341 MICROHEMATOCRIT AS A TOOL IN FISHERY RESEARCH AND MANAGEMENT SPECIAL SCIENTIFIC REPORT-FISHERIES Na 341 1 UNITED STATES DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE EXPLANATORY NOTE The series embodies results of Investigation^, usually of restricted scope, intended to aid or direct management or utilization practices and as guides for administrative or legislative action. It is issued in limited quantities for Official use of Federal, State or cooperating agencies and in processed form for economy and to avoid delay in publication . United States Department of the Interior, Fred A. Seaton, Secretary Fish and Wildlife Service, Arnie J. Suomela, Commissioner Bureau of Sport Fisheries and Wildlife, Eteniel H . Janzen, Director MICROHEMATOCRIT AS A TOOL IN FISHERY RESEARCH AND MANAGEMENT By S. F. Snieszko Assisted by Jimmy E. Camper, Fred J. Howard and Lyle L. Pettijohni./ Special Scientific Report -Fisheries No. 341 )_/ These persons, while in-service trainees at the Leetown Laboratory, assisted in collection of data presented in this report. Washington, D.C. June 1960 / CONTENTS Page Introduction 1 Methods 2 Results 3 Discussion 9 Description of technique 11 Acknowledgment 13 Literature cited 13 ABSTRACT The micro method of hematocrit is rapidly replacing red cell counts in clinical hematology. Observations were made on the value of this method in routine hematological examination of trouts. Under the conditions of data collection, the normal hematocrit values for brook trout were 45 to 50, for brown trout 39 to 44, and for rainbow trout 45 to 53 . There was a close correlation between the hematocrits, red cell counts and hemoglobin. The commercial heparinized capillaries, while excellent for human blood, tend to give somewhat higher readings (7 to 18 percent) with trout, due to incomplete prevention of blood coagulation. The procedure as applied to trout is described in detail. MICROHEMATOCRIT AS A TOOL IN FISHERY RESEARCH AND MANAGEMENT INTRODUCTION Hematology is the study of blood or the sum of knowledge about blood. Much of this in- formation consists of measurements of value of the components of blood under normal and ab- normal conditions. Most readers have had red and white cell counts or hemoglobin determina- tions made during routine medical examinations. Less familiar, perhaps, but even more important is the hematocrit determination. The value and significance of hematologic - al examination, and that of hematocrit in particular, may best be conveyed to readers not familiar with these procedures, by quotations from authoritative sources: "Since a change or lack of change in the blood picture' is a fundamental characteristic of practic- ally every physiologic or pathologic state, hemato- logic findings are among the most valuable and most generally useful of all laboratory diagnostic aids The field of clinical hematology is well within the reach of every practitioner. The laboratory methods are not really demanding, and diagnostic interpretations are generally not diffi- cult" (Wells, 1956). "Determination of the hematocrit reading, the erythrocyte count and the hemoglobin concentra - tion are all used in evaluating the erythrocyte content of blood. The hematocrit determination is the most accurate of these methods inasmuch as it is not subject to the rather large errors in- herent in pipetting and diluting blood according to the other methods" (Strumia fit §1., 1954). "The hematocrit reading, or the percent- age of packed cells in the peripheral blood is one of the most important of all clinical constants . Because of its simplicity and hi^ degrees of re- producibility, this procedure is most useful as a routine for detection of anemia, (Wells, 1956). Some aspects of fish hematology have been reported. Hematocrit determinations have been given in some of the more recent works which are here briefly reviewed. The most complete com- parative study on the blood of vertebrates was made by Wlntrobe (1934) who Introduced hemato- crit to hematology. He found that hematocrit, hemoglobin, and mean corpuscular hemoglobin concentration were uniform among all vertebrates, but that the number of red cells was the most variable . The hematocrit values in fish range from about 5 to about 60. This unusually great amplitude probably results from the fact that many of the examined fish were kept under ab- normal conditions. The author makes it clear, therefore, that his figures should not be con- sidered as representative for the different species of fish he examined. Wlntrobe' s data show that among warm blooded vertebrates the hematocrit values were mostly between 35 and 50 . Studies on marine fishes reported by Klsh (1949) show similar hematocrit values to those found by Wlntrobe, but with a narrower amplitude (20-51). Hematocrit values for freshly caugjit carp (Cyprinus carpio) and northern pike (Esox luclus) are of the same magnitude (Vars 1934) . Similar hematocrit values for carp and eastern brook trout (Salvelinus fontinalis) were reported by Field etal_. (1943) and for sea- lamprey (Petromyzon marlnus) by Thorson (1959). Repeated hematocrit determinations for individual fish dur- ing a period of several months show that these values vary considerably. Whenever a fish sup- posedly became diseased or lost appetite due to undetermined causes, the hematocrit values be- came much lower (Young, 1949). So far as I could establish, Benditt, Morrison and Irwing (1941) were the first to use capillaries for hematocrit determination in fishes . They found for Atlantic salmon (Salmo salar salar) in brack- ish water the mean hematocrit value was 39 and in fresh water the value was reduced to 25. Watson et al. (1956) recorded 47 as the normal mean micro- hematocrit value in fingerling sockeye salmon (Oncorhyncus nerka) . In the same lot of salmon apparently infected with a specific pathogenic virus the hematocrit values were significantly lower . Hