 APPLIED IMMUNOLOGY this time constitutes one of its most valuable uses and will confer immunity. Therapeutic Administration of Tuberculin. — Al- though tuberculin has failed to become the much- vaunted panacea that, at first, it was hoped would be realized, it, at least in most cases, when properly em- ployed, favorably and decidedly influences the tuber- culous process. This has led, in recent years, to a marked revival of interest in its therapeutic employ- ment. It must be clearly and definitely understood by the clinician, as a fundamental premise, that it is not claimed that tuberculin is a specific for, or per se a curative agent in, the treatment of tuberculosis. It is to be regarded simply as an accessory agent of Nature, and when thus utiHzed serves as a most valuable, if not indispensable, adjunct to routine antituberculosis measures, amounting frequently, in localized and non- febrile cases, apparently to curative results. Failure is due oftener to incompetent, careless, or reckless ad- ministration than to tuberculin itself. It must be con- stantly borne in mind that tuberculin, more so than most biological products, is a powerful agent, capable of producing evil, or even disaster, rather than good, unless properly and wisely administered. In experi- enced hands, however, tuberculin therapy is absolutely harmless. Trudeau states: TUBERCULIN THERAPY 195 We have learned the dangers of tuberculin treatment and its evident limitations. We have, however, also in late years learned something about the complex defensive resources of the living organism which tend to the production of immunity, and how to call them into action, though we are evidently as yet only on the threshold of the knowledge of im- munization by vaccines in the treatment of chronic infections. Every- thing we know, however, points to immunization as the goal toward which our efforts should be directed. We have much to learn about tuberculin treatment, but even in the present state of our knowledge, I am inclined to think that the production of tuberculin immunity by the mild clinical method is capable of favorably influencing the course of chronic tuber- culosis, of prolonging life, and in many cases of aborting a commencing infection or extinguishing the smouldering fires of a chronic infection. Available Preparations, — It is obvious, from the great number of tuberculins that have been recom- mended during the past twenty-five years, that none is ideal, particularly for therapeutic purposes. Some have fallen into disuse because of their greater toxic properties; others have gained confidence because of greater immunizing power ; still others have been sub- jected to purification processes, in the hope of elimi- nating toxic reactions and so on. Those preparations which to-day enjoy greatest popularity, in the order named, are Bacillen Emulsion or " B. E,," Tuber- culin Riickstand or " T. R.," Bouillon Filtrate or " B. F.," Old Tuberculin or " O. T.," Spengler's Perlsucht Tuberculin, " P. T. O.," Dixon's Bacillary Extract, and Tuberculinum Purum, " T. P." (see Chapter XV) . The majority of tuberculin therapists favor Koch's so-called new tuberculins, " B. E." and " T. R.," believing that the resultant immunity is 196 APPLIED IMMUNOLOGY greater and the toxic effect is less; some still adhere to " O. T." or its bovine preparation according to Spengler; a few have faith in Dixon's product and many have been impressed by the virtue of " T. P.," which Neumann has shown to be the least toxic, hence to be preferred in febrile cases or when the process of immunization is to be effected in the shortest possi- ble time. The ideal modus operandi would appear to be a combination or alternation of " B. E." and " B. F.," the former consisting of the bacilli and their extractives inactivated by a minimal degree of heat, the latter comprising the unheated filtered toxins of the bacilli; thus the patient would seem to be im- munized against all constituents of the bacillus. A number of attempts have been made to employ so- called modified tubercle bacilli in the treatment of tuberculosis. The most recent failure of this nature was the well-advertised " Friedmann Cure " or inocu- lation with non-virulent turtle tubercle bacilli. Modes of Administration and Dosage, — The usual method of administering tuberculin to patients is by subcutaneous inoculation (for technic see Chapter XV, p. 178). In the case of " B. E." and " T. R.," the common initial dose may be 0.001 milligramme ; ^ with Dixon's tuberculin, the beginning inoculation ^ A few clinicians utilize and recommend the superiority of infinitesi- mal doses in tuberculin therapy, namely, an initial inoculation of 0.0000001 to 0.000000001 milligramme of " B. E." or " O. T." TUBERCULIN THERAPY 197 consists of the extract from 1.0 milligramme of tuber- cle bacilli; with Tuberculinum Purum or " T. P.," the usual primary dose is 0.02 milligramme. In the absence of reactions, the size of these doses is doubled, semi-weekly and later weekly, until the maximum tolerant dose is reached. Dixon advises that the mini- mal dose be repeated five times at intervals of five days, before proceeding to the next higher dose, which is ten times the strength of the first. This is repeated five times before giving dilution No. 3, which is twice the strength of the former. Thus the patient is car- ried through seventeen more dilutions, each being an increase of one-tenth of its predecessor. In children the size of the dose is regulated by the age of the patient, as in any other therapeutic remedy. As a rule children tolerate tuberculin very well. For most tuberculins the technic of making dilu- tions as described in the previous chapter on page 172 will prove satisfactory. Pharmaceutical firms now prepare various tuberculins in serial dilutions, bearing legends explanatory of their use, and rendering their employment a matter of great convenience. If the dilutions are not freshly prepared, caution must be exercised to see that they are not clouded or precipi- tated, hence inert, due to the action of the preservative phenol. Tuberculins for subcutaneous inoculation are thus marketed in vials with hermetically sealed rubber 198 APPLIED IMMUNOLOGY caps, glass ampoules, or in tablet form. Tuberculin tablets, " B. E." and *' T. R.," are now obtainable in six strengths, namely, 0.0001, 0.001, 0.01, 0.1, 1.0 and 10 milligrammes. By dissolving a tablet in one c.c, using a special graduated syringe (Fig. 17) , or in a ten c.c. graduate containing ten c.c. of water or fractions thereof, injecting never more than one c.c, the in- oculations with proper sized dosage can be readily executed. Tablets possess the advantage of being more stable than solutions, although great precautions as to sterility must be observed. Contrary to the researches of PfeifFer and Persch, that pepsin, trypsin and enterokinase destroy the ac- tivity of tuberculin, a number of investigators have administered and recommended tuberculin by mouth, stating that when thus administered it is just as effec- tive as by subcutaneous injection, and carelessly given is equally capable of harm and even fatal results. Oral treatment has been carried out with 0.00001 to 0.001 milligramme of " T. R. " in 10 c.c. of normal horse serum; in the presence of mixed infection, staphylococcic bacterin has been combined with the dose, best given on an empty stomach. Pharmaceu- tists have prepared triturates of "B. E." and " T. R. " for mouth administration, the tablets arranged serially and containing from 0.000001 to 0.01 milligramme each. TUBERCULIN THERAPY 199 Tuberculin has also been given per rectum either in dosage of 0.001 milligramme " T. R." in normal serum or in the form of suppositories as recommended by Lissauer. Suppositories of " O. T." are obtain- able, the doses ranging from one to five hundred milli- grammes. Tuberculin therapy by mouth, and to a less extent by rectum, thus far has not been and probably never will be very favorably received by the profession. The physiological action of tuberculin, locally, focally, and generally, has been sufficiently described in the foregoing chapter (see page 173). Control of Tuberculin Treatment. — Specific ther- apy under no circumstances invites or permits relaxa- tion in the general hygienic and dietetic management of the case. Tuberculin therapy may be controlled by (1) the opsonic index or (2) the clinical symptomatology. The opsonic control of Wright (see Chapter XX) has comparatively few adherents in the treatment of tuberculosis, although Wright and his school have found it particularly well adapted to govern the inocu- lations in surgical tuberculosis. They believe that so long as the opsonic index remains in the positive phase, further and particularly increased dosage is unnecessary and inadvisable. The clinical symptomatology serves as the popular 200 APPLIED IMMUNOLOGY method in the control of tuberculin therapy, not only because of the tedious technic of the opsonic index, but also on account of the inferior results of the latter method, at least in the pulmonary form of the disease. The practitioner should not be led to believe that tuberculin treatment, properly guided by the clinical symptoms, is an easy matter requiring no special in- struction or knowledge. Far better that he realize that tuberculin is a double-edged sword, cutting suc- cess on the one hand and strewing disaster on the other. Due consideration and correct interpretation of trivial symptoms, as malaise, headache, slight fever, weakness, grippy sensations, vague pains, insomnia, anorexia, nausea, loss of weight ; slight focal reactions, as increased cough, expectoration, rales, pleuritic and laryngeal pains, vesical irritability, suppurative and other inflammatory signs; marked tenderness, pain, redness and swelling at the site of injection, are most important in therapeutic inoculations, and indicate that, for the time at least, the patient's tolerance to tuberculin has been reached; a further inoculation at this time may spell disaster. High fever, prostration, marked focal inflammatory signs and prolongation of systemic reactive phenomena mean that the patient has received an overdose of tuberculin and that treat- ment must be indefinitely suspended. Dogmatic in- structions to increase progressively the dose every TUBERCULIN THERAPY 201 three, five, or seven days, without due regard to the physiological effects, are criminal. No man should ever employ tuberculin who is ignorant of its physiologi- cal action. This action may escape notice, except by the skilled observer. The phenomena which do occur often demand keen discrimination as to the size and in- terval of subsequent dosage. The patient should have his temperature, pulse and respirations recorded at three-hour intervals for two or three days before and throughout the course of treatment. His weight should also be noted weekly. If febrile he must remain quietly in bed. In general the initial dose should be quite small in accordance with the directions given above for the particular tuberculin concerned. If no reaction whatever be produced, the dose may be doubled in three or four days. If there be slight evi- dence, general or focal, but nothing definite, the same sized dose should be repeated. If, however, a slight reaction occur, the next inoculation must not be given until all traces of the reaction have disappeared for three or four days, and then not more than half of the previous dose should be given. If, after any in- oculation, a severe reaction be precipitated, no further inoculation shall be given for two or three weeks after the patient's condition has returned to normal, and the dose then must be not more than a fourth or a half of 202 APPLIED IMMUNOLOGY the previous intolerant inoculation. Again the dosage ascends the scale, and this time will probably pass be- yond the size of the former intolerant injection with- out harmful effect. Occasionally, a patient will be en- countered exhibiting tuberculin hyper susceptibility. This does not refer to the ordinary response to a full- sized dose of tuberculin, but such a state as is seen when, after the reduction of the dose to one-half, the reaction reappears, and after a week or ten days with a further reduction to one-tenth, an even greater re- action occurs. This supersensitiveness to tuberculin can be overcome and immunization resumed by sus- pending all inoculations for three or four weeks, then beginning far down the scale of dosage; that is, one- thousandth of the former inoculation. Experience has taught that most patients first show reactions to doses of tenths and hundredths of a milligramme. Consequently, when doses of these or larger sizes are administered, they should be spaced by a week or ten days instead of three or five days as is the flexible rule when administering the early small doses. An all-important thought for the tuberculin thera- peutist to bear constantly in mind is that in the average case it will require six to eight months to reach large immunizing doses; if the patient be hypersensitive the TUBERCULIN THERAPY 203 time must be extended to a year or more.^ Any at- tempt to " push the treatment," by shortening the intervals or carelessly increasing the size of the doses, will result in failure. Limitations and Contra-indications of Tuberculin Therapy. — Two facts must be emphatically and in- delibly impressed in the mind of the tuberculin thera- peutist. They are: First, that tuberculin acts simply as an accessory to nature, and, second, that tolerance, even to large doses of tuberculin, does not necessarily confer immunity against tuberculosis. If these points are fully realized, tuberculin, in capable hands, will measure up to expectations and be accorded its right- ful place in modern therapeutics. Success in tuberculin therapy is directly propor- tionate to the degree of dosage attainable without pro- ducing deleterious reactions or intolerance. In the average case this is a procedure of months' or perhaps years' duration, in the event of the necessity of inter- mittent administration. Obviously, little can be ex- pected from a short course of tuberculin inoculations with a maximum tolerant dose of only a fraction of a milligramme. After the patient has been successfully carried ^ Tuberculinum Purum, " T. P.," is an exception to this doctrine, since the patient may receive the entire series of inoculations in the course of three or four months, due to the lesser toxicity of this preparation, making it possible to ascend the scale of doses rapidly. 204 APPLIED IMMUNOLOGY through a course of treatment and has reached large doses, these must not be continued indefinitely, lest the patient become over-stimulated and thrust into the '' negative phase," hence no longer capable of the pro- duction of specific antibodies. It were better to cease inoculations for several months, when, if indications arise, a new course of treatment may be instituted. As a general rule, tuberculin therapy is contra- indicated in acute, diffuse and chronic affections with acute exacerbations. It is far more applicable to so- called surgical or localized tuberculosis than to the pulmonary form of the disease. In short, it may be stated that tuberculin should not be employed in phthi- sis pulmonalis except in the afebrile or mildly febrile cases. It is definitely contra-indicated when a remedia- ble surgical condition, as caries, sequestrum, tubercu- lous kidney, etc., exists, until after operative interven- tion. The usual acute or advanced case of pulmonary involvement should be invariably treated by rest in con- junction with the well-known climatic, dietetic, and hygienic antituberculosis measures. Subsequently, if there be no contra-indication and the patient is not progressing satisfactorily, tuberculin should supple- ment the treatment. Tuberculin therapy is decidedly contra-indicated in debilitated or wasted individuals or patients exhibiting very active lesions or extensive complications, characterized by considerable and in- TUBERCULIN THERAPY 205 creasing fever, although shght dyspnoea, increased respirations, accelerated pulse-rate and even hsemop- tysic tendencies, with or without evidences of dry pleu- ritis, are not necessarily contra-indications. Extreme caution must be exercised in practicing tuberculin in- oculations on ambulatory patients, and particularly is this so if they are febrile, even though slightly and only occasionally. Menstruation and the supervention of an acute infection as a " cold," tonsillitis, bronchitis, etc., are definite indications for the temporary suspen- sion of bacterial inoculations. A matter of some importance in tuberculin therapy is the prevalence of mioced infection in tuberculous processes, either pulmonary or surgical. The advent of the secondary invading bacterium is characterized by an increased and typical irregularity of tempera- ture. Under such circumstances culture and reculture of the suppuration from time to time and the prepara- tion and administration of autogenous bacterins, pre- ceding or alternating with tuberculin, will produce results not obtainable with tuberculin alone. Indications and Results of Tuberculin Therapy in Tuhercidosis, — Tuberculin is a powerful therapeu- tic agent in all localized, subacute and chronic forms of tuberculosis, not excluding the pulmonary type, provided the patient is not markedly asthenic or feb- rile. The immediate clinical effects of tuberculin 206 APPLIED IMMUNOLOGY inoculations are ofttimes distinctive and impressive. Briefly summarized they are: (1) local, manifested by improvement in healing tendencies at a former opera- tive site, disappearance of inflammatory signs, of cough, expectoration and tubercle bacilli from the spu- tum, although the last is very persistent and may never take place; (2) general, comprising improvement in appetite, digestion, strength, weight, disappearance of fever, etc., and (3) mental, that is, general morale. The various forms of tuberculosis in which tuber- culin has proved useful, if not curative, comprise pulmonar}', genito-urinary, bones and joints, intes- tinal, peritoneal, laryngeal, ocular, mastoidal and lymph-nodal. In most sanitaria, tuberculin is routinely employed in selected cases, namely, afebrile or mildly febrile pa- tients, when an incipient infection may be aborted or the course of a chronic loulmonary process favorably influenced. In 1907, Trudeau stated that during the past 15 years the post-discharge mortality of patients from the Saranac Sanitarium was 18 to 25 per cent, less with tuberculin-treated than non-treated cases. Indeed this deduction is reasonable in view of the fact that tuberculin cautiously and competently adminis- tered is the best expectorant obtainable. Recently, tuberculinum purum, " T. P.," has been extolled in the treatment of this form of the disease (Fig. 18). 208 APPLIED IMMUNOLOGY The forms of genito-urinary tuberculosis amenable to tuberculin include hacillary tuberculous nephritis, where both kidneys are functionally sufficient, but the primary or more affected kidney is indeterminable; bilateral, functionally insufficient, hence inoperable tuberculous kidneys; secondary ureteritis and cystitis following nephrectomy for tuberculosis and primary or secondary epididymitis, prostatitis, seminal vesiculi- tis and salpingitis. Exact discrimination must be exercised in the latter conditions as to whether the tuberculin syringe shall precede or follow the scalpel. Perhaps the best results in tuberculin immuniza- tion have occurred in the treatment of chronic osteitis and arthritis. Frequently there is a superimposed mixed infection in these cases, calling for the pre- liminary or alternative employment of autogenous bacterins ; repeated cultures become necessary at least biweekly, to identify the changeable pyogenic bacteria for preparation of the correct bacterin. We have treated many such cases of knee, spinal, and hip- joint disease with excellent results (Fig. 19). In acute osteomyelitis and arthritis, tuberculin therapy should be condemned. Intestinal and peritoneal tuberculosis offers little promise for tuberculin. We have experienced re- markable results using tuberculin following explora- tory laparotomy revealing extensive nodular tuber- JAN -I* .t'^'Ht .f It -* m tf 1 J.C istered oi TUBERCULIN THERAPY 209 culous peritonitis, and we have seen equally bad cases recover without tuberculin after simple laparotomy. Tuberculous laryngitis is nearly always associated with pulmonary involvement and tuberculin has at- tained but indifferent success, although a few ob- servers have reported excellent results. It would ap- pear that the association of local treatment is a most important part of the management of this affection. Ocular tuberculosis includes chronic conjunctivitis, phlyctcenular and interstitial keratitis, iritis, iridocycli- tis and retinochoroiditis, and seems to be a field wherein the results of tuberculin therapy have been most happy. Remarkable results have been obtained with " B. E." and to a less extent with " T. R." Old tuberculin seems to be definitely contra-indicated in all ocular affections. Chronic mastoiditis and otitis media, at times, have responded brilliantly to tuberculin immunization. Again, when the inoculations have been without effect, operative intervention has revealed carious bone. Tuberculin therapy in chronic cervical lympha- denitis, in the absence of indicated lymphadectomy, that is, when the nodes have undergone marked casea- tion or hquefactive necrosis, and particularly follow- ing the removal of diseased tonsils and adenoids, has enjoyed considerable success and should be employed in the future more extensively than has been the cus- 14 210 APPLIED IMMUNOLOGY torn in the past. The patient's general health im- proves immediately, rapidly and markedly, and if old sinuses are present they close promptly. In our ex- perience, equally good results have been obtained from " T. R.," " B. E.," " O. T." and " T. P." The re- sults of treatment by tuberculin in this form of tuber- culosis show that 75 per cent, are markedly improved, and that 40 per cent, of these will be cm^ed, while 35 per cent, will demonstrate only slight improvement, and 10 to 25 per cent, must be counted as failures. The best results will be observed in children under the age of six. XVII PHAGOCYTOSIS By phagocytosis is meant a property of the leuco- cytes whereby they take up into their substance foreign particles, such as bacteria (see Frontispiece), pigment, carbon granules, etc., thus removing them from the circulation. This power belongs pre- eminently to the polymorphonuclear neutrophiles, though phagocytosis by lymphocytes is occasionally observed. This phenomenon was discovered by MetchnikofT, who regarded it as the principal if not the sole means at the disposal of the body to rid itself of disease-producing germs. According to his teach- ing, phagocytosis included the taking up, the killing, and digestion of living bacteria, as well as absorption of dead bacteria and inert materials. Later studies of this phenomenon have led to modification of this comprehensive view by the majority of observers. It has, for example, been pointed out that the absorption of live bacteria by the leucocytes does not necessarily lead to their destruction, and the pus of gonorrhoea needs only to be cited as evidence. In gonorrhoea the great majority of the gonococci lie within the leuco- cytes and, far from undergoing destruction, rather tend to multiplication, at the same time retaining their highly infectious nature. Experiments with other 211 212 APPLIED IMMUNOLOGY varieties of bacteria have shown that they also may retain their vitahty after being taken up by the leuco- cytes. On the other hand, it has been shown that dead bacteria are taken up and digested by the leucocytes, and that insoluble foreign particles, such as carbon, pigment, etc., are absorbed and deposited in parts of the body where they will do no harm. It would ap- pear, therefore, that this power of the leucocytes, known as phagocytosis, applies to the taking up of all foreign particles, irrespective of whether they are liv- ing or dead bacteria, pigment, etc., thus removing them from the circulation, but in itself has little or nothing to do with destruction of the life of bacteria. Phagocytic action is to be distinguished from the bactericidal action of the leucocytes, which depends upon separated soluble substances and does not take place within the leucocytes themselves. Phagocytosis, therefore, is more or less a passive endeavor to remove foreign particles from the circula- tion, independently of whether they are living or dead, and in this way is to be regarded as one of the de- fensive forces of the organism against disease. The killing and destruction of living bacteria and neutrali- zation of their toxins are carried out by means of bac- teriolysins, opsonins, agglutinins, and antitoxins, in the soft tissues of the body, processes that are quite distinct from phagocytosis. XVIII RECOVERY FROM BACTERIAL INFECTIONS In the combat waged against disease, the body is equipped with natural defences that are called into play by the entrance of bacteria. In addition, each particular form of infection excites the production of antibodies that are specific for the disease in question. By means of these non-specific and specific defences, spontaneous or natural recovery is brought about. In the case of the bacteria that produce their dele- terious effects by means of separated soluble toxins, for example the diphtheria and tetanus bacilli, the body defences take the form of specific antitoxic sub- stances, which, by neutralizing the toxins, cause a secondary destruction of the infecting organisms. Where the invading bacteria act by means of endo- toxins that are inseparably bound up within their protoplasm, the body acts directly against the micro- organisms themselves through various soluble bac- tericidal substances in the blood-fluid. After the bac- teria have been killed, their bodies are taken up by the leucocytes (phagocytosis), and their endotoxins neutralized by non-specific oxidizing substances in the leucocytes. Experimental and clinical evidence leads to the belief that there are in the blood two types of bac- 213 214 APPLIED IMMUNOLOGY tericidal substances, (a) leucocytic bacteriocidin, (b) humoral bacteriocidin, and that certain varieties of bacteria are destroyed by the one, and other varieties by the other. These two types of bactericidal sub- stances are found in the fluid part of uncoagulated blood, that is, in the plasma. If, however, the blood is allowed to coagulate, or is defibrinated, only the humoral substance is found in the serum. From this it is concluded that one bactericidal substance is de- rived from the leucocytes, and is probably identical with fibrinogen, while the serum or humoral bacterio- cidin does not come from the leucocytes. It has been found that streptococci and pneumo- cocci are killed only by the blood-plasma, i,e,, by the leucocytic substances; these organisms multiply in serum alone. On the other hand, typhoid bacilli are destroyed by serum or humoral bacteriocidins. In this way is explained the clinical fact that in streptococcic and allied infections there is a marked leucocytosis, while in typhoid fever the leucocyte count remains normal. It has further been found that the humoral bac- tericidal substances lose their activity by heating the serum to 55^ C, but that they can be reactivated by the addition of fresh normal serum; in other words, their action depends on the presence of complement. On the other hand, the leucocytic substances are not inacti- BACTERLiL INFECTIONS 215 vated by heating to 55° C, and are not dependent upon the presence of complement for their action. It is probable that the humoral bactericidal substances are specific, each one acting against a certain microorgan- ism, while the leucocytic substance is non-specific. Among the natural defences of the body also are substances in the blood known as opsonins, which have the property of preparing bacteria for phagocytosis by the leucocytes. Whether these are distinct soluble substances or only a property of the serum is not de- termined; it is quite likely opsonins are a modified form of the bactericidal substances in the blood-fluid. From the foregoing discussion and from previous chapters, we see that the principal defences of the body against the deleterious effects of bacterial infec- tion include antitoxins, agglutinins, bacteriocidins (bacteriolysins), opsonins and phagocytosis, each of these playing a distinct part in the spontaneous recovery of the organism from disease. By a knowl- edge of the way in which different varieties of bac- teria produce their deleterious effects, and the re- sources present in the body for meeting the attack of the different species, we are enabled to assist nature in combating different forms of infection by artificial stimulation of these natural resources. This is brought about by the various general therapeutic measures, and in a specific manner by bacterin therapy. XIX BACTERIAL INOCULATION PRINCIPLES UNDERLYING INOCULATION THERAPY— PREP- ARATION OF BACTERINS— AUTOGENOUS VERSUS HET- EROGENEOUS PREPARATIONS— CLINICAL SYMPTOMS VERSUS OPSONIC INDEX IN CONTROL OF TREATMENT Principles Underlying Therapeutic Inoculation. — It has been elsewhere stated that bacterial inocula- tions operate by stimulation of tissue cells, after sub- cutaneous, intramuscular, or intravenous injection, to the production of specific antibodies, be they named agglutinins, bacteriolysins, opsonins, or what not. From their source, they are taken up by the Ij^mph iand blood-serum and distributed throughout the body. In the morbid process their action is to sensitize the invading bacterin, whereby they become more readily devoured by certain leucocytes, hence phagocytosis is promoted, or the antibodies by virtue of their lytic properties attack their specific bacteria, resulting in their disintegration (bacteriolysins), and tissue re- pair is favorably influenced. A patient smitten by an acute infection is the victim of an inoculation of living bacteria (antigen). If his resistance be poor and the virulence of the in- vading microbe be great, he is prone to die; if his re- sistance be par or better, and the bacterium relatively 216 BACTERL\L INOCULATION 217 avirulent or the dose small, recovery is to be expected : on the other hand, although his vital resistance be exceptionally good, a severe virulent infection will probably result fatally. In any case it is obvious that the size of the living bacterial inoculation is too large — also more potent than the commonly employed maximum therapeutic inoculation of dead bacteria — for the welfare of the patient and he is temporarily thrust into the so-called " negative phase." Thera- peutically, therefore, bacterin therapy is indicated only in chronic or possibly also subacute localized in- fections. It is impossible to conceive by any stretch of the imagination the rationale or at least the necessity of their employment in acute or diffused infections in which the human organism is already overloaded with the products of a living antigen, or indeed its defences actually demolished and its system over- whelmed by the invading microbes; namely, such states as bactersemia or septicaemia. Indeed, bacterial inoculations in such conditions not only add insult to injury but may be the determining forces for death instead of prolonged recovery. Administered in acute localized aiFections, as a rule unwisely perhaps, and certainly so unless the indications of the opsonic index are followed, either of two possible results may be ex- pected. If the bodily resources are barely able to cope with the situation, and the local infection be very 218 APPLIED IMMUNOLOGY active, additional injection of antigen will turn the tide of battle against the individual and an otherwise acute course be converted into a chronic or vital issue. On the other hand, if the individual's defences are great and his infection slight, an inoculation of bac- terin may possibly increase his specific antibodies, thereby enabling him to abort or at least shorten the attack of his local infection. In the latter case, the probability is that the patient if left to himself will readily conquer his infection in due time. This dis- crimination, in view of the danger involved, between leaving Nature to herself and intervention by bacterin therapy, can be made only by recourse to determina- tions of the opsonic index. As a rule, these acutely localized, particular^ if severe, and bacter^emic con- ditions constitute realms for the wiser and more logical practice of serum therapy or passive immunization. Consensus of opinion generally supports the belief that the bacteria best adapted to bacterin therapy or active immunization are those containing the poison- ous substances within themselves, the so-called " endo- toxins," typified by the staphylococci and strepto- cocci. Such bacteria do not liberate their endotoxins until disintegration or death of their organism occurs. On the other hand, diseases caused by the " exoteric " or toxin-producing microorganisms, typified by diph- o n 3 P o 3 =r r P 3 3 ^^' •3 ^ o o 3 C 0-2. n o 0- . S ° P 3 c S. 3 D. (R p BACTERIAL INOCULATION 219 theria and tetanus, are more appropriately treated by immune sera or antitoxins. Preparation of Bacterins. — The earliest, simplest and commonest method of bacterin preparation con- sists in washing off the appropriate media twenty- four-hour — with certain bacteria the time is much longer — pure cultures of the bacterium, using a plati- num spatula. The growth is suspended in normal saline solution, containing, if desired, 0.25 to 0.5 per cent, phenol. After shaking for one-half hour in a mechanical shaker (Fig. 20), the suspension is stand- ardized to contain from 100,000,000 to 1,000,000,- 000 bacteria per cubic centimetre. This is best accomplished by the hsemocytometer method, first employed by one of the authors (Thomas) in 1906. The bacterial suspension is drawn up in an ordinary red blood-cell counting pipette to the mark 0.5, fol- lowed by freshly filtered carbol-thionin, Leishman's or Jenner's stain, sufficient to stain the bacteria. After mild agitation for two or three minutes the bulb is filled to the 101 mark, and after thorough dissemina- tion of the bacteria a drop of the pipette content is placed on the hsemocytometer stage and the micro- organisms are comited precisely as are red blood-cells, determining the number per cubic centimetre. It is advisable to use a cover-slip not over 0.13 mm. thick; otherwise there may be interference with the oil-im- 220 APPLIED IMMUNOLOGY mersion objective. Recently, Glynn, Rees, Powell and Cox have recommended a special counting-cham- ber 0.02 mm. deep, instead of the common 0.1 mm. depth, with which a heavier, 0.18 mm. thick, cover- slip, suitable for all achromatic oil-immersion lenses, even with a free working distance as low as 0.09 mm., can be used. It is claimed that with the shallower chamber the bacteria settle to the bottom in fifteen minutes, while with the deeper they are still in motion after one-half hour, therefore, with the special hsemo- cytometer they are more easily enumerated for focal reasons, their definition is clearer cut and the heavier cover-slip is more durable. Any hsemocytometer method is more accurate than Wright's method, which underestimates the number of bacteria in suspension and possesses an average and maximum error three times as great as the h^emocytometer. Wright's method consists in mixing a unit volume of the bacterial suspension with an equal volume of normal blood known to contain approximately 5,000,- 000 erythrocytes to the cubic millimetre or 5,000,000,- 000 to the cubic centimetre. This is diluted by the addition of six or seven volumes of 0.15 per cent, sodium citrate solution to prevent clotting. After thorough mixing, fairly thick smears are made on glass slides and stained preparatory to the count. Fields throughout the smear are counted for the sake BACTERIAL INOCULATION 221 of accuracy and the proportionate number of bacteria to the red blood-cells counted, determined as follows : If in fields containing 1000 red blood-cells 500 bac- teria are counted we have the proportion 1000 : 500 :: 5,000,000,000 : x. X =z 2,500,000,000, or the number of bacteria per c.c. of suspension. This suspension is obviously too concentrated and may be conveniently diluted as follows: If it is de- sired to prepare the bacterin in 10 c.c. containers, so that each cubic centimetre shall contain 400,000,000 bacteria, we have the formula 400,000,000 X 10 2,500,000,000 =^ x = 1.6 c.c. Therefore to 1.6 c.c. of the original suspension must be added 8.4 c.c. of the diluting fluid, phenol-salt solution. The bacterin is then sterilized by submer- sion in a water-bath for one-half to one hour at the thermic death-point of the particular bacterium, usu- ally about 60° C. After removal from the bath, a drop of the bacterin is cultured for a day or two in order to test its sterility before employment thera- peutically. Other methods of sterilization occasion- ally utilized include chemical, cold and maceration by prolonged shaking. Preparation of Bacterins by Autolysis. — Certain investigators have attempted to improve bacterins and enhance their immunizing properties by subject- 222 APPLIED IMMUNOLOGY ing the bacteria to a process of autolysis, on the as- sumption that the autolysis contains toxic or anti- opsonic substances, and that the antigenic part of the bacterin depends upon the bacteria themselves minus their extractives. This disintegration of the bacteria is accomplished by treating them with salt solution, alcohol, ether, chloroform, or xylol, followed by filtra- tion and suspension of the residue in normal saline. Serohacterins. — Recently, advantage has been taken of Besredka s assertion, founded upon the dem- onstration of Ehrlich and Morgenroth, that inocula- tions with sensitized bacteria supersede in eiFective- ness, rapidity of action and harmlessness ordinary bacterins or bacterial vaccines. Such sensitized bac- terins or "serohacterins" are prepared by treating bac- teria with their specific immune serum, by which process the bacteria become inseparably joined with their antibodies, thereby in a state, immediately on in- jection, prepared to be acted upon by the complement of the patient's blood. Thus the usual delay of a week or more, required for the patient to form his own anti- bodies— incidentally also the negative phase and local injectional reaction — using the ordinary bacterins, is avoided, and immunity is secured rapidly and inten- sively in twenty-four to forty-eight hours. Finally, by virtue of the absence of " negative phases," the in- oculations may be repeated every day or two, thereby BACTERIAL INOCULATION 223 markedly shortening the course of treatment. Should sensitized bacterins fulfil their promise, they will mark the greatest advance thus far in the history of bacterin therapy. Bacterins, like tuberculins, are best stored in a dry, cool, dark place. As a matter of convenience they are put up in hermetically sealed ampoules or small Fig. 21. — Various forms of containers for storage of bacterins. bottles or vials fitted vi^ith ground glass stoppers or rubber caps (Fig, 21). The standardized strength of the bacterin, that is, the number of bacteria per cubic centimetre, also its date of preparation, should be inscribed on the container. The preservative, phe- nol, usually added to the diluting fluid, insures against contamination or bacterial growth in the preparation. It should be remembered that the bacterin may ap- 224 APPLIED IMMUNOLOGY pear perfectly clear after a certain time owing to digestion of the previously discernible microorganisms. On the other hand, it often happens that the bac- terin becomes clouded or a precipitate forms, due to the chemical action of the contained phenol. Such bacterins should be immediately discarded. As a rule, bacterial suspensions in trikresol-saline solution will keep for months. It is alleged that typhoid bacterin becomes inert after three months. Autogenous versus Stock Bacterins, — By an autog- enous or personal bacterin is meant one that is pre- pared from the particular bacterium or strain of bac- terium cultured from the infected patient. A stock or laboratory bacterin is understood to mean a prep- aration in which the bacterium has been isolated from another patient, who has suffered from a similar in- fection. In view of the diversity of the strains of many bacteria, notably the streptococcus, colon ba- cillus, etc., also of the fact that not a few diseases are caused by different bacteria, namely, abscess, acne, etc., it is natural that the preferable and most scientific procedure is to employ an autogenous bacterin when- ever possible. The authors have often successfully treated cases with an autogenous preparation after stock bacterins have resulted in failure. Not infre- quently it becomes expedient to use a stock prepara- tion, at least while the autogenous bacterin is being BACTERIAL INOCULATION 225 prepared. Under such circumstances, notably in gonorrhoea! and tuberculous affections, it is impera- tive that the infecting germ be accurately determined. Moreover, the bacterin should be polyvalent, that is, composed of as many different strains of the bac- terium as may be obtainable, and in view of the reports of Besredka, Broughton-Alcock, Ardin-Deltiel, Negre, Raynaud, Gordon, Boinet, Cruveillier and others, it would appear advisable to employ so-called sensitized bacterins. Clinical Symptoms versus Opsonic Indeos in Con- trol of Treatment, — Regulation of the size of doses of bacterins and the spacing of the intervals between in- oculations is possible by observations of the clinical symptoms, subjective and objective, and by determi- nations of the opsonic index. Both methods involve special knowledge and are not to be undertaken lightly. The former entails the closest kind of ob- servation respecting local, focal and general responses ( see Chapters XVI and XXI ) . Opsonins are not the only antibodies formed after inoculation of an ani- mal, hence the opsonic index, theoretically, cannot measure the full degree of immunity.^ Practically, the opsonic index in the majority of cases runs par- * There is no such thing as absolute immunity, however, a fact pointed out by Pasteur years ago, for an animal will contract disease, irrespective of the degree of immunization, provided the dose of infective material is suflBciently large and virulent. 15 226 APPLIED IMMUNOLOGY allel with the cHnical symptomatology, and although in many cases it is unnecessary to employ the index as a guide, there are many cases in which dependence upon it is absolutely essential to attain the greatest success. To ignore the index absolutely in all cases will invite disaster. The authors feel, after an ex- perience of many years, that the most brilliant results — often in the most difficult cases — have attended bacterin therapy, wherein the opsonic index was asso- ciated in the management of the case; although they are inclined to attach primary importance to the clini- cal symptomatology, properly interpreted, and to relegate the index to second place. For instance, in the treatment of acne or recurrent furunculosis, we are not infrequently at a loss, after the disappearance of the present lesions, to say whether or not the process of immunization has been carried far enough to insure no recurrence, or in certain other affections, charac- terized by a high fluctuating temperature and tox- aemia, or in deep-seated lesions, such as pyelitis, pye- lonephritis, cystitis, etc., the value of the index in controlling dosage and, in the case of mixed infec- tions, in selecting the needed bacterin, is not incon- siderable. The treatment of this class of conditions lies without the sphere of the general practitioner and he would do well to refer such cases to those more particularly versed in the application of bacterial in- BACTERLVL INOCULATION 227 oculations. On the other hand, physicians generally, at the present day, owing largely to the energy of pharmaceutical firms marketing stock bacterins, em- ploy bacterins controlled or miscontrolled by the clini- cal symptoms in preference to the opsonic index. It is utterly out of the question for the average general practitioner to master the bacteriology and laboratory technic required for the reliable determina- tion of the opsonic index, and experience has already demonstrated that in the majority of patients thus far subjected to bacterial immunization the clinical symptomatology has admirably sufficed to control the inoculations. Consequently, bacterin therapy, in order to enjoy the popularity which is its due, must be governed, within limitations, by the subjective and ob- jective symptoms and signs, observing the former and avoiding the latter. XX THE OPSONIC INDEX DEFINITION OF OPSONINS AND THE OPSONIC INDEX— TECHNIC OF DETERMINATION OF THE OPSONIC INDEX —INTERPRETATION, VALUE AND LIMITATIONS OF THE OPSONIC INDEX Denys and Leclef, Leisliman, Wright and Doug- las and others have demonstrated that the real activity of phagocytes depends upon a specific substance — the exact chemical nature of which has been unde- termined— existing in the serum of the blood. Irre- spective of a certain amount of spontaneous phag- ocytosis, which has been shown may take place, Sir Almroth E. Wright, of St. Mary's Hospital, London, has definitely proved that the subcutaneous injection of dead bacteria is capable of the production of spe- cific antibody, which taken up by the blood-serum markedly augments the ability of the phagocyting leucocytes to devour invading pathogenic bacteria. To this specific substance, capable of preparing bac- teria for ingestion, Wright assigned the name " opso- nin," from the Greek verb bi^ovid, I prepare food for. Opsonins, therefore, are those specific substances or antibodies, not yet isolated, in the blood-serum, possessed of the property to sensitize or prepare bac- teria for phagocytosis. Opsonins are bacteriotropins 228 THE OPSONIC INDEX 229 and should not be confounded with bacteriolysins, another type of antibody. Moreover, it has been shown that opsonins are divisible into thermolabile (the normal opsonin of Wright) and thermostable ( immune opsonin ) . The former are readily destroyed at a temperature of dQ"" C, the latter retain their bac- teriotropic property in spite of this degree of heat. By virtue of the assiduous labors of Wright and his co-workers in evolving a technic (determination of the opsonic index) to measure phagocytosis (degree of immunization), therapy by bacterial inoculations was revived and popularized to an unprecedented ex- tent and has led to the application of active immuniza- tion of far-reaching consequences, and it is for this reason that medicine owes Wright a perennial debt. The opsonic index may he described as the measure of the ratio of the phagocytic activity of neutral or washed leucocytes in the patient's serum for given bacteria, as compared with those in a normal or con- trol serum. Inasmuch as a neutral phagocyte will in- gest the same number of bacteria, provided the two sera possess identical qualities, the normal or base line is arbitrarily taken as one. If, in the ease of the pa- tient's serum, it is found that 100 leucocytes contain 900 bacteria, while the same number of leucocytes treated with the control serum contain 1000 bacteria, 230 APPLIED IlViMUNOLOGY we have the proportion of 900 : 1000 :: tr : 1, in which 00, the opsonic index, equals 0.9. Teclinic of Determination of the Opsonic Index. — The technic of the opsonic index is a rather intricate laboratory procedure, which, to be reliable or trust- worthy at all, involves much practice in mastering the details as stipulated by Wright. It demands that the opsonist shall be a thorough bacteriologist as well as an experienced laboratory worker. Briefly, the pro- cedure may be described as follows: Material and apparatus required, (a) Pure culture of infective bacterium. (b) Specimen of serum of patient's blood. (c) Specimen of serum of normal or control blood. (d) Normal washed leucocytes. (e) Centrifuge, preferably electric. (f) Thermostat or opsonizer. (g) Centrifuge or special test-tubes. (h) Teat and capillary pipettes, glass tubing, slides, absolute alcohol, stains, etc. (i) Microscope. A few drops of the patient's blood and of the nor- mal or control blood are collected in two glass tubes drawn out on either end to capillary size (Fig. 22). The straight or distal end, free of blood, is sealed in a flame, and in a few minutes, after the tube cools, dur- Fig. 23. Fig. 24. ^^^ ¥ Fig. 25. — Electric centrifuge. This instrument is particularly well adapted for the preparation of washed leucocytes by virtue of the easy manner in which it loses speed when the current is broken, thereby avoiding disturbance of the layer of "leucocytic cream." baciena\ 5u5pen5'\onv Fi w \ Fig. 26. — \'arious ingredients neoes-sary for the determination of the opsonir index THE OPSONIC INDEX 231 ing which time the blood coagulates and is spontane- ously drawn toward the sealed end, the curved or inlet end may also be sealed. The serum begins to lose its opsonic content in six hours and should not be utilized for index determination after this time. Ten to fifteen drops of normal blood are then allowed to fall from the finger-tip into a test-tube of normal salt solution containing 1.5 per cent, sodium citrate (Fig. 23). The citrate will prevent the clotting of the blood, and the corpuscles are thor- oughly washed by centrifugation two or three times in normal saline (Fig. 25 ) , to render them neutral and abso- lutely free from serum. The white blood-corpuscles, being lighter than the red, will overlie the latter as a grayish layer of " leucocytic cream " (Fig. 24), whereupon they may be picked up by a pipette and trans- ferred to another receptacle (Fig. A six- to twenty-four-hour culture of the given bacterium is washed off in normal salt solu- tion (Fig. 27), then centrifuged to throw down all bacterial clumps. The supernatant suspension should contain only individual bacteria in not too 1/ Fio. 22. -Capillary glass capsules for the collection of specimens of blood. A, simple straight; B, Wright's. 26 232 APPLIED IMMUNOLOGY great concentration. The best strength would ap- pear to be that rendering a phagocytosis averag- ing about four bacteria pro leucocyte. Two opsoniz- ing capillary pipettes, P (patient) and C (control), are prepared so that the lumina are about the diame- ter of a large sized hat-pin. A distance of one-half Fig. 27. — Washing the culture of the given bacterium from the culture medium, using the platinum loop, in order to obtain the bacterial suspension. or three-quarters of an inch is marked off on the ends of these two pipettes (Fig. 28, 1 and II) . By the use of a small rubber bulb, equal quantities of patient's serum, bacterial suspension and washed leucocytes, permitting a small bubble of air to separate each, are drawn up and mixed by skilful thumb and finger gymnastics, in the pipette designated P. In the same 1^ a ^ p ^ '^ — n >: o — "2 »_ ^ '^ ^ < C >^t3 5 S s « c ^Z' C -I C^ •O ra o p /\ A 3 S.O fie 7~ §■=; ="5 £3 - ^ p c .3 S. L igy— -^ Fig. 29. — Opsonizer or thermostat. This incubator i.s equipped with an automatic heating device and permits of the ready manipulation of pipettes introduced through the side port-holes. THE OPSONIC INDEX 233 manner equal quantities of control serum, bacterial suspension and washed leucocytes are taken up and mixed in pipette C (Fig. 28, III and IV). The tips are sealed in a flame and the two pipettes incu- bated for a few minutes in a thermostat or opsonizer at 37° C. (Fig. 29). Care should be taken to agitate the contents every five min- utes during incubation, otherwise the corpuscles will gravitate and a per- fect admixture of serum, bacteria and phagocytes will not be obtained. At the conclusion usually of fifteen minutes, smears are made of the contents of the pipettes on cover-slips by the usual method or glass slides, employing Kulm- hardt's spreader (Figs. 30 and 31). After drying, the smears are fixed in absolute alcohol and stained with freshly filtered carbolthionin or by the method of Homer Wright. In the determination of the tuberculo-opsonic index, the culture of tubercle bacilli is killed by fractional sterilization on three successive days at 100° C, then thoroughly ground Fig. 30. — Illustrating the construction of Kuhnhardt's spreader. A shows a glass slide properly nicked by a file; B, the slide p.cpcrly broken in a slightly concave line; C, the broken slide with corners clipper" ; D and E, the perfected spreader spliced to a second slide with adhesive plaster. 234 APPLIED IMMUNOLOGY in an agate mortar and suspended in 0.85 per cent, solution of sodium chloride. After fixation of the smears either by ethyl or methyl alcohol or by heat, the usual staining method comprising carbol-fuchsin, nitric acid and methylene blue for tubercle bacilli is employed (see Frontispiece). The number of bac- FiG. 31. — Kuhnhardt'3 spreader properly held at the right angle for the preparation of a satiafactory smear. teria contained in no fewer than one hundred phag- ocytes should be counted and the index determined as noted above; or, in accordance with the method of Simon, the ratio of the number of phagocji:ing leuco- cytes may be determined. The latter method is simpler, less exacting on vision and possibly just as reliable as the method of Wright. THE OPSONIC INDEX 235 Interpretation, Value and Limitations of the Opsonic Index, — Wright contends that the opsonic index, properly done and interpreted, is a reliable ex- pression of an animal's resistance against infection. The effect of a bacterial inoculation, after a transi- tory drop in the opsonic index, is an increase in the degree of immunity of the inoculated animal against the disease caused by the given bacterium. The pri- mary drop or so-called *' negative phase," presumably due to the absorption of preformed opsonins by the injected bacteria or to the systemic effect incident to the cellular stimulation of the organism for the pro- duction of specific antibodies, may be of very short or prolonged duration, and if of any consequence is characterized by the clinical phenomena, general, focal and local, described in the following chapter. In a few hours, after a proper sized dose, the opsonic index rises in the scale. This ascent is termed the " flow " and may fail to reach the normal or soar well above it. Since the effect is but transient, the index will slowly fall and this descent is called the " backflow " or " ebb " or " negative phase," occasionally following an inoculation, if the indices are determined suffi- ciently often, the curve will exhibit a mild " initial rise " preceding the " ebb " or " negative phase," be- fore the real " flow " or " positive phase " sets in 236 APPLIED IMMUNOLOGY ( Fig. 32 ) . In the conduct of therapeutic inocula- tions it should be the purpose of the immunologist to repeat the inoculation in the " positive phase " or as the " ebb " begins. Extremely large inoculations or reinoculations while the index is in the " negative ©• Opsonic Ibwces. X* iNocuuTioitSTAPwno-BAcmiin rurrr FEBRUARY MARCH "**•«. I215114|15!l6p7ll819120tel|22|2a2465»6]27l28jl 2 3 4 5 6 7 8 9 Mil 12pjl4 Sl6|17p8|19j20!21j22j23B4j35tear> Inoo{ _ _ _ __ J_ - -_L % „ " « - _ 5 " 5 ~ 1 1 .as ? S3 _ . , silt II S. 1^ -p i ? Hi t s fg ; E 5. i 1 6 s ~ . i ^ "."S j i it : ^Iss^ jSi2~s ■, s i-^ Si : 1 < I ^ S 1 -<£?-| Sj 1 g If 1 •: « ■: •» B ^•'■« 5S .^i i' = 3 < S 2 5 ££ i 1^ sS ^S "'^■^""~'^'^°" M • ' ' '^5 - ?i b - - -h N *► io - - X i-^ ^^- w %- ^^H^-^ - t! ^ - -^i - i? V^ - ,-•' t \l "u5 -^ 1^ ~ Z S' ^ ? M - ^ ±^,-^ : « ♦ " "^ 4 ^i^ - Ji B ^ ' lij:3^'^C__ ^ \ ^ i .^M. t - ^ - \l ii.«^" t i \,2s^:^ __ . (L _ ._ _ _ w «•« — -^ - -I -?T:i: tl^^ ^ i /~ ^' t5t^ ^ t ^ li.^ t'^ l\~- - ^i , - «I li: 2 J A — ^'2 ~ J ■ - ^ e=^2'' o - o « — i -s -1 .1 __ _ ± _ Fig. 32. — Analysis of curve of opsonic indices. phase " result in an exaggeration and prolongation of the " negative phase " which handicap the patient's recuperative powers and may suffice to turn the tide of battle against him. Inoculations repeated at the correct time will still further raise the opsonic index above the normal, until, as Nature resumes her work, THE OPSONIC INDEX 237 the " ebb " is less conspicuous and the " positive phase " becomes sustained. At this time the patient is said to be on the " high tide of immunity " and further inoculations are contra-indicated. This is ob- served in connection with prophylactic inoculations and where recovery is apparent. Wright's original rule was to take the opsonic index daily and be guided exclusively by its indications ; at present, although the value of the index is not belittled, the clinical course of the case is utilized as an important guide in treat- ment. Inoculations are not repeated until positive phase symptoms are no longer apparent, or in doubt- ful cases determination of the index demonstrates a fall in opsonins. So long as there occurs any benefi- cial response whatever from a bacterial inoculation, its size, whether the " minimum effective " or the " medium or average," is not increased. The " ideal curve " consists of a " negative phase '' of twelve or twenty-four hours, followed by a " posi- tive phase " lasting from three to fourteen days. This curve depends upon two factors, (1) the size of the dose and (2) the vital resistance of the patient. A small dose may have to be repeated frequently, possi- bly every day, owing to the short duration of the " positive phase," whereas a large dose may necessi- tate the suspension of any further inoculations on ac- 238 APPLIED IMMUNOLOGY count of a prolonged " negative phase," or possibly the establislmient of immunity and recovery. Failure OPSONIC INDICL5 HOUR 1 11 ll.is 12 12.22 L3 = 1 GOIVOCOCCUS TUBEJ^CLE BACILLUS 12 < U 0 u ( / I NORMAL / ( .9 / .8 / .7 / /-. ---^^, **/^ .6 / vj Fig. 33. — Case of gonorrhceal arthritis of knee, sho-wing manner of differentiation between the possible causes of synovitis or arthritis. The opsonic indices for the two suspected etiological organisms were determined before the application of passive hy- peraemia to the infected joint. After intermittent application for one and a half hours the indices were again taken, with the result that the opsonic index for the gonococcus, the specific organism in this case, had risen considerably. to evoke a rise in the opsonic index may be attribut- able to (1) too small dosage, (2) reinoculations dur- ing the negative phase, or (3) inoculation treatment THE OPSONIC INDEX 239 of patients, wherein bacterins are contra-indicated, namely, those already overwhelmed by their infection. In addition to its value as a control of treatment in bacterin therapy, the opsonic index, in expert hands. I T 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2 3 4 5 6| 2-7 2-6 25 2-4 2-3 2-2 2-1 20 V9 18 1-7 1-6 1-5 t-4 1-3 12 It 10 •9 •8 •7 •6 •5 •4 :3. 40' 38° 3/ C 36^ — 1 e" ^ * • 1 1 • ' % 1 1 \ / ' ' A \ ^ A ♦ » \l '\ '^ 1 V « ', \ I \ t j \ i 1 1 \ 1 \ •1 \ 1 \ f\ ,*i V ^ y V f ^ » .^, \ / / ^ ^ . / f \ "> — J / /\ W ■■ii" — \ ^ /- A. B. Fig. 34. — Pneumonia. A, recovery; B, death; , curve of opsonins (I); 1 , curve of temperature (T). Observe in the favorable case a rise in the opsonic index coincidently with the fall in temperature. (Gigiioli and Stradotti.) has been utilized both diagnostically and prognosti- cally ( Figs. 33 and 34 ) . An important sphere of its utility may be in the determination of the degree of animal immunization and standardization of sera, and has been so employed by the authors. Its value in a 240 APPLIED IMMUNOLOGY diagnostic and prognostic capacity would appear to be limited, ( 1 ) because other methods, as the cutane- ous allergic reactions, are simpler if not more reliable, and (2) owing to a certain amount of error, even with FiQ. 35. — Showing effect of variable concentrations of bacterial suspension on determinations of opsonic index. . bacterial suspension centrifuged for 10 minutes, then dilution of supernatant suspension so that 1 c.mm. contained approxi- mately 10,000,000 bacteria. , bacterial suspension centrifuged for 10 minutes. .bacterial suspension centrifuged for 10 minutes and supernatant suspension diluted with 3 volumes of 0.85 per cent, saline solution. , bacterial suspension standardized by McFarland's "nephelometer." experienced workers, small variations may not be de- pendable, although it renders great service at times, in cases of mixed infection, enabling the immunologist to choose for inoculation the particular responsible THE OPSONIC INDEX 241 bacterium, or in the course of immunization to select from time to time the bacterin to which the index is low. It is generally admitted that the error due to technic alone, aside from the question of personal equation, amounts to one- to two-tenths. This varia- bility is dependent particularly upon ( 1 ) the number of cells counted and ( 2 ) the concentration of the bac- terial suspension (Fig. 35). 16 XXI PRACTICAL APPLICATION OF BACTERIAL INOCU- LATION IN MEDICINE, PROPHYLACTICALLY AND THERAPEUTICALLY GENERAL CONSIDERATIONS— INDUCED AUTO-INOCULA- TION—DURATION OF ACTIVE IMMUNITY"— MODES AND TECHNIC OF ADMINISTRATION OF BACTERINS— DOSAGE— CONTRA-INDICATIONS, LIMITATIONS AND CAUSES OF FAILURE IN BACTERIN THERAPY— APPLI- CATION AND RESULTS OF BACTERIAL INOCULATIONS IN SPECIAL DISEASES— DISEASES OF THE SKIN AND SOFT PARTS— DISEASES OF THE GENITO-URINARY SYSTEM— DISEASES OF BONES AND JOINTS— DISEASES OF THE EYE, EAR, NOSE AND THROAT— DISEASES OF THE LUNGS— DISEASES OF THE ALIMENTARY SYSTEM- DISEASES OF THE CARDIOVASCULAR, LYMPHATIC AND NERVOUS SYSTEMS, ALSO OTHER ACUTE SPECIFIC FEVERS— MALIGNANT NEOPLASMATA— YEAST AND SOUR MILK General Indications. — Wright has declared that, " we have in the power of increasing the antibacterial power of the blood by the agency of vaccines, and in our power of bringing the antibacterial agencies of the blood into operation in the focus of infection, be- yond all comparison the most valuable assets of medi- cines." It might be wise to modify this assertion to the effect that, in the competent employment of bac- terins tve have one of the most valuable assets, A very important fact ever to be remembered in the practice of medicine — and this includes particularly bacterin therapy — is never to discard or disregard old- established measures of proven worth, for a new idea, 242 BACTERIAL INOCULATION 243 no matter how attractive and possibly of superior value, for, by the skilful association of all, cure will be best promoted and facilitated. Wright's doctrine has become classical: " The medical man who has re- course to vaccine therapy ought to have familiar ac- quaintance with the microbes which affect the human body. He ought to appreciate the fact that vaccines owe their efficacy to the reaction they set up in the tissues, and not to any action exerted directly by the vaccine upon the invading microbe. He ought to have mastered the general principles of immunization. He ought to know in connection with each vaccine the minimum effective dose, i.e., the dose which gives the minimum immunizing reaction without any interven- ing negative phase ; and the medium or average dose, i.e., the dose that gives, after a negative phase, a more powerful immunizing reaction. He ought to know the general conditions which affect the sensibility of the organism. He ought to understand how to adjust the dose to the requirements of the individual patient, and he ought to have a knowledge of the conditions which obtain in the focus of infection, and of the methods of circumventing the difficulties which are introduced by these conditions." It is extremely improbable that bacterin therapy as practiced by the average general practitioner is destined to realize the full measure of its promise. 244 APPLIED IMMUNOLOGY either because he lacks the required bacteriological facilities or is too busy to devote the necessary time and attention to trivial signs and symptoms. It seems not to be appreciated by physicians generally that bac- terins are agents capable equally for good or evil. It cannot be too strongly emphasized that bacterin therapy is merely a valuable accessory to Nature in the art and science of healing and is not a " cure all." Carelessness, ignorance, incompetence or a desire on the part of the therapeutist to push the treatment, when beneficial results become apparent, will lead not only to failure, perhaps disaster, but at all times dis- credits a therapeutic measure of great value, and de- prives the patient of his natural resources for re- covery. Two solutions for the problem are offered the general practitioner contemplating bacterial in- oculations : either to familiarize himself more with im- munology, including bacteriology and laboratory methods, or to refer his patient to or cooperate with an immunologist, precisely as he is accustomed at times to consult an ophthalmologist. Induced Auto-inoculation, — Therapeutic inocula- tion may assume one of two forms, (1) exogenous injection of bacterial suspensions, forming the main theme of this chapter, and ( 2 ) endogenous inoculation or auto-inoculation. The latter is a very common clini- cal phenomenon and may be spontaneous or induced. BACTERIAL INOCULATION 245 Spontaneous auto-inoculation is frequently observed in a patient the victim of subacute or chronic phthisis pulmonalis, and is characterized by the irregular flares of temperature. If the doses of bacteria or their prod- ucts, which at these times are cast off into the general circulation, are too great or repeated too often, the pa- 11 i 5 I s 16- I si 11 ^ Fig. 36. — Case of gonococcal arthritis. Showing the effect of massage on the gono- cocco-opsonic index. I =inoculation of 30,000,000 gonococci. M =massage. (Dr. J. Freeman.) tient is thrust into a profound or permanent " nega- tive phase " and death supervenes. If, on the other hand, the dose of re-infection be of the proper size and occurs opportunely, the patient becomes to a cer- tain extent immunized and at least a temporary re- covery takes place. Induced or artificial auto-inocula- tion is a most interesting problem and at times has 246 APPLIED IMMUNOLOGY proved feasible in the treatment of various infections. It would appear advisable ofttimes when bacterin or vaccine therapy is contra-indicated. It consists in an endeavor to inoculate the patient from his own lesion by purely physical measures. These include massage, exercise, applications of heat, passive hyperaemia, etc.. TUBERCULO- OPSONIC inULA. ~ ■ "" - ""■ '~~ ! iiu < >• CO 1 < IS "5^ 14 / !-3 / \ \ < <0 12 -J \ / c*^ \ L II ? 1 \ / 5 k 5 Y NORMAL 10 / \ 'Jr_ \ — •9 1 \ A *V N, \ 8 / V \ i > h ^- •7 •6 FEB. 21 22 23 24- 25 26 27 28 1 2 3 Fia. 37. — Case of tuberculous cervical lymphadenitis. Showing effects of massage on tuberculo-opsonic indices. (Wright.) and explains wherein lies the diagnostic value of the opsonic index, for example, in gonorrhoeal arthritis (Figs. 33 and 36) , and in tuberculous cervical lymph- adenitis (Fig. 37) . The production of induced auto- inoculations may result in excessive dosage, as we have no means to gauge, at the moment, the size of BACTERIAL INOCULATION 247 the dose cast into the circulation, and in view of the fact that the inoculation consists of living bacteria, not as in ordinary bacterin therapy of measured dead microorganisms, the method is attended by a certain amount of danger and should be utilized only by ex- perienced hands. Duration of Active Immunity, — In general it is admitted, and may be stated, that the greatest and most lasting immunity is produced by inoculations with living bacteria, then with dead bacteria and finally with the products of bacterial growth. This has been quite satisfactorily demonstrated by animal experimenta- tion, and a common procedure is to begin the process of immunization with inoculations of dead bacteria, followed by the living microorganisms. Thus far, the profession has not seen fit, nor would patients sub- mit to injections of living bacteria, although by re- sort to the technic of Williams and Webb — inocula- tions at first with a single living bacterium, then by cautious count progressively increasing the dosage — ■ or supplementing inoculations of dead by living or at- tenuated bacteria, no danger should be incurred, and the day may not be far remote when such a practice in expert hands will be an unobjectionable and com- mon procedure. Immunity is never absolute, but the protection af- forded by bacterial inoculations lasts indefinitely, fre- 248 APPLIED IMMUNOLOGY quently for months or years. There is substantial evi- dence that prophylactic typhoid immunization pro- tects the individual for at least three years. The duration of immunity may be determined by cutane- ous allergic tests and by serological reactions, includ- ing the opsonic index. Modes and Technic of Administration of Bac- terins. — Bacterins have been administered subcutane- ously, intravenously, orally and per rectum. The last three methods are not in general use, in spite of sporadic marvellous results, nor do they appeal strongly on immunological grounds, and consequently seem not destined to become popular. Subcutaneous administration is the procedure usually practiced. In the event of mixed infections, multiple bacterins of the respective pure bacterial cultures may be prepared for alternate or indicated inoculations, or a mixed bacterin proportionately compiled of the various in- vading microorganisms may be employed. The technic consists in sterilizing the skin with a pledget of cotton and alcohol overlying the insertion of the deltoid muscle or elsewhere on the body, as described on page 178. It is recommended whenever possible to inoc- ulate on the distal side of the lesion, thereby giving the patient the immediate benefit of the antibody forma- tion through the local lymph system in addition to the subsequent effect to be obtained through the blood- BACTERIAL INOCULATION 249 serum. The syringe (Fig. 38) constructed entirely of glass or one having an asbestos piston is most service- able, as it permits of ready cleansing and sterilization by boiling. When inoculations are given on a large scale, requiring frequent sterilization, the oil method of Wright is novel, convenient and effective. It con- sists in heating a quantity of olive oil over a gas flame. The oil will heat to 150° C. without boiling. This is one and a half times the temperature of boiling water and instantly sterilizes the syringe when drawn up and forced out after inoculation. From the ampoule after Fig. 38. — All-glass hypodermic syringe. Desirable for bacterial inoculations because of ease of cleansing and surety of sterilization. filing or breaking off its neck or from the small bottles, • — in the case of the rubber-capped one by plunging the needle through a drop or two of phenol, formalin or alcohol placed on the rubber, — the desired dose of bacterin is drawn up into the syringe. In the case of some vials with rubber stoppers it is expedient to with- draw partially the piston of the syringe before thrust- ing it through the cork, then to push it home and as it is slowly withdrawn again the desired quantity of bac- terin readily follows the piston. If the skin is firmly grasped and lifted between the finger and thumb and 250 APPLIED IMMUNOLOGY a perfectly sharp pointed fine needle is employed, the amount of pain occasioned by the puncture is ex- tremely slight. The bacterin, if a large dose, should be slowly injected into the loose subcutaneous tissue to minimize pain. As the needle is withdrawn the site of the pmictui-e should be compressed and the skin gently stroked a couple of times with the pledget of alcoholized cotton. No further dressing is neces- sary or desirable. Dosage. — The size of the dose of bacterin must be cautiously gauged. The two fundamental considera- tions are ( 1 ) the nature of the particular infecting bac- terium and (2) the condition or state of the patient. It should be thoroughly realized that not only bacteria in general but individual strains of the same bacterium differ greatly in virulence and, as a rule, the more viru- lent the microbe the smaller should be the inocula- tion. To determine this and the inmiunizing power of the bacterin, with the greatest precision, various clini- cal laboratory and blood tests are essential. Thus the dose of the more virulent bacteria, as the streptococcus, pneumococcus, gonococcus, colon bacillus, etc., should be considerably smaller, that is, one-half to one-fom'th that of the less virulent microorganisms, as the staphy- lococcus. Again, a most important problem is the de- termination of the viiimnal effective and the average and maocimal dose for each bacterium. In desper- BACTERL4L INOCULATION 251 ately or acutely ill patients, or with patients in whom a beneficial response has been obtained with the mini- mal effective dose, this should not be exceeded. With chronic cases, presenting well-localized lesions, the average or maximal dose may be administered at the start, and subsequently increased. The following table, giving the minimal, average and maximal doses employed in the authors' experience, may prove of service : THERAPEUTIC DOSE TABLE Bacterin Gonococcus Colon M. Catarrhalis Influenza Typhoid Pneumococcus Streptococcus Staphylococcus B. Punctatum B. Proteus Vulgaris. . . B. Friedlander B. Acnes Pertussis Diphtheria Pseudodiphtheria .... B. Pyocyaneus B. Fluorescens B. Pseudotuberculosis Rodentium B. Acidi Lactici B. Lactis Aerogenes. . . B. Mallei... B. Fusiformis B. Koch- Weeks B. Morax-Axenfeld . . . , Tuberculin Actinomycotin . Minimal effective dose Average dose 2 to 5 to 10 to 5 to 5 to 10 to 2 to 50 to 10 to 5 to 10 to 5 to 5 to 5 to 10 to 5 million 10 million 25 million 10 million 10 million 20 million 5 milHon 100 million 25 million 10 million 20 million 10 million 10 million 10 million 25 million 25 million 2 to 10 to 5 million 25 million 0.00001 to 0.0001 milligram 0.001 milligram 50 to 25 to 50 to 25 to 25 to 25 to 25 to 100 to 100 to 50 to 50 to 25 to 25 to 25 to 50 to 50 to 50 to 100 million 50 million 100 million 50 million 50 million 50 million 50 million 400 million 200 million 100 million 75 million 100 million 100 million 50 million 100 million 100 million 100 million Maximum dose 50 to 100 million 50 to 100 million 50 to 100 million 50 to 100 million 50 to 100 million 50 to 100 million 0.0001 to 0.001 milligram 0.01 to 0.1 milli- gram 400 million 400 million 300 million 200 million 500 million 300 milHon 200 million 1000 million 400 million 400 million 300 million 200 million 200 million 100 million 300 million 400 million 400 million 400 million 400 million 400 million 400 million 400 million 250 million 100 milligrams 4 milligrams 252 APPLIED IMMUNOLOGY The physical and clinical state of the patient, apart from the variability in the virulence of bacteria, ofttimes presents a difficult problem as to the correct dosage of bacterin. In this connection, the age, sex, and stature of the patient, the gravity of his illness, the acuity or chronicity of the affection, a non-febrile or febrile condition and his debilitated or toxic state demand the keenest discrimination. The golden rule is the sicker the patient the smaller the dose, but both the clinical symptomatology and the opsonic index may suffice to determine that he is too sick for any dose, however infinitesimal. Subsequent inoculations both as to size of doses and intervals may be governed in most diseases by close and accurate observation of the clinical symptoms or by the revelations of opsonic indices or by the utiliza- tion of both of these controls, an advisable procedure for the best results in no small number of cases. Generally speaking, the clinical symptomatology is of primary importance and the opsonic index takes second place ; at times, however, these become reversed. It is an easy matter to control properly the process of immunization by study of the clinical symptoms, and success will obtain only by an experienced and careful observation of such trivial phenomena as malaise, indisposition to work or play, bodily aches, grippe-like attacks, headache, anorexia, slight fever. BACTERIAL INOCULATION 253 nausea, variations in weight, leucocytic and hsemo- globin determinations, and particularly local manifes- tations in the diseased region as well as at the sites of inoculations. Allusion has previously been made to the fact that there are many diseases in which guidance furnished by determinations of the opsonic index is indispensable for the proper execution of bacterin therapy, and without which bacterial inoculations, per- haps, had best not be practiced. These include espe- cially deep-seated affections, as bronchitis, pneumonia, certain intestinal infections and diseases of the genito- urinary tract typified by pyelitis, cystitis, seminal vesiculitis, prostatitis, etc. The safest rule to be ob- served respecting bacterin therapy, controlled only by the clinical symptomatology, is to begin with a very small or assuredly harmless dose; if there be absolutely no reaction, local, focal or general, in two or three days a second inoculation, twice the size of the first, may be given, and so on with intervals of three to seven days until reactionary phenomena are observed. The slightest reaction is evidence that the dose has been sufficient if not too large and indicates that no further inoculations are to be made until all reactionary signs have completely subsided for several days. Frequently, the next inoculation must be de- ferred for two or more weeks and when administered should not exceed in size its predecessor, commonly 254 APPLIED IMMUNOLOGY being cut down to only one-half of the previous dose. Strict observance of this dictum is imperative, but unfortunately not in accordance with the instructions furnished the general practitioner by all pharmaceuti- cal firms marketing stock bacterins, thus accounting for no small nmiiber of failures in bacterin therapy. No hard and fast rule can be dogmatically laid down, either as to interval or size of the inoculations. Each case is a study unto itself and must be treated ac- cordingly. Contra-indications, Limitations and Causes of Failure of Bacterin Therapy, — By virtue of the theory of biological therapeusis, little should be ex- pected of bacterial inoculations in the acute stages of infectious diseases, indeed they may exert an evil in- fluence. Bacterin therapy, however, is particularly contra-indicated when the individual is overwhelmed by a diffuse infection or when owing to prolonged illness he is prostrated or his tissues are greatly wasted and no longer susceptible to artificial stimulation for the production of antibodies. Such states are bacte- rsemia, septicaemia, pysemia and saprsemia. Among other contra-indications should be mentioned igno- rance and inexperience on the part of the would-be immunologist, and complicating surgical conditions demanding primary and immediate operative inter- vention. BACTERUL INOCULATION 255 Bacterin therapy has definite and, in the minds of immunologists, well-recognized limitations. It is not assumed to be a specific, capable of cm'e in all cases to the exclusion of other important time-honored therapeutic measures, physical and chemical. It pur- ports to be merely the logical and scientific means to assist Natm-e in her struggle against infection, and, with due appreciation and application in this light, it will seldom fail to render due service. It offers no promise to resuscitate a medical derelict. '^4^11usion must be made to certain causes of failure by those practicing bacterial inoculations. Sum- marily, they may be stated to be: (1) utilization of the improper bacterium, whether autogenous or hetero- geneous; (2) routine employment of stock instead of autogenous bacterins; (3) ignorance in administra- tion, either of size of doses or intervals of inoculations, and (4) disregard of commonly associated conditions. The mistake of utilizing the wrong germ could be obviated by the routine employment of autogenous bacterins whenever possible. This implies technical laboratory and bacteriological knowledge by each physician contemplating bacterin therapy or that he consult with or refer his patient to an immunologist. Thus there would arise no shifting of responsibility between the laboratory worker making and stand- ardizing the bacterial suspension and the general prac- ^56 APPLIED IMMUNOLOGY titioner desiring to give the inoculations. Otherwise, in the event of an unhappy result, the former will accuse the latter of incompetence and the latter the former of faulty technic. This seems like stringent doctrine, but we believe in the long run will redound not only to the credit of physicians and bacterin therapy, but also and especially to the better health of patients. Even in the experience of expert bac- teriologists it becomes at times no easy task to isolate from a mixed infection the fundamental etiological bacterium. Obviously, a bacterin prepared from a superimposed or secondary infection, even though that bacterium be preponderant, would produce little or no effect, curatively, upon the primary or underlying morbid process. Stock bacterins, if employed — a pro- cedure at times advisable — should be polyvalent, that is, constituted by as many strains as may be obtain- able of the particular species of bacterium in question. Caution should always be exercised to determine by bacteriological methods the exact identity of the in- fecting bacterium in a given case before proceeding with the '' supposedly correct " stock bacterin. On more than one occasion we have found patients falsely immunized with a stock preparation devoid of the bacterium actually causing the disease. Such failures discredit physicians vastly more than the mode of therapy. The advantage of the autogenous prepara- BACTERIAL INOCULATION 257 tion is, naturally, that the patient will be immunized with the exact strain of the germ by which he has been infected, leading to a more definite and decisive re- sult. The commercial so-called '' mixed bacterins or vaccines " for therapeutic purposes cannot be too highly condemned, simply on scientific grounds; prophylactically their employment would appear rational. It is not true that the injection of a few million extra brands of germs, in addition to that responsible for the main affection or possibly con- comitant infections, is without deleterious effect. Each variety of bacteria thus injected stimulates the tissue cells to the production of its specific antibody. This, certainly, is an unnecessary work or drain on the part of the immunological mechanism, so far as the irrelevant bacteria are concerned, and has no ef- fect therapeutically upon the pathological process. Again, the bacterin, especially if heterogeneous, may be unfit for use, that is, too old or in a state of precipi- tation or autolysis, or chemically contaminated from its container, be it bottle, vial, ampoule or syringe. Finally an item of much importance in the prep- aration of a bacterin, whether autogenous or stock, is that it should not be overheated, thereby destroy- ing its immunizing properties. The correct degree of temperature is merely the thermic death point for the respective bacterium. 17 258 APPLIED IMMUNOLOGY Many failures in therapeutic inoculation result from improper administration of the bacterin. The doses given are either too large or too small, more frequently the former, and the intervals carelessly spaced. This is partially accounted for by the fact that some physicians are gifted with superior judg- ment and possess keen powers of observation, while others are below par, yet overworked and indifferent, or overenthusiastic or prejudiced. Another impor- tant cause of failure in biological therapeusis is dis- regard of associated conditions. These are both general and local. The general comprise diabetes, in- testinal stasis, constipation and toxicosis. In these states phagocytosis is markedly handicapped or im- possible. The local causes responsible for failure include marked suppurative foci, caries, sequestra and other conditions demanding primary surgical interven- tion. Again, owing to the anatomy of the part, as in chronic otitis media, also on account of the pathology as observed in sinuses, fistula and old abscess cavities, where, due to coagulated fibrin and old inflammatory exudate and cicatrization, the diseased area becomes walled off, it is impossible for the systemic blood- serum, even though highly opsonized, to bathe the affected part, hence exert a healing influence. Here the rational employment of hyperemia, by massage, rubefacients, heat and Bier's methods, flushing sinuses, BACTERIAL INOCULATION 259 abscesses, etc., with Wright's solution (one and a half per cent, sodium citrate in five per cent, saline), in- cision, citric acid to decrease the viscosity of the blood, nuclein and eliminants and tonics directed to the in- testinal tract, kidneys, skin and liver, become indis- pensable adjuvants, converting an otherwise sure fail- ure into natural victory. Application and Results of Bacterial Inoculations in Special Diseases. — During the past decade, owing to the opsonic tidal wave and the resultant interest in bacterial inoculation, injections of killed bacteria have been made, both in a protective and curative capacity, in almost all infections or infectious diseases to which man falls heir. There is not a system in the human organism to which bacterin therapy has not been found applicable. Just as was the case with tuberculin therapy in the early nineties, the therapeutic pendu- lum swung too far, but to-day the cordon is being drawn tighter and tighter around those specific dis- eases amenable to this form of therapy and year by year the indications and contra-indications and modus operandi become better known and the doctrine of bac- terial inoculation more intelligently engrafted. Diseases of the Skin and Soft Parts This group, comprising acne, boils, carbuncles, abscesses, ulcers, cellulitis, dermatitis, impetigo, syco- sis, sinus, fistula, tuberculosis, glanders, actinomycosis, 260 APPLIED IMMUNOLOGY smallpox and bubonic plague, by all odds furnishes the most fertile field for bacterial inoculations, thera- peutically and prophylactically. Acne. — Extraordinary results have followed bac- terin therapy in this disease, especially in the pustular variety, when usually the M. alhus, but occasionally the M, aureus and rarely the M, citreus, have been isolated (Fig. 39). Incipient or non-pustular acne, tNDEX OCT NOV H^ U^ NOV 0|C DEC JAN. ™ ?..?. ?> ®« OPSONIC INDEX , /• ■ 2.0- X" INOCULATION M.ALBUS / 1 .1 1 .8 o O O OO OO ooo o -°- -5 S- S- § %—Z' S— S-S- o— 2 ri -n o o ._.o ,-, _o o o . ..^_ - o OO o o OO o_oo o^7 -^ s( ^ j( s S— S ^^2— ci'-:f'^ ^f- I .7 1 .U L.-i 1 .4 J. .a ^y*^ "^ 1 .1 1=^^ \ / 1 . 1 / \ / / \ ^ / \ JL-^ fi.R «r \ .*<>'' y V ^sr O.fc <* \ s^ QjT J Fig. 39. — Harris E. T. Case of long-standing and obstinate pustular acne vulgaris. Yielded to autogenous bacterin therapy only after a prolonged course of treatment. according to the researches of Unna and Sabouraud caused by the Bacillus acnes, is in many cases favor- ably influenced by inoculations with this organism, al- though the results are not nearly so brilliant as with the staphylococci in the pustular form of the disease. Sabouraud contends that the acne bacillus is also the cause of sehorrhoea and alopecia areata, and Fleming has reported signal successes in the treatment of these conditions with B. acnes bacterin. In the treatment of BACTERIAL INOCULATION 261 acne in any form, the accessory or supplemental measures are of prime consideration, and include cor- rection of underlying systemic conditions, notably ali- mentary and genital disorders, lymphagogues as sodium citrate or citric acid internally and hot fo- mentations, the removal of comedones and pustules locally, followed by sulphur, salicylic acid or betanaph- thol ointment, etc. NOEXM 50 1 I S 4 5 t IN JUUY : i 6 10 11 uij 14 15 It in H:0!.1U23M25it2Tyi9 50 51 ^ IVfPSC JIC II jnry y» Nfil: iLAT riN M. IIRFUS : - I - -^ ■ " " "" : :z ~ : : ::z: 1 H Sbi : _: _ 121 - "g 7 __ _ . I ^•n ^ ~ ::.._:-^«_- :.::: _:Z_- :_ ^_- "i i :. _ - I- -§ - i - g ^^ "1 1 g "~"^'v2 _ - Is , = e g :__.:zB ° '- ±:: ±_- .2 Fig. 40. — Annie H. Furunculosis of nostril. _ It will be observed here that the inoculations were governed entirely by the clinical course of the case; the opsonic indices were taken incidentally, merely, to note when the patient reached the "high tide of immunity, " so that further inoculations might be suspended. Furunculosis and Carhunculosis. — None the less remarkable are the effects of bacterial inoculations in these common affections. The infecting bacterium is almost invariably the M, aureus. In the case of the acute primary boil or carbuncle, bacterin therapy need not and should not be used except by an expert pos- sessing a knowledge of the opsonic index, since more harm than good may result (see page 226), whereas 262 APPLIED IMMUNOLOGY in the chronic recurrent form of the diseases it is the most valuable therapeutic agent at our disposal. Here again opsonic indices utilized to guard against over- inoculation, and to govern remote inoculations, will suffice to prevent recurrences (Figs. 40 and 41) . Abscesses. — These may be primary or complicate other diseases, as typhoid fever. The bacteria isolated include 31. aureus, alhus and citreus. Streptococcus pyogenes, Pneumococcus lanceolatus, B. coli, B. ty- INDEX 1.3 l.^ 1.1 1.0 .9 .8 .7 .6 4 ^3 s - 1^ 1 i:MBE 4'i — ; 19 Ik n u n M ?Q 1 0 4 r % 1 s ^ F ^ -^ So g _J -^ ' ■' t1 & r^^ ^ ©= ©•OP.^ONin INHFX y-lNOLSTAPHYLnrQCCl Fig. 41. — Fred. G. Carbuncle of neck. Observe in this case the utilization of the opsonic index to determine the maintenance of immunity in the "positive phase," after the suspension of inoculations. phosus, B, pyocyaneus, B, tuberculosis, B, antkracis, B, mallei and Streptothrioo actinomyces. An autoge- nous bacterin is always preferable to a stock prepara- tion and the therapy when so conducted usually re- sults brilliantly (Figs. 42 and 43). Little should be expected from bacterial inoculations with secondary invaders, as B. pyocyaneus, B, lactis aerogenes, etc., or with B. anthracis, B. mallei and Streptothrix actino- mycosis. FiQ. 43. — Linda c' ""epresents opsonic indices. A, skin grafts to arm and leg; B, develop raei^^ demonstrated M. aureus. The small crosses on the diagonal line indicat°°*^***°° *°^ health of the patient. Note also that no new abscesses formed Fig. 43— Linda C. Euros one-third I dia~gonal line indicate inoculation with M. aureus bacterin. 205.000.000 bacteria, *Note the parallelism existing between opsonic new absoessea formed after the first inoculation of bacterin and the rapidity of disappearance of the recurrent preftxiatent aubcutaneous BACTERIAL INOCULATION 263 Ulcers, — A variety of bacteria have been identi- fied, the majority of which in chronic and intractable OPSONIC - TEIMPERATURE CHART — 0) - < u a. O si r o 6 99 12 ^6991 •) 6 ^T~ 2 3 ef<: 27- P,6- 25- 24- 23- 2Z- 2.1- 20- 1.9- 1.6- 17- 1.6- 15- 14- 13- 12- 11- 10- B- s- 7- 6- 5- 4- 3- 2- 105- 104- 103- 102- 101 - ■ 1 ■ " -■ -p ^ - r n / / A /I ^ 1 I 1 1 1 r ■1 i 1 t fi ' 1 / / / ; ^ 1 "7 ( (^ ^ t ^ 1 / \ 1 ^ / 1 1 , s I ( / y r z! ^ f- '' 1 ■' ' , tv T 1 1 / J . *^ '"7'_. 1 f / •, s. 1 99 ; ■ \- f- ^ • 1 1 :^i^^ T 1 [ _i ^ g - 1 1 / ' 1 1 / 1 ' 1 1 / /^ r / __\ 4 ' . \ 1 \ 98; 97; 96; / \ ^ '■/ V / : U ' / / / / / ^ 1 ■^ ^ — _ _ 4 ~* 1 6^0 Iso PULSE- 1 X 9a l« 102 .» 9B.l»»i Ciiaz M^gcli »« 3.-. 94- 110 90 7« 90 « M 9* HSZ 82 66 \^ 92 K L. «)e8i86 6*7<> ?2!M;80 82 ..[T2«*;72 76 7 Ri5pm*Tio». ;s> ro ^» rs> zs> e^ t^ to («> DMcHtirje freer. temperi&TB (|j\ sUjWly lower. 1^ 5u,p p uT a.tvOTt i jcr e a se i. Free ilsckir^e of pos, temp.fell \y crisis from 105° to gg', tumor maa* smiUer. Sappuration. decreasel teaierness over' kiiney is less , Temjera.ture rea.thesio4. ani IS intermittent. Tem,peratu.Te lower, not over 103". Citric a.cld ititernaUy. Kiiney mass smaller Dlsclvar^e scajity and. watery. Temperature fluctuates between gf* and 103°. Markei improvement. P. improved oeneraUy. Fijtuia iressei temi-weekly. P.stron^er, ooes to jeasKori to recuperate. Kiiney mass sXrinkm^ away. Temperature reatlves 100* 16101° in evening Returns from sea oatti In weiont. 2,3 1 Vistulj ' ^ ' FisXula barely aimiTsjTO CUcharje scanty ft Sterile . Fig 48 —Dorothy W. Pyonephrosis. Nephrectomy in this case was impossible owing to the size and adherent state of the kidney. Nephrectomy temporarily relieved for a few months; then the patient became critically ill and was rescued apparently by autogenous bacterin therapy. Note the unprecedented high flights of the opsomo index, especially that of September 14. BACTERIAL INOCULATION 273 Diseases of the Genito-urinary System Cystitis, Pyelitis, Pyelonephritis and Pyonephro- sis.— Bacterins from the following microorganisms have been of service in certain diseases of the kidney and bladder; B, coli, B. tuberculosis, B, pseudotuber- culosis rodentium, B, of Friedldnder, B. typhosus. =inoculation with colon bacilli; > = inoculations with streptococci. Streptococcus pyogenes. Staphylococcus and M. lan- ceolatus. Great discrimination must be exercised as to whether the particular condition is amenable to bac- terin therapy or should be operated upon. Bacterins were never intended to supplant surgery, but at times 18 274 APPLIED IMMUNOLOGY are wonderful accessory agents in promoting cure, and not infrequently, with proper administration, re- lieve surgery of some of its indications. In colon bacillus infections of the bladder and kidney in chil- dren, also in kidney suppurations where, owing to the size and adherent character of the diseased organ. NDEXi MAY JWl JULY AUQ 9 16 16 23 24 51 1+ ^. 1 1 r 1 1 1 1 i iS - i7 - S.6 - is - 54 - 3J - 3A i.l io Z9 - 55 ■ tn - 2.6 - 2.5 - £.<■ - 2.5 ■ Z2 ■ 2.1 - l.o - 1.9 - 18 • 1 -X'l ' III S 1 1 ~ ¥ 8 *^, s^ y i f \ ~] J s T \ s J l k ] s •j J \ f r / ^ u / \ / s / V j \a P V ^ ••l ^ J / j \ T L5 - I.* - l.i - 1-2 - 11 - i.O . 3 - I T \ r 1 \ 1 k / \ I 1 9 -a . - - Fig. 50. — Margaret B. Pyelitis and cystitis. nephrectomy is temporarily impossible, autogenous bacterins have proved signally meritorious (Figs. 48 and 49 ) . In such deep-seated infections and in cystitis and pyelitis estimations of the opsonic index to con- trol the inoculations have been invaluable (Figs. 50 and 51). UretJmtis, — Compared with the gonococcus of Neisser all other invading microorganisms sink into BACTERIAL INOCULATION 275 insignificance, although occasionally a non-specific in- fection arises, due to the 31, catarrJialis or other pyo- genic bacteria. Gonococcus bacterin has been utilized both prophylactically and therapeutically against gonorrhoea. As a protective measure, Douglas, Wright's associate, recommends an initial dose of 100,000,000. If this is followed by no reaction a second inoculation of 200,000,000 is administered in a week or ten days. The adoption of this procedure is HirSiBElQSQiBQElIEiniSIBIQIBISIBIISIS^Ql^SBlSSSSlSD^EEnQB ^ t i 1 1 1 ^ Jt 2 S 5 a ^^*v^ t ± ^^ ^^ * ^^ ^^ ^c 15- ^^ ^ 2 / ^^ 7 ••■ ^ / / ®= OPSONIC INDEX ,»---^ --' X- I^OCULATO^S b.cc .1 ^^^^■^- - -- - -U-^^ 444--- Fig. 51. — Cystitia and toxic neuritis. advisable, although unattended with any absolute as- surance, in certain marital relations when the disease is innocently acquired, for the protection of girls in an institution in the event of an outbreak of gonorrhceal vulvovaginitis, and perhaps to safeguard innocent newlyweds, under peculiar circumstances, when one of the contracting parties has recently convalesced from an attack of urethritis. Again, it must be borne in mind that the discharge 276 APPLIED IMMUNOLOGY of an acute gonorrhoea, sooner or later, invariably contains bacteria other than the gonococcus, namely, the Streptococcus pyogenes, the Staphylococcus, the Pneumococcus, the Colon bacillus, the Micrococcus catarrhalis, etc. Thus the prophylactic effect of early immunization in a case of gonorrhoea against these complicating germs, in the prevention or ameliora- tion of such conditions as gleet, prostatitis, seminal vesiculitis, etc., should not be underestimated. The authors feel that in many cases in their experience the cautious administration of mixed gonococcic bacterin has sufficed to shorten convalescence and to prevent or reduce the severity of certain complications, notably inflammation of the prostate and seminal vesicles and stricture formation. It is a difficult or impossible task to gauge the effect of treatment upon any disease prone to run a definite course, consequently the value of bacterins in urethritis, in nmnberless instances, has been immeasurable, and although certain investiga- tors have reported favorably on biological therapeusis in acute gonorrhoea, consensus of opinion discourages the procedure. In this connection reference must be made to Bruck's " Arthigon," a suspension of dead gonococci, standardized to contain twenty millions per cubic centimetre. Trustworthy evidence has accumulated to the effect that the intravenous injection of this APRIL MAY JUNE JULY AUGUST FEBRUARY APR L V.AV 'tMTE fc K » 2 ■* ' 8 !.0 |.2 |l* |.6 fB s 2$ P' a !» 1* " -' " "i " » * IT ^ • 1 : ' M M , ■ . (;>NdlC>NS .NDE* as- " 1 ■ ! 1 ; 1 . i M i- 25- 2.- 15- 1- J- ; / = / s i 1 ? 3 ! I ; 1 ii - - i 1 ! ! J n i * I / s; 5 ? f - I - - - s j f V 5 - - - - 7W / 1 J A ^ / ? 1 i 3 ^ - - ¥ / ^ha / / ~ ■~ '- J *- _ _ _, J f^ - - - _ J L •-^ .>-■••■** > - 7' ^ — _ _ _ _ _ — _ _ _ _ _ _ _ _ _ ^ t = = = >. p •^ ii. -^2ti44- .^ ^ .u _ -J441- >-..>^ - - ^ - — - — - - - - - ^H M = ^ - - = ^h^ = - - - - ^ - #- - - - - - - - - '- t - - -1 - - - - -Pff* K; ^ - - - ; ^y ir ^ ^ _L _ _ U J Jc __ - J. i. k i I CuLTuR£ . B Pyocyaweus _ , Culture.M.Onoicans - CULTURE. MCflNWCANS CuLTu—B Colon Cin.Timt.M.*o«ei.i c.iA.TuRe . n. Aureus CuLTUitfSmu u □ UJ □ D □ QJ _ -a E« KYANEUS ^ su :i. _ _ B.Pyocyame « _ B.CANDICAN* Cjy. ~« »» :_ .. Canchcans abk-w 1 1 1 1 1 1 ! 1 Fio- 52 — fiobert C. Cbrooic proBtetttu. Observe the cbaoge i .ct«rial flora ob demooBtratcd by repeated ( tini altcmatioo of the baetertal i Note the seoerAl trend o( th«o BACTERIAL INOCULATION 277 preparation in quantities of 0.05 to 0.5 c.c. produces a reaction which is invariably constant in gonorrhoeal subjects. A rise of temperature above 1.5° C. after an injection, of 1 c.c. may be regarded as pathogno- monic. Therapeutically, with increasing dosage, the re- sults have been no better than with the subcutaneous administration of bacterins or sero-bacterins, and it is extremely doubtful if intravenous injection will super- sede the older method, properly interpreted, in a diag- nostic capacity. Prostatitis and Seminal Vesiculitis, — The writers believe that these complications, in addition to the gonococcus, are commonly precipitated and perpetu- ated by a mixed infection due to the Streptococcus, PneumococcuSj Colon bacillus, M, catarrhalis, etc. They have obtained in many instances gratifying re- sults by alternating a decivalent gonococcus stock and autogenous bacterins, the latter prepared from urine cultures after massage of prostate and seminal vesi- cles, when, in addition to the above, other organisms, as the Micrococcus alhus, aureus, citreus, candicans, Candidas and orbicularis, the Bacillus typhosus, pyo- cyaneus and acidi lactici and the Cory neb acterium pseudodiphtheriticum, have been isolated. The therapy has been conducted only in the subacute and chronic cases, preferably in the presence of suppura- tive inflammations ( Fig. 52 ) . 278 APPLIED IMMUNOLOGY Epididymitis, — Gonococcus bacterin is probably of value in acute epididymitis (Fig. 53), although the disease is prone to run a definite short course under proper treatment without immunotherapy. It would appear, however, that the employment of bacterin di- 102 99 98 % 10 11 \z MEMEME l\ w w a Fig. 53. — Typhoid fever complicated by epididymitis. Observe transient slight fall in temperature a few days after first inoculation of typho-bacterin, and the fall by lysis after the second dose of bacterin. minishes the likelihood of a resultant inflammatory nodule of the epididymis, at the same time reducing the liability to sterility. In the chronic form due to tuberculosis, tuberculin is of unquestionable value, both diagnostically and therapeutically, although in no small numbers of cases epididymectomy must be BACTERUL INOCULATION 279 performed, and this applies particularly to dispensary patients. Vulvovaginitis, — In children inoculations of a polyvalent gonococcus bacterin, in the acute stage, have resulted brilliantly, causing the early disappear- ance of gonococci and shortening the usual course of treatment by months. Five million gonococci consti- tute the average dose. From three to a dozen inocula- tions are required. Benefit is also observed from autog- enous bacterins in the chronic form of the disease due to other pyogenic bacteria, as the Streptococcus, Staphylococcus, Pneumococcus and Colon bacillus. Vaginitis in adults, in the experience of the authors, has been absolutely uninfluenced by bacterial inocula- tions. Cervicitis, Endometritis, Metritis and Salpingitis, — The following bacteria have been isolated: 31, gonorrhoece, B, coli, Pneumococcus, Streptococcus, Staphylococcus and Tubercle bacillus. This category of diseases needs further investigation respecting the value of bacterins, since the studies to date have been inconclusive. In general the results have been very indifferent, scarcely warranting the procedure. This is certainly true in the case of cervicitis. In the treat- ment of salpingitis a number of remarkable results have been reported. An essential to success lies in the 280 APPLIED IMMUNOLOGY selection or preparation of the appropriate bacterin, dependent upon the determination of the invading microbe. Puerperal sepsis is entitled to special and serious consideration. In our opinion the applicability of bacterins is directly dependent upon the presence or absence of a general bacteremia, determinable by blood culture. If the former exists, active immuniza- tion is definitely contra-indicated ; if the septic process is localized to the pelvis, bacterial inoculation, cau- tiously employed, may prove serviceable. We have been gratified on several occasions by the excellent results of bacterins supplementing or alternating passive immunization (serum therapy). In any event, this is a condition wherein opsonic indices will render important service in the control of therapy. Diseases of Boxes and Joints Osteitis, Periostitis and Osteomyelitis, — Infections from the following bacteria come into consideration: Staphylococcus, Streptococcus, Pneumococcus, Gono- coccus. Typhoid hacillus. Colon bacillus and Tubercle bacillus. In the acute stage of an osteoperiostitis, if bactersemia is not present, bacterin therapy is very beneficial, especially in pneumococcic, gonococcic, typhoid and colon infections ; in early spur formation BACTERIAL INOCULATION 281 from the os calcis (osteophytes, exostoses) the ad- ministration of gonococcus bacterin has rendered operative intervention unnecessary, or at least has eliminated toxins and insured a more satisfactory post-operative result. In chronic cases it is even more ^"uz APRIL 1 13 24 li 26 U 28 29 JO' \ , 5, 5 4 5 6 T « 9 10 n u MAY 13 14 15 16 n 18 15120 21 27[23|24 25 26 n. 28'l29 30 . - j^ ^, :: it :: 1 - -f^ 1 «- r - "I » - ■ T"' ~ ■■■ 1 1 1 1 lit It It : :: ie: - - Rt --t - . 4llL . - JILIL, 1 1" ■■ + ■-■ j;^ 1 jTL 3 If 1 1 11 1 U 1 1 Ml' Jji[-L 99 ■-^-\\- VM -H t il_ |Lji!uuuii4 TTFlf 1 Liii ;iLj«! i! n'" r at 1 ' i a ■ I \w in' i!^ ijF 3^1 -I- :_ -I .:.-4-";±:; I"' : : : .1: Fig. 54. — Sam. S. Acute osteomyelitis of tibia followed by septictemia. The arrow indicated when a blood culture demonstrated M. aureus; the crosses represent the times of inoculations with M. aureus bacterin. Observe that the patient, as indi- cated by the increased fluctuations of temperature, progressively grew more septic, and that bacterin therapy was not only useless, but possibly harmful. The substitution of bacterin at the juncture by antistaphylococcic serum apparently exerted a definite beneficial influence for recovery. meritorious, if caries and sequestra are excluded and hypergemia used to augment the treatment. In our experience bacterins and tuberculins have done more harm than good in acute osteomyelitis (Fig. 54). Synovitis and Arthritis, — Associated with these diseases the Streptococcus rheumaticus, Gonococcus, 282 APPLIED IMMUNOLOGY Pneumococcus, Streptococcus pyogenes and Staphy- lococcus should be mentioned. Bacterins compiled from the first-named organism, whether isolated from the joint, gmns, tonsils or feeces, have in a number of instances rendered therapeutic service in the acute, subacute and chronic forms of disease. We have never observed any remarkable benefit by the employment of the common pyogenic streptococci and staphylo- cocci, nor is much to be expected in chronic cases where hypertrophic osseous changes have occurred. On the other hand, this is a field wherein gonococcus bacterin enjoys much distinction. Indeed, biological inoculations furnish the treatment pai' eoccellence for gonorrhoeal synovitis and arthritis. Many patients, with limitation of motion in subacute and chronic gonorrhoeal joints, are best treated by preliminary inoculations of Neisser bacterin a few weeks or months prior to forcible manipulation under general anaes- thesia, thereby eradicating the cause of the inflam- matory exudate and preventing its re-formation after operation. It should also be observed in this connection that gonococcus bacterin is of exceptional value in the differential diagnosis of many obscure joint and other gonorrhoeal afflictions, used precisely as is tuberculin in a diagnostic capacity. BACTERL\L INOCULATION 283 Diseases of the Eye, Ear, Nose and Throat Singularly perhaps, but nevertheless fortunately, infections of the eyes have, in the majority of in- stances, demonstrated the great value of bacterin therapy ; not so much can be claimed in the treatment of diseases of the ear, nose and throat. There are certain anatomical facts explanatory, at least in the case of the ear, of these variable effects. Conjunctivitis and Dacryocystitis, — The follow- ing bacteria have been isolated: Pneumococcus, Streptococcus, Staphylococcus, Gonococcus, M, catar- rhalis, Friedldndefs bacillus, Koch-Weeks bacillus, Moraoj-Accenfeld bacillus, Pyocyaneus bacillus and Tubercle bacillus. In many acute infections, notably that due to the bacillus of Koch- Weeks, the usual ophthalmological treatment will prove all-sufficient, while in chronic types, as that caused by the bacillus of Morax-Axenfeld, frequently little response fol- lows ordinary treatment and the effect of bacterins appears to be specific. Between these are a number of infections due to the gonococcus, pneumococcus, streptococcus, etc., which may do well with ordinary measures, but in which the accessory employment of bacterins hastens convalescence, diminishes the in- roads of the pathological process, prevents sympa- thetic ophthalmia and avoids the loss of vision. In 284 APPLIED IMMUNOLOGY dacryocystitis bacterin therapy may obviate surgical extirpation of the lachrymal sac. Corneal Ulcer and Hypopyon. — The Diplococcus pneumonke, the Streptococcus mucosus, the Strepto- coccus pyogenes, the Staphylococcus, the Gonococcus and Tubercle bacillus have been identified. Autog- enous bacterins prepared from the pneumococcus and streptococcus in cases of ulcus serpens of the cornea, with and without hypopyon, notably in one instance in which panophthalmitis threatened com- plete destruction of the eye, have produced incredible results (Fig. 55). Care must be observed not to employ too small dosage in the treatment of these affections. The initial inoculation should be at least 100,000,000 bacteria. Iritis and Uveitis. — Here as in corneal ulcer the same organisms are found at work. If the infecting bacterium be determinable the results are invariably excellent. Especially noteworthy is bacterin therapy in gonorrhceal involvements. In these conditions, as in practically all gonococcic complications, practi- cability demands that a polyvalent stock preparation be employed. Otitis Media and Mastoiditis. — An extensive array of bacteria as the Streptococcus, Staphylococcus^ Pneumococcus, Colon bacillus, B. proteus vidgaris, B. lactis aerogenes, B. pyocyaneus, B. fluorescens, B, BACTERIAL INOCULATION 285 diplitlience and pseudodiphtherice, 31. catarrhalis, B. influenzce, B, typhosus and B, tuberculosis have been found. Due to the fact that in the majority of cases the cultured organism is a secondary invader, also JANUARY FEMP. 10 11 12 13 U 15 16 17 18 L9 20 21 22 23 24 Z5 26 Zy\ZQ .., ;c.]^ c Lirons S-rei^(c c:o- itczw IIW ~'o'~S^~ o § -tg § o o § «« oo oggoggg lot OCMt^Niool- QO _i K - --.4 4 \- i^ » X NflSMAjIX-- ^-- t\ - ,-J^^tltt mm Z^ZZ 1^.1' IZ T^^ll J> \ on h^ \- L^ S*^ ^-^* ^L _ : 1,7 : ^±" : :_ : s^i: ^ X-i \/k JL ^'^ > 4- FiG. 55. — V. S. Corneal ulcer with hypopyon. Culture of pus from hj-popyon demonstrated the Streptococcus mucosus, from which an autogenous bacterin was prepared. We believe that this patient was over-treated, that is, inoculated at too short intervals. Although he made a satisfactory recovery, it is probable that the result would have been more brilUant with less frequent inoculations. that the infected area is encased by bony walls limit- ing the supply of blood, although highly opsonized, the results of bacterin therapy have been poor, espe- cially in chronic cases. Nevertheless, in the early stage of otitis media complicating scarlet fever, the sup- 286 APPLIED IMMUNOLOGY puration seems to be markedly curtailed and convales- cence proportionately shortened. On the other hand, tuberculin in carefully selected cases of ear and mas- toid tuberculosis has proved of great benefit. Rhinitis and Sinusitis. — The bacterial flora of the nasal passages and associated sinuses differ little from those of the auditory apparatus. The results follow- ing the use of bacterins, however, are much better, due probably to the greater vascularity of the parts affected. Indeed, in suppurative sinusitis the em- ployment of autogenous bacterins, aside from any surgical indication, should constitute the sheet-anchor in treatment (Fig. 56) . The chief offending bacteria are Streptococci, Pneumococci and Micrococci catar- rhalis. The dosage frequently must be increased over the average. Laryngitis and Tracheitis, — The following bacteria are commonly demonstrable: 3/,. catarrhalis, D. pneumonice. Streptococcus pyogenes. Staphylococcus, M, paratetragenus, B. diphtherice, B, influenzcB and B, tuberculosis. Occasionally, excellent results have attended the use of bacterins in these affections, par- ticularly with the M, catarrhalis, Pneumococcus, Streptococcus and Staphylococcus, Diphtheria. — In recent years active immunization against diphtheria has been studied extensively by several observers. If those who are naturally immune^ BACTERIAL INOCULATION 287 as determined by the Schick reaction (see p. 191) , are eliminated, the results of active immunization, em- ploying either autolyzed diphtheria bacilli themselves or toxin-antitoxin mixtures, have not been brilliant and do not by any means warrant its substitution for OP50N1C-TEMPERATURE CHART m^ __. < u ll htoculstioni XXX XXI Month. c ober Novombep I Date^ zz Z3 Z4 ?5 li Z7 Z8 Z3 30 1 2 3 4 5 6 7 8 9 to II 12 13 14 15 16 17 18 19 20 21 n Z3 ?4 25 26 ?7 Z8 29 30 31 1 z 3 4 5 6 u u • ' M a f »■ a- 105 loi 103 102 tOh I0« 99 9& o _ — o o o § o q o i — 1 ^ % § 1 — 1 1 , , — _ 1 1 _ . _ _ 1 o — o o — — § — — — 1 — — — — — — — — — — — - — — ' — ' i' — C— — 1 — — — — 1 — — — = ;^ i ^ KI 1 CLnicaJ Notes AnW,» •J::,Zi ,7/ ..crrtlon- COM '.mm',;' f.^.',^ 0.«h.r,«J J Fig. 56. — Cultures both from ethmoidal sinuses and bronchial expectoration dem- onstrated the Streptococcus pyogenes in preponderance, together with the pneumococcus, staphylococcus and M. catarrhalis. _ Opsonic indices were taken only for the strepto- coccus, although therapy waa carried out by inoculations with the autogenous mixed bacterin. antitoxin. In the first place, the formation of pro- tective antibodies is slow, seldom before the second week, hence the superiority of immune serum in epi- demics, both prophylactically and therapeutically. Park and Zingher point out that there is lack of a sufficient response to active immunization in at least 288 APPLIED IMMUNOLOGY fifty per cent, of patients susceptible to diphtheria and that the immunity conferred lasts not more than a year or two. An important conclusion of their work is that the Schick test will demonstrate the futility of immunization in approximately two-thirds of those exposed to diphtheria. Respecting the value of treatment of so-called " diphtheria carriers " by bacterins, opinion is divided. Some remarkable results have been reported and the procedure deserves further trial before conclusions are drawn. Scarlet Fever, — Opinion has grown to the effect that if the streptococcus is not the cause of, it is ma- terially concerned in the pathogenicity of the com- plications of scarlatina. Experience has shown that bacterins of the Streptococcus conglomeratus, what- ever may be their effect curatively in the acute course of the disease, have unquestionably favorably in- fluenced the complications, as angina, suppurative rhinitis, lymphadenitis, nephritis, otitis, mastoiditis and arthritis. INIost remarkable are the prophylactic inoculations used by Gabritchewsky in Russia, where in thousands of cases specific immunity has been se- cured on the average for eighteen months. Donilow states that only 1.1 per cent, of inoculated persons contracted the disease, including those already in the incubation stage; furthermore, that the mortality BACTERIAL INOCULATION 289 among the inoculated group was only 0.123 per cent, as compared with 11.1 per cent, among the uninocu- lated. For protective inoculation three doses of 250, 500 and 1000 million, from a week to ten days apart, should be administered. Hay Fever, — An allied treatment by antigenic inoculations is that of hay fever by injections of the extracted pollen of rag-weed, on the assumption that this affection is a pollen toxicosis and not an anaphy- lactive expression, a thought that has gained credence recently. Noon and Freeman gauged dosage by the conjunctival reaction and injected their patients at in- tervals of three to ten days with units representing the amount of extract from 0.001 milligramme of pollen. Clowes was able to raise the resistance a thousandfold, although it persisted but five months, by inoculations of one cubic centimetre of 1 : 5,000,000 to 1 : 500,000 suspensions. Recently, Lowdermilk reported astonishing re- sults with pollen toxin, prepared as follows: One gramme of mixed pollen (250 milligrammes Ambrosia artemisicefolia, 250 milligrammes Ambrosia tiifida and 500 milligrammes of various varieties of soli- daco) was mixed with ten grammes of sterile sea- shore sand in a mortar, moistened with a part of a solution of 100 c.c. physiological saline contain- ing 0.5 per cent, phenol and ground for several 19 290 APPLIED IMMUNOLOGY hours, slowly adding the remainder of the saline- phenol solution until the sand was reduced to an im- palpable powder. The whole was transferred to a sterile flask and allowed to stand with frequent shak- ing, at room temperature, for twenty-four hours. After decantation and centrifugation the toxin was sealed in glass ampoules, containing one cubic centi- metre each. This product was standardized so that the unit represented the quantity of toxin extracted from 0.000,001 gramme of pollen. Thus each cubic centi- metre contained ten thousand units. Six to eight inoculations were administered at intervals of one to ten days apart, representing from 25 to 1000 units each. The typical reactionary phenomena simulate the symptoms of an acute attack of hay fever. It is wise to reinforce the pollen toxin injections with inocu- lations of autogenous bacterins prepared from the complicating infection. Such treatment has been ap- plied therapeutically as well as prophylactically. It is, however, of such recent date that no definite con- clusions should be drawn. It may share the same fate as Dunbar's " pollantin " used to produce passive immunity in pollenosis. Diseases of Lungs Bronchitis. — The following bacteria may be identi- fied: M, catarrhalis, M, paratetragenus, B, influenzcB, Streptococcus, Staphylococcus, B. typhosus, B, coli, Z),. BACTERIAL INOCULATION 291 Pneumonice, Diphtheroid bacilli, B, tuberculosis, Fried- lander's bacillus and Streptothrix actinomycosis. Pul- monary diseases offer an exceptional field for bac- terin therapy, owing to the unusual vascularity of the tissues, permitting the opsonins to become effective. Consequently, brilliant results are achieved routinely both curatively and prophylactically (Fig. 5Q) , This, of course, does not apply to tuberculosis and actino- mycosis nearly to the degree that it does to other infections. Pneumonia, — Essentially the same bacteria are to be found as in bronchitis. Keen discrimination must be exercised not to employ bacterins if there exists a doubt of bactersemia, otherwise the employment of autogenous bacterins in adequate dosage will lessen mortality and shorten convalescence. The opsonic index here as in bronchitis should be utilized to secure the best results. In unresolved pneumonia and bronchopneumonia of childhood a special field of use- fulness is offered autogenous bacterins. Prophylactic immunization applies in pneumonia the same as in bronchitis and in the event of epidemics should be routinely practiced. The protective dose is 1,000,- 000,000 pneumococci. Purulent Bronchiectasis and Pulmonary Ab- scesses, — The bacteriology differs in no respect from that of bronchitis and pneumonia. The expectoration 292 APPLIED IMMUNOLOGY must be cultured and recultured frequently for the preparation of the correct autogenous bacterins. The opsonic index will render service in the proper manage- ment of the case. Although some benefit attends the use of bacterins, the results are not brilliant, owing to the poor drainage in these chronic conditions. Whooping-cough, — Although the bacillus of Bordet-Gengou is generally admitted to be the cause of pertussis, early in the attack, even before the second week, other bacteria as M, catarrhalis, B, influenzce, Pneumococcus and Streptococcus complicate the in- fection. Sufficient data have been gathered to demon- strate the value, prophylactically and therapeutically, of mixed bacterins in this disease. Indeed the attacks have not only been ameliorated but the period of con- valescence shortened fifty per cent. Diseases of Aumentary System Pyorrhoea Alveolaris and Tonsillitis, — The or- ganisms usually found include the Pneumococcus, Streptococcus, Staphylococcus, M, catarrhalis, spiro- chcetce, vibrios and Tubercle bacillus. The results of bacterin therapy have not been such as to place it as a recommendable procedure of any great moment in these diseases, unless supplemented by adequate local treatment, in the case of pyorrhoea alveolaris requir- ing the cooperation of a dentist. Some form of the streptococcus can be cultured in at least 75 per cent. BACTERIAL INOCULATION 293 of cases and when remote secondary joint involve- ments arise and anaemias supervene, autogenous bac- terial inoculations assisting effective oral treatment are of great value. It will be remembered, however, that reinfections are prone to occur, also that unless the patient is willing to submit to dental cooperation and prolonged treatment, inoculations of bacterins will prove of little or no value. In advanced disease, when the gums are spongy and necrosed, the teeth loose and the alveolar tissue in a carious state, benefit may accrue, but cure, in its strict sense, is no longer possible.* Acute tonsillitis furnishes no particular oppor- tunity for bacterin therapy. The importance of the tonsils as atria of infection in certain synovial arthritic and other affections must not be overlooked. Con- sequently in subacute and chronic disease, in peri- tonsillitis or in aborting attacks of quinsy autogenous bacterins may render signal service ( Fig. 45 ) . Sur- gery, however, is indicated in many instances, in preference to bacterins. Enterocolitis, — Exclusive of typhoid fever, dys- entery and cholera, the bacteria demonstrable in this condition, and more particularly mucous colitis, are the colon group, the Streptococcus^ the Pneumococcus, * Recent evidence points to Entamoeba buccalis as an etiological factor in many cases of pyorrhcea alveolaris. In these cases the local and hypodermic use of emetin hydrochloride has a markedly beneficial effect. 294 APPLIED IMIVIUNOLOGY the B, 'pyocyaneus, the B, acidi lactici and rarely the Bacillus of Friedldnder. Bacterin therapy, especially using autogenous suspensions of the first three named organisms, has in many cases been productive of good results. The initial dose had best be smaller than the average and here the utilization of the opsonic index as a control of treatment will prove service- able. In not a few cases no material benefit has followed the use of bacterins. Typhoid Fever. — Inasmuch as the lesions of the intestines stand out conspicuously in the pathology of this disease and are the organs first concerned by the invading microbe, the B, typhosus, it may be permissible to discuss its therapy under this heading. In the first place, it will be recalled that not infre- quently by bacteriological study the fever will be found to be due to an organism closely allied to the bacillus of Eberth, namely the B, paratyphosus A or B or the paracolon bacilli, hence the advisability of accurate bacteriological investigation if this disease is to be treated biologically. A number of observers have reported favorably as to mortality complications and relapses by bacterial inoculations. In spite of the fact that after the first two or three days a bac- tersemia supervenes, enduring for a number of days or weeks, a strong movement is in progress relative to the routine employment of typho-bacterin in BACTERIAL INOCULATION 295 typhoid fever. The argument is not altogether con- vincing, but the adoption of this mode of therapy is entitled to serious consideration. In ten days or two weeks, after the bacilli are no longer demonstrable in the blood, the expert administration of typhoid bac- terin may result advantageously. It would appear that a stock bacterin prepared from an old culture of high antigenic properties is preferable to an autog- enous preparation. There exists a danger of the em- ployment of too large dosage. A primary inoculation of 50,000,000 bacilli is proper. The opsonic index will render service in the control of subsequent in- jections (Fig. 53). The bacterin treatment of " typhoid carriers," whether the infection lurk in the gall-bladder or urinary tract, has at times resulted brilliantly, at others failed. Antityphoid inoculation stands out as one of the particular bright lights in bacterial immunization. In the armies of England, France, Germany, Japan, and the United States, adopting prophylactic inocu- lations, the fall in incidence and mortality rate from typhoid fever has been remarkable (Fig. 57). Were the practice universal typhoid fever would soon cease to exist. In 1913, in the army of the United States, of 90,646 inoculated officers and enlisted men, only three 296 APPLIED IMMUNOLOGY developed typhoid fever with no fatahties. Anti- bodies reach their height usually within two weeks after the first inoculation and immunity is alleged to be absolute for one or two years and may endure for many more, although it is advisable to reinoculate after one year if an epidemic threaten or the individual be exposed. The most approved doses for protective inoculation and those adopted by the United States Fig. 57. — Illustrating typhoid-fever rates in United States Army. Shaded columns rep- resent decrease in incidence of typhoid; black areas, the decrease in death-rate. Army are: First dose 500,000,000; second and third doses 1,000,000,000 each. Intervals of a week to ten days must separate the inoculations. In over 500,000 inoculations in the Army and Navy no bad results have been reported. Clinically, the majority of those inoculated develop fever rarely over 101° F., but at times exceeding even 103° F. Headache, malaise and muscular aches are commonly observed; rarely chills, nausea and vomiting occur. Agglutinins and BACTERIAL INOCULATION 297 opsonins are readily demonstrable (Fig. 58). The time is ripe when it is imperative that physicians, nurses, ward attendants and all those coming in con- tact with typhoid patients should be immunized. This JUNE JULY I TEMP 10€> 105 104 103 >02 101 IOC 99 98 INm ?.^ ?4 ?!» 26 27 28 29 30 :* 4 ■ 5 i 1 7 8 9 1) iiri2! -5 t te> o n ^-^ o o o ^ H H ^•^ § o ,^ o V y - Q ^' ^ ^ er ■^ =OPSONIC IND :X 1 ■^ 1 M 1 1 1 1 1 1 1 : 1 1 1 1 ■ = TE ^PERATUR ilURVEl " !» / ) / ^ 1 / 1 1 i / * 1 / >( /* |1 /I (Pi 1 1 1 \ \ \ I l\ -/i i- 1 V / 1 ' 1 i V f \\ I' \' V ./ /I V A T V ' V 1 W 1/ 1 ' 1/ / f V . i \\ / \ \, . . _ _ J Fig. 58. — Antityphoid inoculation or immunization. Observe the rapid rise in antibody formation as demonstrated by the opsonic index curve. rule should include all individuals travelling in and residents of typhoidal districts as well as the members of families and pupils of schools in which the disease has made its appearance. 298 APPLIED IMMUNOLOGY Dysentery. — Bacterin therapy concerns only the bacillary type of this disease. Closely allied bacilli have been described by Kruse, Shiga, Flexner, Hiss and Strong. Bacterial inoculations, employing mostly the Kruse- Shiga type of organism, have been extensively used in all save the acute gangrenous form of the disease, with the result that the mortality has fallen from 6.3 to 0.9 per cent. It should be noted that in acute cases bacterin therapy is contra-indicated from the fourth to the twenty-first day. During this period Kruse and Shiga's anti-endotoxic serum should be administered in full doses. Cholera. — Bacterin therapy in this disease is limited entirely to preventive inoculation; it is value- less in a cm-ative capacity. Since the introduction of prophylactic inoculations with the vibrio cholerse, the incidence of the disease in a study of thousands of uninoculated and inoculated cases has fallen from 3.6 to 0.66 per cent. Three immunizing doses of 500, 1000 and 1000 million spirilla respectively are administered at intervals of about ten days. Diseases of Cardiovascular, Lymphatic and Nervous Systems, also Other Acute Specific Fevers Bactercemia, Septicoemia and Pycemia, — It is our belief, for reasons previously stated under " Contra- indications," that bacterial inoculations, in these con- BACTERIAL INOCULATION 299 ditions, are not only valueless, but harmful, and should the patient recover, he does so not because of, but in spite of, bacterin therapy. Endocarditis, — The streptococcus of the mitior or viridans, foecalis or salivarius and pyogenes longus types, the pneumococcus, staphylococcus, gonococcus and B. influenzce are held to be the responsible in- fective bacteria. The results of bacterial inoculations have been far from promising, although a few suc- cesses have been reported in the chronic and even in the subacute forms of the disease. Treatment in this disease, if conducted at all, must be with the utmost caution. In the acute form, it is certainly condemn- able. Rheumatic Fever and Articular Rheumatism, — The Streptococcus rheumaticus is accepted, in many quarters, to be the causative organism. The results of Buchanan in the acute stage of these affections and those of Veitch in the chronic form, where entirely satisfactory results were obtained in 50 per cent, of cases treated, are worthy of consideration, but as yet have not been duplicated by confirmatory in- vestigation. Malta Fever. — The M, melitensis is the offending bacterium. Although the authors have had no ex- perience, it is claimed on good authority that small doses in the acute stage and larger dosage in the SOO APPLIED IMMUNOLOGY chronic stage have been followed by undoubted benefit in the treatment of this disease. Cerebrospinal Meningitis, — It is probable that the value of meningo-bacterin is to prevention as anti- meningitis serum is to cure. As a result of the im- munization of over 10,000 persons, none receiving full immunizing dosage developed epidemic spinal- meningitis. Albright states that " meningo-bacterin is of as much value as a prophylactic in meningitis as typho-bacterin is in typhoid fever." For purposes of protection, three doses of 500,000,- 000, 1,000,000,000 and 1,000,000,000 meningococci respectively are administered at intervals of a week or ten days. Hydro phobia, — Although the exact nature of the virus of rabies has not been definitely determined, antirabic inoculation as suggested by Pasteur has been extensively practiced and its value universally recognized. In a curative capacity rabies vaccine seems to be ineffective. After immediate and thorough cauterization of the wound, inflicted b}^ an animal having or suspected of having rabies, the individual should waste no time in undertaking the " Pasteur Treatment." In the mean- time, the animal should not be killed, but securely confined and observed; if rabic, it will die in a few days manifesting symptoms of the disease. If the BACTERIAL INOCULATION 301 animal has been killed, as is commonly the case, the head and attached neck should be sent to a State or municipal laboratory for examination for the char- acteristic " negri bodies." Antirabic inoculation consists of a series of daily subcutaneous injections of virus prepared by emulsi- fying the specially dried spinal cord of rabbits, dead of rabies from a fixed virus. Rabies vaccine is best pre- pared in a laboratory particularly devoted to that pur- pose. A number of pharmaceutical firms and rabies institutes, to-day, on request, make daily deliveries in caloris bottles of the amount for administration, making it unnecessary for the patient to patronize an " institute " and rendering it perfectly feasible for the family physician to conduct the treatment at home. All that the practitioner is obliged to do is to note the age of the patient, the date and hour of the bite, its location and extent, if possible whether or not the animal surely had rabies, and furnish these data to the expert or firm about to produce the vaccine. In gen- eral the " Pasteur Treatment " comprises twenty-five inoculations administered over a period of three weeks. On the first day three inoculations are given four hours apart, on the second and third days two in- jections are made at intervals of six hours; on the fourth and succeeding days only one inoculation is administered. 302 APPLIED IMMUNOLOGY Allusion, however, should be made to the more recent work of Semple and Harris, who have prepared efficient vaccines from the medullas of rabbits, which will keep for long periods and are constantly ready for use, thereby obviating the elaborate methods of Pasteur. Harris in particular has reported on the in- transpinal injection in animals of 0.1 milligramme of powder obtained from the pulverized frozen brain and cord of animals killed by a fixed virus. This prepara- tion is dried in vacuo over sulphuric acid at a low tem- perature. It is claimed to produce immunity in a few hours or days, instead of weeks, and can be preserved in sealed tubes indefinitely. Further investigation is necessary to determine whether or not Harris' preparation is to supplant the more familiar " Pasteur Treatment." Malignant Neoplasmata All attempts to produce an efficient tumor extract or emulsion for the treatment of carcinoma and sar- coma have proved futile. A few years ago Doyen proclaimed the specificity of the Micrococcus neo- formans in the etiology of cancer. This organism is midoubtedly an accidental invader, and to the best of our knowledge no case of carcinoma has been or will be cured by the agency of neoformans bacterin. Coley's treatment of sarcoma is worthy of mention. BACTERIAL INOCULATION 303 Coley's fluid, so-called, is a mixed bacterin of the streptococcus of erysipelas and the Bacillus prodigi- osus. It is indicated (1) in all cases of inoperable sarcoma, excepting the melanotic type; (2) for two or three weeks even in operable cases, if there be a chance of saving a limb, that is when the giant-celled type of tumor exists; (3) as a prophylactic against recurrence after operation, and (4) post-operatively, even against carcinomatous recurrence. Apparently, a large number of properly selected cases have been cured by administering daily inoculations, starting with one-quarter minim in the gluteal or pectoral region, and increasing the size of the dose one-quarter minim daily until a reaction is evoked, that is a tem- perature of 102° to 104° F. The dosage must not be increased and may have to be diminished on the super vention of reaction. It is recommended that the inocu- lations be made subsequently in the tumor if the pa- tient be not too susceptible. The initial tumor in- jection should be only one-quarter of the previous inoculation into the gluteal or pectoral region. Coley states that carcinoma is not influenced by the treat- ment. In the treatment of 430 cases of inoperable sarcoma he reports 47 cases of complete cure, in 28 of which there was no recurrence for three to fifteen years. He asserts that as a prophylactic after opera- 304 APPLIED IMMUNOLOGY tion the use of the bacterial toxins has reduced the percentage of recurrence from 75 to less than 25 per cent. Yeast and Sour Milk Brewer's yeast {Saccliaromyces cerevisice) has been a popular remedy in the treatment of furuncu- losis. In many cases it appears to be without effect and to-day is being supplanted by staphylococco-bac- terins. The employment of yeast in tuberculosis and cancer has been a failure. MetchnikofF observed that the peasants in certain parts of Bulgaria, whose staple diet consisted in the consumption of peculiar soured milks, lived to an advanced old age. A peculiar lactic acid bacillus, the Bacillus lactis Bulgaricus or Bacillus of Massol, was shown to be the effective organism. Accordingly, tne ingestion of sour milk ha§ become a world-wide practice in the hope of avoiding senility, correcting putrefactive intestinal processes and a host of other affections, as infantile diarrhoea and enterocolitis, intestinal indigestion and auto-intoxication, diabetes, rheumatism, gout, arteriosclerosis, etc. Obviously, the results have not measured up to expectations, although benefit seems to have attended the use of artificially prepared soured milk in many cases. There are many tablets on the market for imme- diate ingestion or for making buttermilk, purporting BACTERIAL INOCULATION 305 to fulfil MetchnikofF's assertion, but none of these equal in therapeutic value the living liquid cultures of the B. Bulgaricus. These cultures are best prepared in tubes as is done by certain pharmaceutical houses. The content of one tube is poured into three table- spoonfuls of sweetened water and drunk, or it may be poured into a glass or half a glass of milk and drunk immediately. Fresh cultures should always be ob- tained and stored in a refrigerator to maintain activity. They may be taken indefinitely without harmful effect. 20 APPENDIX PART A SERUM TREATMENT OF HEMORRHAGE NORMAL FRESH SERUM— PRECIPITATED HORSE SERUM- TRANSFUSION OF BLOOD Normal Fresh Serum The most frequent cause of persistent hemorrhage from small vessels is a defect in the coagulation of the blood. Without going into the details of the theory of coagulation of the blood, it is now believed that delayed coagulation is generally due to a deficiency of thrombin or fibrin ferment in the blood-serum. In cases of pathological hemorrhage this deficiency may be supplied by the administration of normal blood-serum, and obstinate and persistent hemorrhage has frequently been checked by this means after all other agencies had failed. This is now recognized as the best form of treat- ment for persistent capillary hemorrhage whether inherited or acquired, such as that due to haemophilia, hemorrhage of the new-born, puerperal hemorrhages, pulmonary, intestinal, nasal, and renal hemorrhage, and traumatic hemorrhage. While theoretically human serum should be more effective in treatment, yet Clowes and Busch (N. Y, Med. Jour.y Jan. 4, 1913) have shown that, practically, horse serum answers the purpose just as well, and is more available. In emergencies, when normal horse serum cannot be quickly obtained, diph- theria antitoxin may be employed with good results. Administration and Dosage. — The serum is usually ad- ministered hypodermically, but may also be given intraven- 306 APPENDIX 307 ously or by mouth. The dose depends upon the severity and cause of the bleeding, age of the patient, etc. The average adult dose is SO c.c. hypodermically or 10 c.c. intravenously. If necessary, a larger dose, up to 50 c.c, may be given in two or three days, and this may be succeeded by 100 c.c. a few days later. There is no hard and fast rule as to the size and repetition of doses. One must be guided entirely by the effects. Large doses can be given without injury. The dangers of anaphylaxis may be avoided by repeating the injections at intervals of less than 10 days. In children it is well to begin with a dose of not more than 10 c.c. Normal horse serum may be applied locally for the control of hemorrhage from wounds. It may be injected through a ureteral catheter in cases of renal hemorrhage. Precipitated Sebum Clowes and Busch (loc. cit,) first suggested that precipi- tated horse serum be employed in place of the less convenient fresh serum. To quote from their conclusions : " Blood serum precipitated by means of a suitable mixture of acetone and ether is fully as effective as fresh serum, if not sup- x'ior to it. Precipitated serum is freely soluble and possesses the advantage over fresh serum of being sterile, always available, and retaining indefinitely its capacity to stimulate coagu- lability of the blood." Precipitated horse serum may now be obtained in the form of sterile powder under the name of " Coagulose." This is easily dissolved in sterile water, and is then ready for injection. The powder itself may also be applied to wounds and bleeding surfaces. This powder retains its coagulating properties almost indefinitely, while fresh serum loses its effectiveness in a short time. 308 APPLIED IMMUNOLOGY Transfusion of Blood There are many conditions under which transfusion of blood from one individual to another is of great value. Among the commonest of these are: Loss of blood follow- ing operations and accidents, severe ansmia from various causes, hemorrhage in typhoid fever, gastric hemorrhage, hemorrhage of the new-born, and postpartum hemorrhage. The introduction of new blood fulfils three functions : ( 1 ) It stops the hemorrhage; (2) replaces lost blood; (3) assists in overcoming any infection present by supplying fresh complement and antibodies. In transfusion the person yielding the blood is known as the donor, wliile the person receiving it is known as the recipient. In selecting a donor, it is important that his blood be compatible with that of the recipient. Incompatibility of the two bloods may be manifested in haemolysis or agglu- tination of one case by the other, producing grave symptoms or even fatal results. Haemolysis of the blood of either the donor or the recipient in transfusion is a pathological phe- nomenon and causes haemogiobinuria which may be fatal. Th*. 'e is evidence that cases in which haemolysis occurs in vitro will also show haemolysis in vivo, and a prehminary test of the bloods is therefore important in the selection of a donor. It has not been proved that agglutination of the red blood-cells of the donor by the serum of the recipient, or vice versa, gives rise to serious results, and this is therefore not regarded as a contra-indication to the transfusion. In case, however, there should be a considerable range of selection of donors, one should be selected if possible whose blood is not agglutinative. In every case where practicable, the blood of the donor APPENDIX 309 should be subjected to the Wassermann test, to avoid the introduction of syphilitic infection into the recipient. Simple clinical tests for haemolysis and agglutination have been described by Epstein and Ottenberg (Arch. Int. Med., May, 1909). They have devised a technic in which only very small quantities of blood are required, and which is easy of performance. The blood may be obtained by punct- uring the finger as in the case of the Wassermann test. One or two c.c. of blood are allowed to drop into a tube containing an excess of 1 per cent, sodium citrate in normal salt solu- tion. This is centrifuged to wash the red cells, which can be made up to any desired percentage. Another cubic centimetre of the blood is collected, allowed to coagulate, and the serum to separate. In studying haemolysis, the mixture of serum of the donor and red cells of the recipient, and vice versa, must be made within 12 or at most 24 hours of collecting the blood. One volume of corpuscle suspension and one or more volumes of serum are drawn up into a Wright capillary pipette, thoroughly mixed, and incubated in an upright position for two hours. At the end of this time the occurrence of haemolysis can be easily noted if it has taken place. It is found that a near blood-relative (sister or brother) of the patient is the most satisfactory donor, with the smallest chances of untoward results. A robust person should of course be selected if possible. Many methods have been devised for the transmission of blood from one person to another. Some of these are direct, i.e., by the anastomosis of a vessel of the donor with that of the recipient. Other methods are indirect, i.e., by collec- tion of blood from the donor in a suitable vessel, and sub- sequent introduction of it into the recipient. Brewer and 310 APPLIED IMMUNOLOGY others have devised paraffin-lined glass tubes for the passage of blood from the vessel of the donor to that of the recipient. Crile was one of the first to employ the direct method of transfusion extensively, and he unites the radial artery of the donor to a superficial vein in the arm of the recipient by means of a special clamp {Keen's Surgery, Vol. V, p. 616). Numerous other instruments have been recommended for this purpose, among others, those of Landon {Jour, A, M. A,, Aug. 16, 1913) and McGrath {Jour. A. M, A,, Jan. 3, 1914). The difficulties of blood-vessel anastomosis have led many surgeons to devise various forms of apparatus for removing blood from the donor, preventing it from clotting, and subsequently introducing it into the vein of the recipient. An advantage of indirect transfusion is that the amount of blood utilized can be exactly measured. Kimpton and Brown {Jour, A, M, A., July 12, 1913) have devised a glass cylinder lined with paraffin, into which they withdraw the blood, which is considerably delayed in clotting by the paraffin. Probably the simplest method of indirect transfusion is that recommended by Dorrance {Penna, Med, Jour., Sept., 1914). The blood is removed from a vein of the donor by means of a 50 c.c. all-glass syringe, in which has been previously placed 10 c.c. of a 10 per cent, solution of sodium citrate to prevent clotting. After withdrawal of a syringeful of blood, the syringe is detached, the needle re- maining in the vein of the donor in case more blood is re- quired, the lumen being closed with a stilette. The blood mixed with sodium citrate solution is then injected through a needle similarly introduced into a vein of the recipient. Dorrance states that with experience this operation can be APPENDIX 311 performed so rapidly that no fluid to prevent coagulation need be added. Two technical procedures can be carried out to guide the operator in the quantity of blood to be transfused. One of these is the estimation of the haemoglobin percentage of the blood of the recipient from time to time during the operation, and the other is noting the coagulation time of the blood. Many methods have been employed for the determination of the coagulation time, that of Dorrance {Am. Jour. Med. Sc, Oct., 1913) being accurate and practical, PART B ORGANOTHERAPY THYROID GLAND— ADRENAL GLAND— PITUITARY BODY- OVARY— CORPUS LUTEUM— THYMUS GLAND It has been definitely established that the so-called '' duct- less glands " furnish secretions that have important func- tions in the development and metabolism of the body tissues, some of them being essential to life. In the case of certain of these glands, the function of their secretion is known with some degree of accuracy. In the case of others, our knowl- edge is indefinite or unknown. It is proposed here to give brief mention to the various derivatives of the ductless glands, with indications for the therapeutic use of those which have proved of value. Thyroid Gland For many years it has been well known that atrophy or removal of the thyroid gland causes marked disturbances of nutrition, producing characteristic symptoms grouped under the names cretinism (congenital absence of thyroid gland) and myxoedema. This disturbance is believed to be due to defective tissue oxidation, depending upon the absence from the circulation of the internal secretion of the gland. It has been shown that the thyroid tissue contains more iodine than any other tissue of the body, and that its activity is directly proportional to the amount of iodine present. This iodine is present in the form of iodized proteid (iodo- thyrin, thyroprotein, etc.). Abnormal activity of the gland- secreting substance produces the symptoms found in the acute stage of exophthalmic goitre or Graves's disease. For therapeutic purposes sheep's thyroid is employed. 312 APPENDIX 318 Extract of thyroid gland tissue is indicated in cases of undeveloped thyroid (cretinism) and in all conditions where the normal activity of the gland is impaired (myxoedema following atrophy from disease or loss of the gland by operation). The most brilliant results in the whole domain of organotherapy have been seen in thyroid extract feed- ing in cretinism. In these cases it is customary to begin with a dose of 5 grains of the crude extract three times a day in capsule form. This may be gradually increased accord- ing to the effects produced. Thyroid extract has also been employed with success in a great number of other conditions in which defective oxidation is supposed to be a factor, especially obesity, certain skin diseases, and joint affections. It may be used sometimes with good results in the second or atrophic stage of exophthalmic goitre, where there is a functional insufficiency of the thyroid gland, but never in the acute or early stage. Numerous attempts have been made to isolate the active principle of the gland in a more or less pure form, leaving behind the toxic products. Beebe has succeeded in obtaining a thyroprotein or concentrated extract of the thyroid gland which is standardized to con- tain 0.33 per cent, of iodine. By the use of this standardized product the dosage can be more accurately controlled than in the case of the crude extract. The principal symptoms that the therapeutic limit in thyroid administration is being approached are rapidity of the pulse, overactivity of the sweat glands, and marked loss of weight. Adrenal GiiAND The functional activity of the adrenal glands is due to a substance known under various names, such as adrenalin, suprarenin, epinephrin, etc. This substance is a heart stimulant and powerful vasoconstrictor. For therapeutic pur- 314 APPLIED IMMUNOLOGY poses it is obtained from the ox and sheep, and is also pre- pared synthetically. It is questionable whether the sjTithetic product is superior to the natural organ extract. Adrenalin chloride is the preparation usually employed, and is generally supplied in a 1-1000 solution. It may also be obtained in powder or tablet form. Adrenalin, on account of its vaso- constrictor effects, is chiefly employed to control hemor- rhage from small vessels, and is useful as a topical applica- tion in the treatment of various nasal conditions, such as epistaxis, conditions requiring shrinkage of the turbinates, hay fever, etc. The constricting effects of adrenalin are very transitory, and are usually followed by marked dilatation. Adrenalin is used extensively as an adjuvant to cocaine, novocaine, and other local anaesthetics. The effects of these drugs are considerably enhanced and prolonged by the vaso- constrictor effects of adrenahn. In local anaesthetic mixtures, adrenalin chloride is generally used in the strength of 1-10,000. The cardiac stimulant effects of adrenalin are made use of in the treatment of shock. In this condition it may be given intravenously in normal salt solution in the proportion of 1 drachm of adrenalin chloride to a pint of salt solution. Suprarenal gland feeding has been employed without benefit in Addison's disease, which is due to atrophy or disease of the adrenals. PiTuiTAEY Body Much knowledge of the pituitary gland or hypophysis and its secretions has been gained within the past few years. Experiments have shown that the anterior lobe of the pitui- tary body secretes a substance concerned in vital processes, and which is essential to life. The secretion of the posterior lobe or pars nervosa, while not essential to life, contains a APPENDIX 315 pressor substance, which has a marked action in maintaining continuously high blood-pressure by virtue of its production of peripheral vascular constriction and augmentation of the force of the heart-beat. The effects of this substance upon the circulation are more powerful and lasting than those of suprarenal extract. Pituitary extract in addition produces diuresis by a specific action on the renal epithelium, and is a powerful stimulant to involuntary muscle. Therapeutic Uses. — Extract of the posterior lobe of the pituitary body is used extensively as a stimulant to uterine contractions in the second stage of labor, and is superior to ergot and other oxytocics. It is very effective in cases of uterine inertia, often rendering unnecessary the use of low forceps, and hastens the course of labor in cases of moder- ately narrow pelvis. It checks any tendency to severe post- partum hemorrhage. The administration of pituitary ex- tract does not suffice to induce labor or abortion. The dose of the extract is 1 c.c. intramuscularly, and repeated in one hour if necessary. In addition to its uses in obstetrics, posterior lobe extract is administered in various conditions. It is used by Cohen as a cardiovascular support in pneumonia. He employs it hypodermically in doses of 1 c.c. every three or four hours. Pituitary extract is said to be useful in preventing abdominal distention due to temporary paralysis of the bowel after abdominal operations, in surgical shock, and various other conditions. Contra-indications. — Pituitary extract on account of its marked tendency to increase blood-pressure is to be used with great caution in myocarditis, arteriosclerosis and nephritis. In obstetrical cases it should not be employed in abnormally narrow pelvis, or in threatened rupture of the uterus. In cases with symptoms of glandular deficiency, such as acromegaly, etc., prolonged feeding with powdered extract 316 APPLIED IMMUNOLOGY of the whole pituitary gland is indicated, as in these cases probably both lobes of the gland are functionally insufficient. It is stated that moderate doses can be given for a long period without harmful effects. Ovary Ovarian substance for therapeutic use is a dry powder prepared from the entire fresh ovary of the hog. It is employed with some success in functional dysmenorrhoea, disturbances of the menopause, etc. In dysmenorrhoea, 2 to 4 grains may be given every four hours until the symptoms are relieved. COEPUS LUTEUM The corpus luteum of the ovary produces an internal secretion whose function appears to be to stimulate the menstrual flow and also to sensitize the uterine mucosa for placental formation. Powdered corpus luteum has been extensively used in various forms of dysmenorrhoea, neurasthenia, functional disorders associated with the natural or artificial menopause, and functional amenorrhoea. It has been found useful in vomiting of pregnancy. The average dose of corpus luteum is five grains of the dried powder taken three times daily, during meals. Thymus Gland The thymus gland is a mass of lymphoid tissue situated in the upper anterior part of the thorax. It is well developed at birth, and then under normal conditions gradually dis- appears. Little is known of the function of the thymus, but it is believed to be concerned in growth and development and to be closely associated in function with the thyroid. APPENDIX 317 Thymus gland has been used empirically in a number of conditions, among which may be mentioned exophthalmic goitre, rickets, tuberculosis, haemophilia, marasmus, etc., with varying success. Calf thymus is supplied in desiccated form, and is employed in doses of 2 to 4« grains three times daily. In addition to those mentioned, preparations from several other organs of animals have been used for therapeutic pur- poses, such as mammary gland, testicle, parathyroid, parotid gland, spleen, etc., the results of which have not been of sufficient importance to warrant further consideration at this time. PARTC CHEMOTHERAPY ADMINISTRATION OF SALVARSAN AND NEOSALVARSAN, INTRAVENOUSLY, INTRAMUSCULARLY AND INTRA- SPINALLY— AUTOSALVARSANIZED AND ARTIFICIALLY SALVARSANIZED SERUM The relationship existing between the Wassermann reac- tion and the treatment of syphilis is so closely interwoven as to warrant a chapter on the subject of chemotherapy in a treatise on serology. It is not our purpose to present a de- tailed consideration of chemotherapeutics, but simply to refer to this recent branch of scientific medicine, particularly with respect to the administration of salvarsan and neosal- varsan. It is generally conceded, all things being equal, that im- munotherapy, theoretically, is superior and should take pre- cedence to chemotherapy. The reason for this is at once apparent, due to the fact that the former is parasitotropic and not at the same time organotropic. Unfortunately bi- ologic therapeusis has not been applicable to syphilis, con- sequently the treatment of this disease devolved upon drugs or chemicals, some of which, in spite of the time required to effect cure, have come to be known as " specific," for example mercury. HisTOEY OF Salvarsan and Neosalvarsan Ehrlich, attracted by the alleged value of atoxyl in trypanosomiasis, and mindful of the possibility of increas- ing the antiseptic effect of drugs, at the same time diminish- 318 APPENDIX 319 ing the toxic, by the introduction of the halogen group into the benzine ring, conceived the idea that arsenic in certain combinations could be so compounded that its administra- tion in a sufficiently large dose would not be injurious or toxic to the organism and would at the same time destroy all spirochsetse, sterilizing the system. Repeated trials en- abled Ehrlich and his collaborators to produce a synthetic drug, No. 606 in the experimental series, later styled " sal- varsan," the use of which was claimed to be a " therapia sterilisans magna " for the cure of syphilis. Subsequently, further refinement led to the production of neosalvarsan or No. 914, apparently less toxic but likewise less efficient. Be it understood that the treatment of syphilis by arsen- ical preparations is not by any means a new idea. Syph- ilologists for decades have recognized the benefit to patients when arsenic supplemented or alternated mercury and iodine, but owing to the toxicity of the trioxide of arsenic, it has been necessary to administer it in minimal dosage. Consequently, during recent years other forms of arsenic, less toxic and of higher drug content, have been synthesized, recommended and utilized. Among these arsenical preparations may be men- tioned the arylarsonates (soamin and orsudan), atoxyl, arsacetin, arsenophenylglycin paramidophenylarsenoxide and sodium cacodylate. Some of these have fallen into almost complete disrepute owing to gastro-intestinal disturbances, nephritis and neurological manifestations, including blind- ness, occasioned by their administration, and all have failed to measure up to the virtue of salvarsan and neosalvarsan. Valuable and important as Ehrlich's discovery has already proved itself to be in the treatment of syphilis, ad- mittedly the sheet-anchor in the pharmacology of that dis- ease, it is extremely doubtful if salvarsan will prove to be 320 APPLIED IMMUNOLOGY the universal, omnipotent, all-sufficient panacea for lues originally claimed by its advocates, although the authors have repeatedly observed, in the primary stage of the disease before the advent of a positive Wassermann, a single inten- sive dose of salvarsan suffice to effect a cure, thereby ful- filling the dictum of Ehrlich. Possibly the cure not attained by a single dose may be accomplished by repeated injections or a combination with mercury and iodine. This much only is assured at present, that in the great majority of patients the immediate effect of salvarsan properly administered is a symptomatic cure; concerning the remote results, addi- tional time must elapse before definite conclusions can be drawn. Chemico-physical Properties. — The Ehrlich-Bertheim- Hata arsenical preparation No. 606, synthetically known as dioxy-diamido-arseno-benzene-dihydrochloride, may be repre- sented by the following structural formula : HO. ,0H ^CeHa — As = As - Ceiiz<^ HCINh/ ^NHaHCI It is commercially known in the market at present as " sal- varsan," and appears as a light yellowish powder, markedly acid in reaction and very unstable on exposure to the atmos- phere. For these reasons the drug must be neutralized and prepared in solution strictly fresh each time before its ad- ministration. Ehrlich observed that formaldehyde-sulphoxylates of so- dium possess the property of restraining for a time the auto- oxidation of solutions of dioxy-diamido-arseno-benzene, and by the interaction of the two evolved dioxy-diamido-arseno- benzene-monomethane-sulphinate of sodium or No. 914, com- APPENDIX 321 mercially known as " neosalvarsan." It may be represented by the following structural formula: As As As NH, I 1 = 11 NH, + HO.CHASONa = NH, | | = As I NaCHjO.SONa-f HjO OH ( Neosalvarsan appears as an orange-yellow powder of pecu- liar odor, dissolving very easily in cold water with completely neutral reaction. Neosalvarsan is even more unstable than salvarsan, and although slightly less trouble to prepare for administration, requires even greater precaution as to freshness and tem- perature of solution, owing to its greater proclivity to oxidi- zation and the formation of products of a high degree of toxicity. Unfortunately, its ease of solution in water, at once in a neutral state ready for administration, and better toleration by patients, does not outweigh the greater effec- tiveness and spirochaeticidal power of salvarsan, the injec- tion of which with proper care and technic is attended with a minimum degree of reaction. Indications. — Salvarsan or neosalvarsan is indicated in all stages of syphilis, and should be the first thought in treat- ment, provided no contra-indications exist. Indeed, in the early primary stage cure may often be obtained by the ad- ministration of salvarsan alone, particularly before the ap- pearance of a positive Wassermann reaction. It seems es- pecially effective in those cases where mercury and iodine have failed. In cerebrospinal syphilis the effects are vari- able. For instance in gummatous meningitis, whether diffuse 21 322 APPLIED IMMUNOLOGY or localized, with expressions as epilepsy, monoplegia, etc., remarkable results are observed. In tabes dorsalis, partic- ularly if the intravenous administration of the drug be sup- plemented with intraspinal injection of autosalvarsanized or artificially salvarsanized serum, there is reasonable hope of arresting the disease if attacked in its infancy. In paresis and encephalitis the authors have never observed noteworthy benefit, the diseases exhibiting continuous retrogression. The drug is of great value in hereditary lues and in pregnant syphilitics. The provocative employment of salvarsan and neosalvar- san is a matter of considerable importance. The arsenical preparations similar to mercury may be utilized to provoke a positive Wassermann reaction in a syphilitic patient other- wise exhibiting a negative serological reaction. Thus in patients who have previously received antisyphilitic treat- ment, or in whom the clinical evidence for syphilis is strong and the ordinary Wassermann reaction has resulted nega- tively, the so-called provocative dose of salvarsan, neosalvar- san or mercury should be employed, and the blood-serum again tested at the end of twenty-four to forty-eight hours. It has been our practice to use 0.1 gramme of salvarsan or 0.15 gramme of neosalvarsan, administered intravenously, for this purpose. The explanations offered for the employ- ment of arsenical and mercurial preparations in this role are that the positive serological reaction is due to the liberation of endotoxins from the killed spiroch^etse or to the stimula- tion of the latent spirochastse by a dose of the drug in- sufficient to effect a complete destruction. The phenomenon or biochemical reaction is doubtless closely related to the Herxheimer reaction. In non- syphilitic diseases, salvarsan enjoys an extensive employment. Indeed in relapsing fever, tertian malaria, APPENDIX 323 framboesia (yaws or pian), filariasis and Vincent's angina, salvarsan appears to be equally as specific as in syphilis. It may be emplo3'ed in nervous disorders in which arsenical medication is indicated and exerts either a beneficial or cura- tive effect, according to Best, in acanthosis nigricans, ulcus tropicum or phagedoenicum, variola, verrucae planae, Syden- ham's chorea, scurvy, dermatitis herpetiformis, quartan and tropical malaria. Thus in many diseases the tonic, stimula- tive and alterative action of arsenic is linked with the germi- cidal effect of the drug. Good or indifferent results have been reported in Aleppo boil (Oriental sore), ansemia, kera- tosis follicularis, leprosy, lichen planus, lupus vulgaris, my- cosis fungoides, pellagra, pityriasis rubra, tuberculosis and experimental tick fever. In chancroid, bilharziasis, Hodgkin's disease, psoriasis, scarlet-fever, trichinosis, sarcoma, carcinoma and trypano- somiasis, the drug appears to be without appreciable effect. In explanation of the last it is alleged that the trypanosomes are more susceptible of being rendered arsenic-fast than are the spirochaetse. In veterinary medicine salvarsan has rendered signal ser- vice and a specific effect, particularly in pleuropneumonia of horses and African glanders (lymphangitis epizootica). Acquired Resistance of Spirochcetce to Salvarsan. — It is generally known that trypanosomes possess the power of adapting themselves to circumstances, for instance preser- vation against injurious influences. In other words, they may become immune to their own antibodies or chemicals di- rected against them, as arsenical preparations. This resis- tive property then becomes a characteristic of the organism and may be transmitted from generation to generation. Al- though this characteristic has never been demonstrated to obtain for the spirochaeta pallida, it is presumed from anal- S24 APPLIED IMMUNOLOGY ogy and clinical experience that spirochaetae pallida may also exhibit drug-fast properties. Thus are explained the failures of mercury to influence syphilis after the first few months in many cases. In like manner, the spirochaetae may become tolerant or resistant to the influence of arsenic when ad- ministered in salvarsan and neosalvarsan. It would appear that early, small or sublethal doses of these drugs may be responsible for the production of arsenic-fast or immune spirochaetae. Consequently, the inference is strong that the treatment of syphilis should be intensive from the start, thereby avoiding relapses, significant of immunity on the part of the spirochaetag. This is best accomplished by repeated full-sized intravenous injections of salvarsan, the number being controlled by the stage of the disease and the Wasser- mann reaction. Contra-indications and Precautions. — Salvarsan is con- tra-indicated in advanced degenerative diseases of the central nervous system, in severe non-syphilitic retinal and optic disease, in marked disturbances of the cardiovascular sys- tem, as acute endocarditis, myocarditis, with or without nephritis, extensive degeneration of the blood-vessels, and angina pectoris, in any form of non-luetic nephritis, in dia- betes, in aneurism independent of lues, in pronounced foetid bronchitis and pulmonary tuberculosis, and in persons mani- festing an idiosyncrasy for arsenic. Chronic valvular heart disease, syphilitic endarteritis, aneurism and endocarditis are not contra-indications. In cases of malnutrition, ca- chexia and infantile congenital syphilis great care as to dosage must be exercised. In incipient tabes, early paralysis and epilepsy of syphilitic origin, salvarsan can be employed successfully only when administered early. Certain precautions are imperative and must be observed APPENDIX 325 for the proper, safe and best administration of salvarsan and neosalvarsan. They are as follows : 1. An exact technic. 2. Observance of the integrity of the hermetically sealed ampoule and the normal physical characteristics of the con- tained drug. 3. Immediate and fresh preparation of the solution just prior to administration, avoiding improper degree of tem- perature. 4. Selection of the appropriate dose for the individual case. 5. Previous examination of the patient to receive the drug, particularly investigation of the renal, cardiovascular and nervous systems. 6. Assurance that any previous administration of mer- cury has not caused kidney irritation. Wechselmann, who has given over 30,000 doses of salvarsan, cautions against the combined use of salvarsan and heavy mercurial treatment, and protests vigorously against employment of salvarsan after a course of mercurial treatment. 7. Careful notation after injection of a patient of reac- tionary phenomena, with respect to repeated injections, both as to time and dose ; the most important consideration is ob- viously the possibility of kidney irritation. 8. Caution as to the resumption of vocational activities by the patient too soon after the administration of salvar- san and neosalvarsan. Dosage. — Michaelis has stated the average dose of salvar- san to be 0.01 gramme to every kilogramme of body weight. Salvarsan is marketed in original packages of hermetically sealed ampoules containing 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1.0, 2.0 and 3.0 grammes ; of these the average adult dose is 0.6 gramme, which at times may be advantageously dimin- 326 APPLIED IMMUNOLOGY ished by 0.1 gramme for women. In children the dosage should be proportionate to the age. Syphilitic new-born infants should receive from 0.02 to 0.1 gramme of the drug. Neosalvarsan, although less toxic, is unfortunately also less effective than its predecessor, salvarsan. The average adult dose is 0.9 gramme, corresponding in arsenical con- tent to 0.6 gramme of salvarsan. Commercially it is marketed in ampoules with the following proportionate dosage: Dose Dose 0.15 gramme neosalvarsan = 0.1 gramme salvarsan 0.3 K = 0.2 te 0.45 ii = 0.3 t( 0.6 ti = 0.4 " 0.75 (C = 0.5 (C 0.9 (C =z 0.6 (I 1.5 grammes fC = 1.0 (( 3.0 " = 2.0 grammes (( 4.5 (( = 3.0 a The last three doses are for veterinary purposes only and have no place in human therapeutics. The repetition of the dose is dependent upon the method of administration (intravenous or intramuscular), the or- ganic condition of the patient and his susceptibility to reac- tionary phenomena. Usually when injected intravenously, the doses are repeated in five to ten days in the absence of reactionary contra-indications. In some cases weekly ad- ministration of 0.6 gramme of salvarsan intravenously for several weeks has been unproductive of harmful effect. The policy should be, particularly in early syphilis, to produce an intensive or specific effect by repeated intravenous injec- tions until a negative Wassermann reaction is obtained. It is seldom that less than three administrations are necessary APPENDIX 327 and often a dozen or more injections are required to render the Wassermann reaction negative. Obviously, with feeble or prostrated patients of those manifesting a cardiovascular or central nervous s^^stem lesion, the initial dose, at least, must be smaller than the average. When a tonic and alterative effect, in conjunction with specific action, is desired, full doses of the drugs at intervals of several weeks, may be administered intramuscularly, or, if merely the tonic, alterative effect of the drugs is desired, small and frequently repeated intramuscular injections may be desirable. An intramuscular method of administration is that by fractional doses. 0.1 to 0.2 gramme of salvarsan suspended in oil is injected every other day until a total of 1.2 grammes has been given to the patient. This mode of therapy applies more particularly to the treatment of non-syphilitic diseases. Small intramuscular injections, lack- ing sufficient remedial action, may invite relapses, conse- quently it is advisable either to precede or supplement the in- tramuscular injection by a full dose of the drug intra- venousl}^ In all stages of syphilis, ^'ith the possible excep- tion of the early primary, it is imperative to supplement sal- varsan with mercury or mixed treatment. Methods of Preparation and Administration. — Time and experience have evolved four methods -^ for the employment of salvarsan and neosalvarsan : the subcutaneous, the intra- muscular, the intravenous and the intraspinal or intra- or subdural. Following the successful original experiments on animals by Hata, Hoppe, in the Clinic of Professor Konrad Alt in Uchtspringe, on the suggestion of Ehrlich, in September, ^ The administration of arseno-benzol by mouth is unworthy of con- sideration. The same objection obtains respecting its use as a dusting powder for the chancre on account of cost if for no other reason. S28 APPLIED IMMUNOLOGY 1909, was the first to try salvarsan on the human subject. The preparation used was an alkaline solution of the disodium salt. Shortly afterward Michaelis, Wechselmann and Lange devised the method of neutral suspension injections and prac- ticed the same subcutaneously and intravenously in thou- sands of cases. Other notable methods that have been ad- vised are Junkemann's and Lesser's modification of Alt and Hoppe's alkaline method and Kromayer's suspension of sal- varsan in paraffin. About this time Schreiber, associated with Hoppe, described the method by intravenous administra- tion. Wechselmann appears to be the first to have employed salvarsan by intraspinal injection. Subsequently, Marie and Levaditi likewise injected neosalvarsan directly into the spinal canal. While the technic of direct intraspinal injec- tions of salvarsan and neosalvarsan was being perfected, even before the use of hypertonic solutions of neosalvarsan as practiced by Ravaut and Wile, Swift and Ellis recommended the intraspinous injection of auto-salvarsanized serum. This method is less irritating to the nervous system and less dangerous and consequently is the most popular form of sub- dural arsenic medication. Recently Fordyce has recom- mended adding minute quantities of salvarsan or neosalvar- san to the auto-salvarsanized serum prior to intraspinal injection. Subcutaneous Administration. — The subcutaneous admin- istration of salvarsan or neosalvarsan, either the injection of the full therapeutic dose at once or repeated injections of fractional doses over a period of time, is practically an obsolete method. This has been due to great and long per- sistence of pain, induration, non-absorption of the chemical and necrosis of the skin. Intramuscular Administration. — The intramuscular in- jection is superior to the subcutaneous inasmuch as the above APPENDIX 329 noted objections are not so marked, although not infre- quently encountered, subsequent pain and induration being the most objectionable. Occasionally the indurated area may undergo liquefactive necrosis, resulting in sinus for- mation. Owing to the fact that all the arsenic, detectable in ex- creta, is eliminated in three to six days after intravenous ad- ministration, while it persists for a much longer period fol- lowing intramuscular injection, it has been recommended and is practiced by some clinicians to supplement the intravenous in the course of a week by intramuscular injections. The rationale of this modus operandi is not without reason and is deserving of consideration. It can be explained on the as- sumption that by the intravenous injection the drug is at once conveyed to the remotest parts of the body, where it is needed to destroy the spirochsetse, thus producing a so-called " intensive " action in contradistinction to the " perma- nent " action when the drug is exhibited locally and an intra- muscular depot is established. The technic of intramuscular injections is somewhat vari- able. Three solutions have been extensively employed: (1) the alkaline solution, (2) the oily suspension and (3) the neutral suspension. Of these the alkaline solution method is to-day probably most commonly utilized. The preparation of the solution may be similar to that described under Intra- venous Administration, differing only in that for intramus- cular injection the drug is dissolved and injected in 20 c.c. of fluid, one-half the quantity being injected on either side.^ A common procedure for the preparation of the alkaline solu- * For the intramuscular injection of neosalvarsan a five per cent, solution is employed, since one gramme of the drug dissolved in twenty- two cubic centimetres of water makes an isotonic solution. Therefore, for each 0.15 gramme of neosalvarsan about three cubic centimetres of freshly distilled water should be added. 330 APPLIED IMMUNOLOGY tion is to triturate 0.6 gramme of salvarsan in a sterile mortar with twenty-three drops of fifteen per cent, sodium hydroxide solution, then to dilute with distilled water to the desired volume. More recently intramuscular injections have been exten- sively made with simple suspensions of salvarsan and neo- salvarsan in oils, 1 : 10 dilution, such as sesame, olive sweet almond and paraffin. Certain pharmaceutical labora- tories market the neutral, water-free solidified fat suspen- sions, liquid at body temperature, in sterile ampoules, requiring merely a syringe for their administration. The neutral suspension, at one time largely used, both in- tramuscularly and subcutaneously, at present enjoys a very restricted emplo3"ment. It is prepared by triturating care- fully 0.5 or 0.6 gramme of salvarsan with eight or ten drops of fifteen per cent, caustic soda solution in a sterile porcelain dish. To this is added at first drop by drop with constant trituration the required quantity of sterile water (5 to 10 c.c). The fine suspension thus produced is tested exact- issme with litmus paper for neutral reaction, and a drop of the soda solution or hydrochloric acid added in accord- ance with the reaction. The upper outer quadrant of the gluteal region is the most desirable locality for intramuscular injection, by virtue of comparative freedom from nerves and blood-vessels (Fig. 59). The vicinity of the sciatic nerve must be care- fully avoided. The injection should be given deeply and slowly, thereby obviating hemorrhage and rupture of the mus- cular tissue. The skin is conveniently disinfected at the site of injection by three to five per cent, tincture of iodine. After injection the fluid is distributed as widely as possible by careful massage, and the needle puncture sealed with col- lodion. In sensitive patients the area to be injected may be Fig. 59. — Site for deep intramuscular injection. The needle is plunged inward and downward at a point selected anywhere on the line C Z>, which indi<-ate.s a region in the upper outer quadrant of the buttock free of large ve.ssels and nerves. The line C D les two finger breadths below the iliac crest; the point C located on the vertical line -4 tf erected midway between the tuberosity of the ischium and the great trochanter of the femur. APPENDIX 331 ana?sthetized, preliminaril}^, by injecting through the needle, in situ, two cubic centimetres of a one per cent, or five cubic centimetres of a one-half per cent, novocain solution. Post- ing ectional pains or reactive painful infiltrations may be combated locally with hydrothcrapeutic measures, as the hot- water bottle, hot compresses, etc., and internally the adminis- tration of pyramidon has proved very effectual. Patients had best remain in bed for some time after the injection. Unfortunately, no patient who has ever received salvar- san or neosalvarsan intramuscularly desires it a second time by that method, and any patient who has had the drug ad- ministered both intravenously and intramuscularly invariably prefers the former because of the lesser pain. Certainly there are occasions when, by force of circumstances, it be- comes necessary or expedient to use the intramuscular method, but these exceptions must be few in number. Intravenous Administration. — The intravenous adminis- tration of salvarsan and neosalvarsan in dilute solution is the best method and the mode of therapy of the future. The consensus of opinion among those of widest experience is that salvarsan is more effective than neosalvarsan. A rou- tine procedure with the authors is to commence treatment with neosalvarsan, which, if well tolerated, is supplemented by salvarsan. We firmly believe in the intensive form of treatment, with proper observance of precautions and con- tra-indications, attempting thereby to eradicate the disease ; particularly is this true in the primary stage of syphilis, when the indication for the intravenous administration of arseno-benzol is just as acute as the scalpel in a well-defined case of appendicitis. In the early days of the chancre, es- pecially before a positive Wassermann is obtainable, it is possible to cure syphilis by one or more injections of sal- varsan alone; in the late primary usually, and throughout 332 APPLIED IMMUNOLOGY the secondary, latent, tertiary, nervous and hereditary forms of the disease, mercury or mixed treatment must supplement the administration of arseno-benzol. We seldom administer less than three intravenous injections, usually at intervals of a week. Then, after a respite of three weeks, the blood is tested by the Wassermann reaction. If the result still be positive, three more injections are given and so on until in a few cases a dozen or more doses have been administered. As soon as a negative Wassermann is obtained the patient is subjected to a vigorous course of mercurial or mixed treat- ment for three months to a year. Treatment is then sus- pended and the further conduct of the case may depend upon Wassermann reactions of the blood taken at three-month intervals, associated in certain cases with the Wassermann reaction and cytological examination of the spinal fluid. Although no absolute law may be promulgated, no case should be pronounced cured until consecutive negative Was- sermann tests are obtained for a period of at least two years after the suspension of all treatment. (See technic de- scribed in Chapter XII.) Preparation of Patient. — After determining the organic fitness of the candidate, the patient to receive an intravenous injection of salvarsan or neosalvarsan should receive a cathartic the night before the injection of the drug. He should take no food at the meal immediately preceding the introduction of the remedy, but may be allowed limited quan- tities of liquid. He should be placed in the supine position on a table, the arm (preferably the left in right-handed in- dividuals) slightly abducted, the forearm supinated and resting on a support, the elbow very slightly flexed, as shown in Fig. 60. While an assistant sterilizes the region of the elbow, with soap and water, alcohol and a solution of bichloride, the operator, always with the strictest observance Fig. 60— Position of patient for intravenous injection of salvarsan. Note the easily applied and released rubber dam tourniquet shown in the corner sketch; also the manner ot fixing the skin overlymg the vein with the thumb as the needle is introduced into the vein. ■'I^.^' r ^ M >^ ^ -^ -5 "3 a to ° c =3 „ APPENDIX 333 of the chemical and aseptic precautions, prepares the solution for intravenous administration. Preparation of Salvarsan Solution. — In Fig. 61 are shown the various reagents, solutions, glass vessels and ap- paratus useful for the preparation and administration of salvarsan intravenously. All apparatus, as described in the outfit (Fig. 62), here recommended, including thermometers, burette, etc., may be sterilized by boiling. The ampoule of the drug is best antisepticized by submersion in a cold solu- tion of bichloride or formalin, after noting that it is intact and its content in no way oxidized. The water and saline solution employed for solution and dilution of the salvarsan must be sterile and freshly distilled. Chemically pure sodium chloride must be used in making the saline solution. The common laboratory or commercial distilled water is usually not sterile. Clinical experience has demonstrated that reac- tions following the administration of salvarsan are more common when spring, tap or stale water is used than when freshly distilled water is the diluent. Whether or not this increased toxicity is due to the interaction of the endotoxins of the contained flora and arseno-benzol or is referable to other causes, it is advisable to employ freshly distilled water. This may be readily obtained in sufficient quantities by an apparatus (Fig. 63) capable of being installed in the physi- cian's office. Sterilization may then be assured or completed in an Arnold sterilizer for one-half hour. The use of sterile freshly distilled water and chemically pure salt solution and the preparation of the salvarsan solution immediately before intravenous injection are imperative conditions. Twenty to forty cubic centimetres of warm (110° to 120° F.) sterile freshly distilled water, (W, Fig. 61) are placed in the graduated glass stoppered cylinder or mixer (M) containing a dozen or more small glass balls. The 334 APPLIED IMMUNOLOGY neck of the ampoule (A) containing salvarsan (usually 0.6 gramme) is nicked with the small file accompanying the commercial package and the neck easily broken. The con- tent is emptied into the mixing cylinder and the substance en- tirely dissolved by shaking, producing a clear light-yellowish solution of a strongly acid reaction. In order to fit this solution for human administration it must be neutralized. Accordingly 1.14? cubic centimetres or approximately 2S minims of a freshly prepared fifteen per cent, solution of purified sodium hydroxide (C) are added. For this purpose a medicine dropper (D) is convenient. The following table, dependent upon the quantity of salvarsan utilized, may be of service : Salvarsan 16 per cent. Solution of Sodium Hydroxide 0.6 gramme requires 1.14 c.c. or approximately 23 to 24 minims. 0.5 0.95 " " 19 ' 20 0.4 0.76 " " 15 ' 16 0.3 0.57 " " 12 0.2 0.38 " " 8 0.1 0.19 " " 4 The addition of the caustic soda, which should be in one quantity and not added slowly drop by drop, produces a heavy whitish yellow precipitate, redissolved in excess on shaking; if necessary another drop or two of the caustic soda solution may be added. At this juncture the solution should be neutral and perfectly clear, resuming its original light yellowish coloration. It is advisable now to add just one more drop of the soda solution, which will render the solution very faintly alkaline. Slight turbidity or cloudiness of the solu- tion is an indication that insufficient sodium hydroxide has been added. If in doubt the reaction should be tested with litmus paper (L) and the neutrality, weak alkalinity or acidity of the solution definitely determined. If decidedly alkaline, a drop or two of the dilute hydrochloric acid (H) Fig. 64. — Illustrating method of eliminating air from tubing. The burette and needle are alternately elevated and lowered, and w.hen the tubing is entirely free of ail air bubbles, the two stop-cocks, one in the needle and the other in the burette, are turned off, leaving the tubing and needle filled with normal saline solution. Fig. 65. — Thomas' salvarsaii and neosalvarsan burette. The length of the rubber tubing between the needle and transfusion thermometer should not be more than eight inches, and that between the thermometer and burette proportionately increased in length. APPENDIX 335 must be added. The additions of caustic soda or hydro- chloric acid can best be controlled by taking them with pipettes or droppers (D) from the medicine glasses (G). The rubber tubing incorporating the transfusion thermom- eter (T) and fitted with the platinum-iridium needle (P) or the blunt infusion needle (N), rarely necessary, is adjusted to the burette, which with the tubing is thoroughly rinsed out and the tubing and needle filled with warm normal salt solution (S), the tubing being freed of air by alternately elevating and lowering the burette and needle, as shown in Fig. 64. The weakly alkaline solution of salvarsan is then filtered through sterile cotton contained in the funnel (F) into the graduated burette (Fig. 65). Sufficient warm (110° to 120° F.) sterile 0.5 per cent, saline solution made from freshly distilled water is then passed through the cotton filter, filling the burette to the 300 cubic centimetre mark.^ This is the proper dilution for 0.6 gramme salvarsan. The quantity is diminished by 50 cubic centimetres for each 0.1 gramme less of salvarsan. With the patient supine and the arm moderately abducted and forearm supinated, the region of the elbow is antisepti- cized with soap and water, alcohol and bichloride or a three * In view of the recommendation that the preferable diluting fluid for salvarsan is 0.5 per cent, saline, while for neosalvarsan it is 0.4 per cent, salt solution, the authors have found it practicable and con- venient from the standpoint of preparation of solutions to utilize two standard solutions — 0.9 per cent, chemically pure sodium chloride solu- tion and freshly distilled water. From these any desired strength saline solution can be readily prepared. For instance, in diluting the salvarsan solution, distilled water is added until the quantity in the burette reaches 135 cubic centimetres, then 0.9 per cent, salt solution is added until the amount equals 300 cubic centimetres. In the case of the full-sized dose of neosalvarsan, the dilution is made first with distilled water to the 85 cubic centimetre level, then 0.9 per cent, salt solution is added imtil the quantity in the burette stands at 150 cubic centimetres. 336 APPLIED IMMUNOLOGY per cent, tincture of iodine, and a tourniquet of rubber dam (R) is placed around the arm, sufficiently taut to render the superficial veins about the elbow prominent, care being exercised not to obliterate the radial pulse (Fig. 60). The median cephalic or basilic vein is preferable to a vein exactly in the cubital fossa, because, should inflammation in or about the vein supervene, the patient will experience less discomfort on flexion and extension of the elbow. The bu- rette containing the required quantity of salvarsan solution is given to an assistant to hold or it is suspended on a stand adapted to the purpose (Fig. 60). At this point it is usually possible to plunge the sharp pointed platino-iridium obliquely through the skin into the vein, holding the needle with the eye directed downward, at the same time fixing the skin in the axis of the vein about two inches distant from the site of puncture with the index finger or thumb of the hand not holding the needle. As the needle is slowly and steadily ad- vanced at an angle of about thirty degrees, always keeping it at the plane of the axis of the vein, it will usually be possible to see a slight dimpling of the skin overlying the vein at the point where the needle will pierce the vein. Just as the sen- sation, imparted to the fingers, of decreased resistance is experienced, as the tip of the needle enters the lumen of the vein, the skin dimple likewise disappears. Rarely, it will be necessary in the case of young individuals, some women and those exhibiting a thick layer of panniculus adiposis, when the veins are extremely small or obscured by fat, to infiltrate the skin with a few drops of a one per cent, novocain solution, and then make a cutaneous incision one-half to three-quarters of an inch in length, exposing the vein. The vein may or may not be lifted from its sheath, but in any event it is then possible to introduce the needle without difficulty, using preferably the sharp-pointed one or occasionally the well- APPENDIX 337 known dull-tipped intravenous variety (N, Fig. 61). If it be necessary to cut the skin, the wound must be closed by a suture. Great care should be exercised to see that the tip of the needle lies within the vein, because the escape of the salvar- san solution into the subcutaneous and perivascular tissues is productive of great pain, cellular infiltration and pro- longed induration and nullifies the advantage of the intra- venous over the intramuscular method of administration. For this reason, it is well to have the needle and rubber tubing filled with normal salt solution, which will not produce irritation should it be allowed to escape into the subcutaneous areolar tissue. If, by accident, a quantity of salvarsan solu- tion should be allowed to infiltrate the perivascular tissues, it is advisable to incise the overlying skin immediately, using an anaesthetic of novocain or eucaine, wash out the salvarsan with normal saline, and then to puncture the vein directly. So soon as the vein has been entered, the tourniquet is loosened, the cocks of the burette and needle turned on, and the solution of salvarsan permitted to flow very slowly from the burette. The temperature of the solution just before entering the vein can be read from the thermometer and should be blood heat. The introduction of the solution should not take less than 10 minutes. Just before the sal- varsan solution in the burette falls to the level of the stop- cock, it should be turned off, cr the rubber tubing tempo- rarily compressed and about twenty-five cubic centimetres more of salt solution filtered into the burette, when the com- pression is released or the stop-cock again turned on. This procedure permits of the patient receiving the full amount of salvarsan and at the same time suffices to wash the vein free of salvarsan, thereby avoiding the possibility of phle- 22 S38 APPLIED IMMUNOLOGY bitis, etc. A dry sterile gauze dressing following the removal of the needle completes the operation. A too rapid introduction of the solution naturally might cause dilatation of the right heart with its attendant re- sults. Too much care cannot be exercised to exclude air bubbles from the apparatus, although the danger from air embolism is not great — incomparable in comparison with the advent of air through the great veins of the neck, where a negative pressure exists — it is nevertheless a serious concern. Similarly, the filtration of all solution entering the vein is a matter of importance, lest solid particles of matter, as glass, acting as emboli, might exert a harmful effect. Throm- bosis is an accident alleged to have occurred. This must supervene only as a result of faulty technic or in the presence of an infection and emphasizes the necessity of thorough sterilization of all apparatus and material employed. It is assuredly more likely to ensue when the vein is exposed, due to atmospheric influence on the blood and to metabolic changes in the vein wall. In an experience covering several thousand administrations, it is a complication which we have never encountered. Preparation of Neosalvarsan Solution. — The solution of neosalvarsan is slightly less trouble to prepare than salvarsan, since when dissolved it is at once neutral and needs no addition of alkali to counteract the acidity. It is far more unstable than salvarsan, consequently must be used immediately after preparation. Moreover, the solution must never be heated. In fact, it is emphatically stipulated that the temperature of the freshly distilled water or salt solution must be that of the room, that is, 68° to 7^° F. This had best be determined by an appropriate thermometer (T, Fig. 61). Undue shaking is also to be avoided. The content of the full adult dose, 0.9 gramme, is dissolved APPENDIX 339 in 15 to 20 cubic centimetres of sterile freshly distilled water. This may be injected intravenously, using an appropriate syringe, or preferably it is filtered through cotton into the burette and diluted up to 150 cubic centimetres, using a 0.4 per cent, saline solution or freshly distilled water. In the case of neosalvarsan, since the quantity of solution is only one-half that of salvarsan, it is allowed to enter the vein in five minutes. In ail other respects the technic of administra- tion differs in no way from that described for salvarsan. Intraspinal Administration. — It was soon recognized after the advent of salvarsan that its administration intra- muscularly or intravenously in certain forms of syphilitic disease of the nervous system was not so effective as in other manifestations of the disease. This observation was confirmed by the Wassermann reaction, inasmuch as the reaction of the spinal fluid in certain neurological cases per- sisted positive long after the reaction of the blood-serum became negative. Moreover, it is a common observation to find in S3'philitic neurological patients a negative blood and a positive spinal fluid Wassermann or a weakly positive blood reaction and a medium or strongly positive spinal fluid result. Communication between the lateral ventricles of the brain and the subarachnoid spaces of the brain and spinal cord exists through the foramina of Magendie and Luschka. The nervous choroid plexuses and the velum interpositum, possibly assisted by the blood-vessels of the meninges, act as the chief dividing plane or filtration plant between the blood and the spinal fluid. Owing to the fact that the specific gravity of the blood is greater than that of the cerebrospinal fluid, also that the pressure of the cerebro- spinal fluid is greater than that of the intracranial venous blood, osmosis takes place in the direction of the venous blood. This fact may account for the failure of salvarsan, 340 APPLIED IMMUNOLOGY administered intravenously, to reach the spinal fluid in any considerable quantity. In any event, so far as the effective treatment of cerebrospinal lues, especially paresis and notably tabes dorsalis, is concerned, it prompted a number of investigators to apply both salvarsan and neosalvarsan di- rectly to the subarachnoid spaces by spinal puncture. Wechselmann,^ Marinesco,^ Marie and Levaditi ^ were prob- ably the first to employ this method. The practice was attended frequently with such marked reactions, and even fatalities, that it has already become obsolete. The modi- fications as suggested by Ravaut" and Wile ^ using hyper- tonic solutions of neosalvarsan are likewise not beyond re- proach and must be regarded as dangerous procedures, not to be recommended for the average case. Autosalvarsanized Serum, — Swift and Ellis ^ have per- fected and recommended the intraspinal injection of the patient's own salvarsanized serum, irrespective of the fact that it is impossible by the Marsh test to demonstrate the presence of arsenic in the serum of patients shortly after the intravenous injection of an adult dose of salvarsan, where- fore if present it must be in minute quantity, rendering the modus operandi of the apparently beneficial effect of this method of treatment difficult of comprehension. Possibly the antibodies in the serum itself may be a most important factor. It is unquestionably true that autosalvarsanized serum is much less irritating and dangerous than direct sub- dural injections of salvarsan or even hypertonic solutions of neosalvarsan. Consequently, this method is deservedly *Deutsch. med. Wochenschr., 1912, 38, 1446. = Zeitschr. fiir Phys. und Therap., 1913, 17, 194. « Bull, et Soc. med. d. hop., Paris, November 18, 1913. ^ Ann. de Med., 1914, 1, 49. "Jour. Am. Med. Assn., 1914, Ixii, 1165; ibid., 1914, Ixiii, 13T. » New York Med. Jour., July 13, 1912, 53. ■^^ i. h I \ A, Fig. 6G. — Showing position of patient for spinal puncture. The skin has been antisepticized with iodine tinr-ture and the needle introduced through the first inter- vertebral space above the level of the iliac crests. /^ / f* rt ^ \ r Fig. 67.— Lumbar puncture with Strauss needle, illustrating the method of intra- spinal injection by the gravity method. After introduction of needle, the obturator, O, is withdrawn to point shown in cut. This permits of the spinal fluid to rise in attached pipette, P, graduated in hundredths of a cubic centimetre, thereby permitting of the determination of the degree of intraspinal pressure. The desired amount of spinal fluid may then be collected in a test-tube held under the exit, M, by removing the metallic plug. The plug is then reinserted, the pressure noted and the obturator partially replaced. The graduated pipette or tube, P. is then disconnected from the needle at C and the rubber tubing, T", leading from the re.?ervoir containing the serum attached to the needle. The obturator is again withdrawn as far as it will go and the serum allowed to flow in. An assistant with his arm around the patient's neck, as illus- trated, and his fist in the epigastric region can do much to steady the patient and assist him to properly arch his spine. ^\ 't A APPENDIX 341 the most popular and best form of intraspinal treatment for syphilis thus far proposed. The technic, slightly modified from the original of Swift and Ellis, is as follows: The patient should receive the same preliminary preparation as for salvarsan or neosalvarsan, of which the former is preferable and more effective. Usually the full adult dose of salvarsan is administered intravenously. Prior to the injection of salvarsan it is assumed that suffi- cient spinal fluid has been obtained by lumbar puncture (Fig. 66) for the various tests necessary in the control of the number of intraspinal injections, namely, the Wasser- mann reaction, the lymphocytic count, Noguchi's butyric acid test for globulin or Nonne's phase reaction. One-half to one hour after the intravenous injection of salvarsan, about 40 cubic centimetres of blood are taken in centrifuge tubes from a vein of the opposite arm. This may be allowed to stand until the following day for the separation of the serum or, as is the practice of the authors, immediately centrifuged and IS cubic centimetres of the serum pipetted off and di- luted with 18 cubic centimetres of sterile normal salt solution. This forty per cent, dilution of serum is activated by heat- ing at 56° C. in a water-bath for one-half hour. On the occasions of the second and third treatments, the strength of the serum is commonly increased to 50 and 60 per cent, dilutions. The 30 cubic centimetres of diluted serum are then carefully and slowly injected by lumbar puncture after ap- proximately an equal amount of spinal fluid has been with- drawn. Theoretically, the intraspinal injection is best done by the gravity method (Fig. 67) ; practically, it can be and is properly accomplished by the faultless use of a syringe, injecting very slowly (Fig. 68). The patient may be per- mitted to lie on his side or sit with his back arched. Im- mediately after the injection, the patient must lie down and 342 APPLIED IMMUNOLOGY the foot of the bed be elevated 14 to 16 inches for four hours. The patient should continue in bed for two or three days. Repetition of the treatment depends upon the extent and duration of any reactions incurred. As a rule the intra- spinal injections are repeated at intervals of two weeks. The actual number of treatments should depend upon the analysis of the spinal fluid, the object being to obtain a negative Was- sermann reaction, a reduction in the number of lymphocytes to five per cubic millimetre, and a normal globulin content. Artificially Salvarsanized Serum, — Attempts have been made to salvarsanize serum in vitro, in the hope of increas- ing the arsenical content in view of intraspinal injections. The results have been disappointing and in the employment of neosalvarsan, notably in Los Angeles, disastrous. Recently Fordyce,^*^ adopting the technic of Ogihde, in which a known amount of salvarsan is added to human serum, recommends the following technic : " Fifty c.c. of blood are drawn into a centrifuge bottle and centrifuged twice. It is important to have the serum clear and free from fibrin and blood-cells. To obtain the requisite amount of the drug, old salvarsan is mixed in the usual way in the proportion of 0.1 gm. to 40 c.c. of fluid, care being taken not to over-alkalinize ; 0.4 c.c. of this solu- tion is the equivalent of 1 mg., and is taken as the standard for measuring the dosage. For this purpose a 1 c.c. pipette graduated in hundredths should be employed. The desired amount of salvarsan is added to from 12 to 15 c.c. of the serum, shaken to and fro to mix thoroughly, and then placed in the incubator at 37 C. (98.6 F.) for one hour, after which it is inactivated for half an hour at 56 C. (132.8 F.). The latter is a most important step in the technic, as Swift and ^^Jour. Am. Med. Assn., Aug. 15, 1914, 555. APPENDIX 343 Ellis demonstrated that the splrochaeticidal properties of the serum were markedly increased by heating. " Salvarsanized serum prepared according to the method of Ogilvie must be used fresh, that is, within three hours of the time that it is made up." Although a few investigators have added as much as 12 mg. of neosalvarsan to the serum in vitro prior to intra- spinal injection, with salvarsan an initial dose of 0.1 to 0.25 mg., increased perhaps to 0.5 mg., should not be exceeded. Pordyce regards the limit of safety to lie within 0.5 mg. After-care of the Patient. — Following a full intravenous injection of salvarsan or neosalvarsan, particularly the former, the patient should lie still for one-half hour. Indeed, it were better for him to be confined to bed over night, and longer if reactions make it desirable. The practice of per- mitting patients to go home alone immediately after an in- travenous injection of the drug is reprehensible, although in many clinics and offices it is commonly done, especially after neosalvarsan. It is our practice both in the hospital and in the office to detain the patient for four to six hours, when if he exhibits no reaction, or any toxic effects of the drug have disappeared, he is allowed to go home. If the reaction is persistent in any way, the patient is advised to remain over night. After an injection of arseno-benzol, the patient is not allowed to have anything by mouth, even water, for two hours. If, then, he is not nauseated he may be given all the water he cares to drink. Food is usually not craved, but should the patient become hungry during the afternoon or evening a glass of milk, broth or consomme with a slice of bread or a cracker is all-sufficient. As a routine practice, it is unnecessary to treat headache, nausea, vomiting, fever, diarrhoea, etc., which occasionally supervene. Rarely 344 APPLIED IMMUNOLOGY pyramidon for headache, a mustard plaster to the epigas- trium for gastric disturbance, bismuth subnitrate for diar- rhcEa and an ice-cap for fever may be of some value or at least pacify the patient. Most important is it that the urine be examined the following day, in order to learn the extent of renal irritation, if present. If albumen or casts are found, urinalyses should be performed daily to determine the duration of their persistence, in view of subsequent adminis- tration of the drug. If the evidences of kidney irritation are marked or prolonged for more than 24 to 48 hours, the repetition of the injection becomes a serious consideration and as a rule is inadvisable. Arsenical intoxication calls for sweat baths, either electric or hydrothermic. Caution should be enjoined that the patient not exert himself severely in view of cardiac strain for a few days, particularly in the advent of severe reactions. Clinical Reactionary Effects of Salvarsan. — As a rule clinical reactions are commoner and severer after salvarsan than neosalvarsan. The primary dose, whether the former or the latter, is usually attended with greater reactions than subsequent injections, although the converse of this is fre- quently observed owing to the cumulative effect of closely repeated administrations. The reactions probably occur by virtue of two facts : Firstly, the endotoxins arising from the destruction of myriad numbers of spirochsetae, and, secondly, the toxic effect of arsenic itself, based upon personal idio- syncrasies. During the administration of salvarsan, the patient not infrequently experiences a sensation of warmth, fulness or throbbing in the head, occasionally complaining of aching of the teeth and a metallic taste in the mouth; his face is usually more or less flushed. If reactions supervene, they are likely to be ushered in by chilliness or a definite chill APPENDIX 345 about an hour and a half or two hours after the injection of the drug. This is followed by headache, nausea and a slight rise in temperature to 99-99 Mi degrees F. Vomit- ing may occur, after which headache and nausea may dis- appear. In severer types of reaction, nausea and vomiting may persist and recur for several hours, associated with a temperature rise as high as 104 degrees F. Diarrhoea is a common occurrence. Rarely a cutaneous eruption of an erythematous type due to arsenic may be observed. This is distinctive from the Jarisch-Herxheimer phenomenon, since it may occur in non-syphilitic cases. Loss of appetite for a few hours or even a day or two may be observed, and occa- sionally irritation of the kidneys, manifested by a cloud of albumen and a shower of hyaline casts, may occur. This last is a most important warning prohibiting in a few in- stances the repetition of the intravenous injection of the drug. Careful studies have never revealed injury to a normal heart, kidneys or eyes. It is decidedly reprehensible to administer salvarsan to a patient evidencing renal irrita- tion from mercurial treatment. Wechselmann has ascribed a number of fatalities to this procedure and insists that sal- varsan should never immediately follow a vigorous course of treatment by mercury. In the vast majority of patients, receiving either salvar- san or neosalvarsan, we have observed no sign or symptom other than a trivial transient rise of temperature. Usually all symptoms disappear in six to twelve hours. As a result of treatment, in two or three weeks, the patient invariably feels better physically and mentally; his ansemia improves; he gains in weight ; all lesions rapidly disappear and the pa- tient is inclined to construe the symptomatic into a per- manent cure. The Jarisch-Herffheimer Reaction. — This phenomenon, 346 APPLIED IMMUNOLOGY originally associated only with a characteristic cutaneous eruption following antisyphilitic treatment, at one time — before the discovery of the treponema pallidum and the Was- sermann reaction — enjoyed considerable importance as a diagnostic aid. The skin and mucosa reactions are mani- fested by oedema, swelling, redness, pain and other inflam- matory signs. To-day the Herxheimer reaction is regarded to be much broader in its scope and may be defined as a cu- taneous eruption, an aggravation of a pre-existent syphilitic roseola or any inflammatory reaction in syphilitic tissue pro- voked hy the administration of salvarsan, neosalvarsan or mercury. This is to be distinguished from the arsenic rash in non-syphilitics receiving salvarsan. Various manifestations of the reaction are apparent. The increased redness of the roseola, the inflammatory reaction in mucous patches, the swelling, exudation and occasional ulceration of gummata observed a few hours or a day or so after medication, the lancinating pains of tabes dorsalis, augmented or relighted, as observed following the Swift-Ellis treatment, the so-called neuro-recurrences and the provocative positive Wassermann reaction are all evidences of the Herxheimer reaction in whole or in part. According to Ehrlich the occurrence of these phenomena is due to treatment dosage insufficient to destroy completely the treponemata pallida whereby the escaped viable or- ganisms are sensitized or stimulated to increased activity. Excretion of Salvarsan from the Body. — The elimination of salvarsan from the system has been carefully studied both on the human and on lower animals. It has been determined by urinalysis and gastric analysis that the elimination of the drug occurs almost immediately following its intravenous administration, while after intramuscular injection it is sel- dom detected for an hour, although after subcutaneous in- APPENDIX 347 jection it has been observed as early as twenty-five minutes. Fischer and Hoppe claim that after intravenous injections no arsenic is demonstrable after two to three days, while after subcutaneous injections the time limit is four to five days; after intramuscular injections the urine may show the pres- ence of arsenic for six to ten days. Consequently it is the natural supposition and the case that tlie duration of elimina- tion following intramuscular and subcutaneous administra- tion is longer than after the intravenous injection. It is alleged that, after the last method of therapy, arsenic has been found in the blood at the end of two days, and absent at fourteen days ; in the urine for two or three days ; in the stools for five or six days. By the Marsh test the authors have been unable repeatedly to demonstrate the presence of ar- senic in the blood-serum thirty, forty-five and sixty minutes after the intravenous injection of salvarsan or neosalvarsan, nor have they succeeded in demonstrating traces of the drug in the spinal fluid one and a half to two hours following intra- venous administration. It is possible that the mould test of Gosio, the most delicate qualitative test for arsenic that we have, may throw new light on this subject. The test depends upon the fact that a certain mould {Penicillmm hrevicaule) when cultured on a medium containing arsenic produces a distinct odor of garlic. Examination of human milk at the end of three and twenty-four hours demonstrated the absence of the drug in that secretion. From animal experiments it is proper to infer that the drug is entirely eliminated from the liver and bone marrow in ten to sixteen days. Further ex- periments show that mercury delays the excretion of arsenic and explains why and how mercury ably supplements and supports salvarsan and neosalvarsan in the treatment of syphilis. Potassium iodide, on the other hand, accelerates the elimination of arsenic. 348 APPLIED IMMUNOLOGY Results of Specific Treatment in Syphilis, — We have four ways of measuring the effect of salvarsan in the treatment of syphilis: (1) the disappearance of treponemata pallida, (2) the disappearance of lesions, (3) the recurrence of lesions and (4) the Wassermann reaction. Of these, the first, constitut- ing the clinical symptomatology, is the most important. How- ever, the value of the Wassermann reaction, correctly and competently performed, in the diagnosis and control of the treatment of syphilis, must not be underestimated, since, by its use, the diagnosis in doubtful cases may be determined and by its periodic utilization, recurrence of lesions may be avoided. The earlier and more intensive the antisyphilitic treat- ment, whether by salvarsan or mercury, the sooner and more lasting are negative results to be expected. A single negative result serologically does not signify cure and is commonly observed in the treatment of early syphilis; the Wassermann reaction may rapidly return to positive if the amount of treatment has been insufficient. On the other hand, the duration of negative reactions is directly propor- tional to the thoroughness of treatment. Mcintosh and Fildes state that positive reactions in the secondary stage of syphilis may be expected to become negative after a single course of mercury in 66 per cent, of cases ; in tertiary syph- ilis in 33 per cent, of cases. One-half to two-thirds of patients have negative reactions after ten courses of mercury or two years of pills. It is safe to conclude that one-third to one- half of all patients treated by the old-fashioned method, although exhibiting no signs or symptoms, judged from the serological standpoint are or were not cured of syphilis. May this large percentage not include many of those patients who later in life develop tabes dorsalis and paresis.'' What has salvarsan added to the curability of syphilis? APPENDIX 349 Mcintosh and Fildes state after the intramuscular and sub- cutaneous administration of salvarsan (0.3 to 1.2 gm.) in primary cases all showed negative Wassermann reactions on an average of five weeks ; in the secondary stage many be- came negative on an average of eight weeks; in the early latent form the average was ten weeks ; in the tertiary stage little or no effect was observed so far as negative reactions were concerned. After the combination of intramuscular with intravenous injections (0.8 to 1.7 gm.), however, a much more pronounced beneficial effect was observed. In secondary syphilis no case failed to become negative and did so with great regularity in six and a half weeks. In the tertiary stage of the disease the intravenous method again proved its superiority over the intramuscular and was pro- ductive of a high percentage of negative reactions. The dictum of Ehrlich, that in the use of salvarsan there exists an agent capable of curing syphilis by a single dose — a " thera- pia sterilisans magna " — is true. Our experience has shown that if the diagnosis of the chancre be made sufficiently early, using the dark field ultra-microscope, before the Wasser- mann reaction becomes positive, the disease may be cured by a single dose of salvarsan. The hope inspired by that dic- tum, however, has not been fully realized, in view of the fact that only a small percentage of cases, untreated with mer- cury, yield a permanently negative Wassermann reaction even after repeated injections of salvarsan. It is the con- sensus of opinion to-day that, as a general rule, the most appropriate, efficient and speedy cure of syphilis is the association of salvarsan, preferably intravenously, with mer- cury or mixed treatment. GLOSSARY Active Immunization. — The process by which the body cells of an animal are stimulated by a toxin or foreign body (antigen) to the production of antibodies specific against the given foreign substance. Agglutination. — A phenomenon characterized by clumping and loss of motility of bacteria, brought about by ag- glutinins. Agglutinins, — Antibodies of the second order of Ehrlich, producing agglutination. Allergy. — The altered condition of an animal into whose tissues has been introduced an antigen or foreign cell product. Amboceptor. — Specific antibody of the third order of Ehr- lich, which acts only in conjunction with non-specific substance or complement. Anaphylaxis. — A series of apparently deleterious effects pro- duced by a second injection of specific protein material into an animal that has been previously " sensitized " by a prior injection of the same material. Antigen. — ^A foreign substance, usually of protein nature, capable of exciting the formation of specific antibodies. Antiserum. — Serum containing specific substances whereby the action of bacteria or their toxins is antagonized. Antitoxin. — Specific substance produced in the blood-serum whereby the action of bacterial toxins is antagonized. Autogenous Bacterin. — Therapeutic suspension of bacteria prepared from the particular strain cultured from the infected patient. Autolysis. — Disintegration of bacteria by treating them with salt solution, alcohol, ether, chloroform, etc., theoret- ically to remove toxic or antiopsonic substances. 350 GLOSSARY 351 Bacterin. — A suspension of bacteria prepared for therapeu- tic purposes. Bacteriocidin. — Substance in the blood-serum, capable of destroying bacteria. Bacteriolysin. — Specific antibody of third order of Ehrlich, concerned in the dissolution of bacteria. Bordet-Gengou Phenomenon. — Complement-fixation reaction as first applied to infection with cholera spirilla. Complement. — Non-specific substance normally present in all blood-serum, acting in conjunction with antibodies of third order of Ehrlich. Complement-fixation. — The using up, and rendering unavail- able for further use, of complement, in the reaction be- tween antigens and their specific antibodies (ambo- ceptors). Cytolysin. — Specific antibody of third order, capable of dis- solving foreign cells. Endotoxin. — Toxin bound up in the bacterial protoplasm, and only set free by disintegration of the bacteria. Hcemolysin. — Specific antibody of the third order of Ehrlich, capable of disintegrating foreign red blood-cells. Hcemolysis. — Disintegration of red blood-cells, setting free the haemoglobin. Hoemolytic Amboceptor. — Synonymous with hgemolysin. Immunity. — The resistance manifested by man and various animal species to infectious microorganisms or other foreign proteins. Immunization. — The process by which the state of immunity is attained. It comprises two forms, active and passive. Lysins. — Specific antibodies of third order of Ehrlich, com- prising cytolysins, haemolysins, bacteriolysins, etc. Opsonic Index, — Measure of the ratio of the phagocytic activity of neutral washed leucocytes in the patient's 352 GLOSSARY serum for given bacteria, as compared with those in a normal or control serum. Opsonins. — Specific substances in the blood-serum possessed of the ability to sensitize or prepare bacteria for phago- cytosis. Organotherapy. — Therapeutic administration of products of ductless glands and other organs. Passive Immunization. — The process by which immunity is acquired when artificial antisera are injected into the animal body. Phagocytosis. — Property of the leucocytes whereby they take up into their substance foreign particles, such as bac- teria, pigment, carbon granules, etc., thus removing them from the circulation. Precipitins. — Antibodies of the second order of Ehrlich, formed in the blood in response to unorganized protein material. Sero-hacterin. — Bacterins prepared by treating bacteria with their specific immune serum, thus " sensitizing " them so that they are acted upon by the complement in the patient's blood immediately after injection. Stock Bacterin. — A therapeutic suspension of bacteria which have been isolated from another patient, who has suf- fered from a similar infection. Toxins. — The soluble products of bacterial and plant growth, whereby their deleterious effects are brought about. Tuberculins. — Various preparations from tubercle bacilli for therapeutic and diagnostic purposes. Vaccination. — Protective immunization to smallpox by in- oculation with cow-pox virus. Vaccine. — Term properly reserved for cow-pox virus ; now frequently applied to therapeutic suspensions of bacteria or bacterins. INDEX Abderhalden-Fauser reaction, 163 Abderhalden reaction, 157 dialysis method, 157 optical method, 160 Pearce and "Williams' modi- fication, 160 Abscess, bacterin therapy in, 262 bronchial, bacterin therapy in, 291 Absorption of complement, 90 Acne bacillus, 260 bacterin therapy in, 260 Acromegaly, pituitary gland in, 315 Actinomycosis, bacterin therapy in, 267 Actinomycotin, 267 dosage, 251 Adrenal gland, 313 Adrenalin, 314 Adulteration of meat products, detec- tion of, 82 Agglutination, clinical application of, 72, 81 Agglutinins, 20, 72 Alexins, -i Allergic reactions, 167 Allergy, 18, 22 Alopecia areata, bacterin therapy in, 260 Amboceptor, 26, 86 Amenorrhoea, corpus luteum in, 316 Anaphylaxis, 23, 27, 167 mechanism of, 29 passive, 31 precautions against, 32 reaction in diagnosis, 31 symptoms of, 30 Anti-anaphylaxis, 31 Anti-anthrax serum, 63 Antibacterial sera, 34, 55 preparation of, 38 Antibody, 19 Antibotulism serum, 53 Anticarcinomatous extracts, 67 Anticholera serum, 62 Anticolonic serum, 61 Antidiphtheritic serum, preparation of, 35 Antidysenteric serum, 52, 62 Antifcrment, 22, 65 Antigen, 8, 16, 89 definition of, 19 titration of, 111 Antigonococcic serum, 51, 59 Antileprosy serum, 64 Antimelitensic serum, 63 Antimeningococcic serum, 60 Antiphytotoxic serum, 53 Antipneumococcic serum, 58 Antirabic inoculation, 301 serum, 64 Antisera, preparation of, 34 therapeutic use of, 40 Antistaphylococcic serum, 55 Antistreptococcic serum, 58 Antitetanic serum, 46 Antithyroid serum and extracts, 68 Antitoxic sera, 34, 42 preparation of, 35 unit, 37 Antitoxin, 20 diphtheria, 42 method of injection, 43 preparation of, 35 protective use of, 42 tetanus, 46 therapeutic use of, 47 Antituberculosis serum, 52 Antityphoid extract of Jez, 65 inoculation, 296 serum, 61 Antivenin, 53 Arthigon, 276 Arthritis, bacterin therapy in, 281 tubercuhn in, 208 Arthus' phenomenon, 28 Arylarsonates, 319 Atoxyl, 319 Aural tuberculosis, tuberculin in, 209 Auto-antibodies, 22 Autogenous bacterins, 224 Auto-inoculation, 6 induced, 244 Autolysates, 7 Autolysis in preparation of bacterins, 221 Autosalvarsanized serum, 340 353 354 INDEX Bacillus acidi lactici in bacterin therapy, 277, 293 acnes in bacterin therapy, 260 anthracis in bacterin therapy, 262, 266 coli in bacterin therapy, 262, 266, 273, 276, 277, 279, 280, 284, 290 diphtherise in bacterin therapy, 285, 286 dysenterise in bacterin therapy, 298 fluorescens in bacterin therapy, 266, 284 influenzae in bacterin therapy, 285, 286, 290, 292, 299 Koch- Weeks in bacterin therapy, 283 lactis aerogenes in bacterin therapy, 266, 284 lactis bulgaricus in bacterin ther- apy, 304 mallei in bacterin therapy, 262, 268 Morax - Axenfeld in bacterin therapy, 283 of Bordet-Gengou in bacterin ther- apy, 292 of Friedlander in bacterin therapy, 273, 283 pestis in bacterin therapy, 271 prodigiosus in bacterin therapy, 303 proteus vulgaris in bacterin therapy, 266, 284 pseudodiphtherise in bacterin therapy, 285, 291 pseudotuberculosis rodentium in bacterin therapy, 273 pyocyaneus in bacterin therapy, 262, 266, 277, 283, 284, 293 tuberculosis in bacterin therapy, 262, 266, 273, 280, 283, 284, 285, 292, 293 typhosus in bacterin therapy, 262, 273, 277, 280, 285, 293 Bacterisemia, bacterin therapy in, 298 Bacterial infections, natural recovery from, 213 Bacterial inoculation, 216 general indications for, 242 Bacterial suspensions, standardization of, 219 Bacterin therapy, accessory measures in, 258 causes of failure, 255 Bacterin therapy, contra-indications, 254 in various diseases, 259-298 limitations, 255 opsonic control of, 225 results of, 259 stock versus autogenous bac- terins, 256 Bacterins, autogenous vs. stock, 224 autolysis of, 221 clinical symptoms in administra- tion of, 225 containers for, 223 dosage of, 250 standardization of, 219 technic of administration of, 248 Bacteriocidin, 214 Bacteriolysins, 21, 86 Blood, medico-legal identification of, 83, 85 Bordet-Gengou phenomenon, 92 Bubonic plague, bacterin therapy in, 271 Cancer, inoculation treatment of, 302 Carbunculosis, bacterin therapy in, 261 Carcinoma, meiostagmin reaction in, 163 sera and extracts in, 67 Cellulitis, bacterin therapy in, 265 Cerebrospinal meningitis, bacterin therapy in, 300 Chemotherapy, 318 Cholera, bacterin therapy in, 298 Cholesterinizod extracts in Wasser- mann reaction, 96 Coagulose, 307 Coley's fluid, 302 Complement, 86 fixation reaction, 16 gonococcus, 141 in echinococcus disease, 153 in proteid differentia- tion, 155 in tuberculosis, 154 in typhoid fever, 154 Complement, preparation of, 116 titration of, 117 Corneal ulcer, bacterin therapy in, 284 Corpus luteum, 316 Corynebacterium pseudodiphtheriti- cum in bacterin therapy, 277 Cretinism, thyroid in, 312 INDEX 355 Crotalin, 69 Cutaneous reactions, 189 Cystitis, bacterin therapy in, 273 Cytolysins, 21, 86 Dermatitis, bacterin therapy in, 265 Diphtheria antitoxin, 35, 42 method of injection, 43 preparation of, 35 protective use of, 42 bacterin therapy in, 286 carriers, 45, 288 curative treatment of, 44 prophylaxis, von Behring's method, 43 toxic skin reaction, 191 unit of, 36 Dose table of bacterins, 251 Dysentery, bacterin therapy in, 298 Dysmenorrhoea, corpus luteum in, 316 ovarian substance in, 316 Echinococcus disease, complement- fixation in, 153 Ehrhch's side-chain theory, 24 Endotoxins, 38 EnterocoHtis, bacterin therapy in, 293 Epididymitis, bacterin therapy in, 278 Epilepsy, crotalin in, 69 Epiphanin reaction, 166 Erysipelas, bacterin therapy in, 265 Exophthalmic goitre, thyroid in, 313 Eye diseases, bacterin therapy in, 283 Fixation of complement, 90 Furunculosis, bacterin therapy in, 261 Genito-urinary diseases, bacterin therapy in, 273 tuberculosis, tuberculin in, 208 Glanders, bacterin therapy in, 268 Gonococcus complement-fixation test, 141 antigens, 143 technic, 142 in bacterin therapy, 275, 277, 278-281, 283, 284 Gonorrhoea, cutaneous reaction in, 189 Hsemocytometer in standardization of bacterial suspensions, 219 Hemolysin, natural, 91 Hsemolysins, 21, 86 Haemolysis, in transfusion of blood, 309 mechanism of, 91 non-specific, 90 Haemolytic amboceptor, preparation of, 106 titration of, 108 Hay fever, active immunization in, 289 Hecht- Weinberg reaction, 130 Hemorrhage, serum treatment of, 306 transfusion of blood in, 308 Horse serum in treatment of hemor- rhage, 306 Hydrophobia, treatment of, 300 Hypersusceptibility, 27 Hypophysis, 314 Hypopyon, bacterin therapy in, 284 Identification of blood, 83, 85 Immune body, 20 Immunity, acquired, 5 active, duration of, 247 definition of, 1 local, 2 mechanism of, 8 natural, 1 passive, duration of, 41 Immunization, active, 5, 35 passive, 8, 35 Immunology, history and develop- ment of, 10 Impetigo, bacterin therapy in, 265 Infections, bacterial, recovery from, 213 Inoculation, bacterial, general indi- cations, 242 therapeutic, principles of, 216 ^ Intestinal tuberculosis, tuberculin in, • 208 Iritis, bacterin therapy in, 284 Isocytolysins, 21 Jarisch-Herxheimer reaction, 345 Jez, antityphoid extract of, 65 Kuhnhardt's spreader, 233 Labor, pituitary extract in, 315 Leprosy, Wassermann reaction in, 132 Leucocytic extract, 65 Ludwig's angina, bacterin therapy in, 265 Luetin reaction, 186 356 INDEX Lymphadenitis, bacterin therapy in, 265 tuberculous, tuberculin in, 209 Lymphangitis, bacterin therapy in, 265 Lysins, 21, 86 Malaria, Wassermann reaction in, 132 Mallein, 268 Meiostagmin reaction, 17, 163 Meningococcus in bacterin therapy, 300 Menopause, corpus luteum in, 316 ovarian extract in, 316 Micrococcus albus in bacterin therapy, 260, 262, 277 aureus in bacterin therapy, 261, 262, 266, 277 candicans in bacterin therapy, 277 catarrhalis in bacterin therapy, 275-277, 283, 285, 286, 290, 292 citreus in bacterin therapy-, 262, 277 melitensis in bacterin therapy, £99 neoformans in bacterin therapy, 302 Myxcedema, thyroid in, 312 Neisser bacterin, 275, 277-281 Neisser-Sachs reaction, 155 Neosalvarsan, chemical formula, 321 dosage, 326 indications, 321 preparation of solution, 338 provocative employment of, 322 Neurasthenia, corpus luteum in, 316 Noguchi modification of Wassermann reaction, 129 Obesity, thyroid in, 313 Ocular tuberculosis, tuberculin in, 209 Opsonic index, 15 apparatus for, 230 definition of, 229 in diagnosis and prognosis, 229 interpretation of, 235 in tuberculin therapy, 199 limitations of, 235 negative phase, 235 positive phase, 235 preparation of smears, 233 Simon's method, 234! technic of, 230 Opsonic index, tuberculo-, 233 value of, 235 Opsonins, 21, 215 definition of, 228 immune, 22, 229 normal, 22, 229 Organotherapy, 312 Osteitis, bacterin therapy in, 280 tuberculin in, 208 Osteomyelitis, bacterin therapy in, 280 Osteophytes, bacterin therapy in, 281 Ovarian substance, 316 " Paras\'philitic " affections and Was- sermann reaction, 136 Paresis and Wassermann reaction, 137 treatment by autosaivarsanized serum, 341 Passive immunity, duration of, 41 Pasteur treatment of rabies, 300 Pemphigus, bacterin therapy in, 265 Periostitis, bacterin therapy in, 280 Peritoneal tuberculosis, tuberculin in, 208 Pertussis, bacterin therapy in, 292 Pfeiffer's phenomenon, 87 Phagoc3'tosis, 211 spontaneous, 228 Phylacogens, 70 Pituitary body, 314 Pneumococcus in bacterin therapy, 262, 273, 276, 277, 279, 280, 282- 284, 286, 291-293 Pneumonia, bacterin therapy in, 291 Poisons, difference from true antigens, 26 Pollen toxin in hay fever, 289 Precipitin reaction, 82, 83 Precipitins, 20 Pregnancy, Abderhalden's test for, 157 Prostatitis, bacterin therapy in, 277 Protein differentiation, complement- fixation in, 155 Provocative employment of salvarsan, 322 Puerperal sepsis, bacterin therapy in, 280 Pulmonary tuberculosis, tuberculin in, 206 Pyaemia, bacterin therapy in, 298 Pyelitis, bacterin therapy in, 273 Pyelonephritis, bacterin therapy in, 273 Pyocyanase, 68 INDEX 357 Pyonephrosis, bacterin therapy in, 273 Pyorrhaa alveolaris, bacterin therapy in, 292 Rabies, treatment of, 300 Reaction, Abderhalden, 157 Abderhalden-Fauser, 163 Calmette, 185 cutaneous, in gonorrhoea, 189 in tuberculosis, 180 in typhoid immunity, 190 Schick's diphtheria toxin, 191 epiphanin, 106 gonococcus complement-fixation, 141 Jarisch-Herxheimer, 345 luetin, 186 meiostagmin, 163 Moro, 183 Neisser-Sachs, 155 precipitin, 82 sero-enzyme, in mental diseases, 162 in pregnancy, 157 in syphilis, 161 tuberculin, 173 von Pirquet, 180 "VN'assermann, 95 Widal, 73 AVolff-Eisner, 185 Receptors, 24 Recovery from bacterial infections, 213 Rhinitis, bacterin therapy in, 286 Salpingitis, bacterin therapy in, 279 Salvarsan, acquired resistance to, 323 chemico-physical properties, 320 clinical reactionary effects of, 344 contra-indications, 324 dosage, 325 excretion of, 346 history of, 318 indications, 321 in non-syphilitic diseases, 322 intramuscular administration of, 327 intraspinal administration of, 339 intravenous administration, 331 after-care of patient, 343 preparation of patient, 332 preparation of solution, 333 Salvarsan, methods of preparation and administration, 327 precautions, 324 provocative employment of, 143, 322 subcutaneous administration, 328 Sarcoma, Coley's fluid in, 302 Scarlet fever, AVassermann reaction in, 132 Schick's diphtheria toxin skin reac- tion, 191 Seborrhea, bacterin therapy in, 260 Seminal vesiculitis, bacterin therapy in, 277 Septicaemia, bacterin therapy in, 298 Sera, antibacterial, 34, 55 preparation of, 38 antitoxic, 34 Serobacterins, 222 Sero-enzyme reaction, mental diseases, 162 pregnancy, 157 syphilis, 161 Serum, anti-anthrax, 63 antibotulism, 53 anticholera, 02 an ti colonic, 61 antidiphtheritic, 35, 42 antidysenteric, 52, 62 antigonococcic, 51, 59 antileprosy, 64 antimelitensic, 63 antimeningococcic, 60 antiphytotoxic, 53 antipneumococcic, 58 antirabic, 64 antistaphylococcic, 55 antistreptococcic, 58 antitetanic, 46 antithyroid, 68 antituberculosis, 52 antityphoid, 61 normal, in treatment of hemor- rhage, 306 sickness, 33 Side-chain theory, 24 Simon's method for opsonic index, 234 Sinus, bacterin therapy in, 266 Sinusitis, bacterin therapy in, 286 Skin diseases, bacterin therapy in, 259 Smallpox inoculation, 11 vaccination, 268 technic of, 269 358 INDEX Staphylococcus in bacterin therapy, 262, 266, 273, 276, 279, 280, 282-284, 286, 290, 292 spray for diphtheria carriers, 45 Stock bacterins, 224, 256 Streptococcus in bacterin therapy, 262, 266, 273, 276, 277, 279, 280, 282-284, 286, 290, 293 rheumaticus, 281 Streptothrix actinomyces in bacterin therapy, 262, 291 Swift-Ellis treatment, 341 Sycosis, bacterin therapy in, 265 Synovitis, bacterin therapy in, 281 Syphilis, chemotherapy in, 318 latent, and Wassermann reaction, 134 luetin reaction in, 186 results of specific treatment in, 348 stages of, and Wassermann reac- tion, 133 Tabes and Wassermann reaction, 137 treatment of, by auto-salvarsan- ized serum, 341 Tetanus antitoxin, 46 therapeutic use of, 47 Thymus gland, 316 Thyroid gland, 312 Tonsillitis, bacterin therapy in, 293 Toxin, diphtheria, 36 Toxins, 20 Transfusion of blood, 308 direct, 310 indirect, 310 TubercuUn, 168 as diagnostic agent, 175 Denys', 170 Dixon's, 171 dosage, 196 focal reaction, 175 general reaction, 174 hypersusceptibility, 202 local reaction, 175 modes of administration, 196 new, 169, 170 old, 168 oral administration, 198 physiological action of, 173 reaction, Calmette, 185 Detre, 182 intradermic, 180 Moro, 183 Tuberculin reaction, mucous mem- brane instillation, 185 percutaneous, 183 scarification, 180 subcutaneous, 176 von Pirquet, 180 Wolff-Eisner, 185 rectal administration, 199 Spengler's, 171 subcutaneous injection, 176 technic of making dilutions, 172 therapeutic administration, 194 therapy, 193 available preparations, 195 clinical symptomatology^ 199 control of, 199 indications and results, 205 in various diseases, 206-209 limitations and contra-indica- tions, 203 mixed infection and, 205 opsonic control of, 199 Tuberculinum purum, 172 Tuberculo-opsonic index, 233 Tuberculosis, aural, tuberculin in, 209 bone and joint, tuberculin in, 208 complement-fixation in, 154 genito-urinary, tuberculin in, 208 intestinal and peritoneal, tubercu- lin in, 208 ocular, tuberculin in, 209 of lymph-nodes, tuberculin in, 209 prophylaxis, 193 pulmonary, tubercuUn in, 206 Trypanosomiasis and Wassermann reaction, 132 Typhoid carriers, treatment of, 295 bacterin therapy in, 294 complement-fixation in, 154 immunity, cutaneous reaction in, 190 Ulcer, corneal, bacterin therapy in, 284 Ulcers, bacterin therapy in, 263 Urethritis, bacterin therapy in, 263 Uterine inertia, pituitary extract in, 315 Uveitis, bacterin therapy in, 284 Vaccination against smallpox, 11, 268 Vaccinia, characteristics of, 270 Vaccinoid, 270 Variola, 268 INDEX 359 Vibrio cholerae in bacterin therapy, 298 Von Behring's method of prophylaxis in diphtheria, 43 Von Pirquet reaction, 180 Vulvovaginitis, bacterin therapy in, 279 TN'assermann reaction, 95 alcohohsm and, 135 antigens in, 96 apparatus for, 102 cholesterinized extracts in, 96 chnical apphcation of, 131 collection of patient's serum, 114 effects of treatment on, 138 htemolytic system, 103 Hccht - Weinberg modifica- tion, 130 inactivation of patient's serum, 121 in framboesia, 132 in inlierited sjphilis, 136 in latent syphilis, 134 in leprosy, 132 in malaria, 132 in parasvphilitic affections, 135 in paresis, 137 in scarlet fever, 132 in tabes, 137 Wassermann reaction in trypanoso- miasis, 132 in yaws, 132 modifications of, 128 Noguchi modification of, 129 preparation of amboceptor, 106 preparation of complement, 116 preparation of corpuscle sus- pension, 116 provocative treatment, 140 quantitative, 125 reading of results, 123 sheep's blood in, 103 technic of, 102 titration of antigen. 111 of complement, 117 Whooping-cough, bacterin therapy in, 292 Widal reaction, 73 macroscopic method, 79 microscopic method, 73 significance of, 80 technic of, 77 use of dried blood in, 76 Wright's capsule, 74, 231 method of standardization of bacterial suspensions, 220 Yaws and W^assermann reaction. 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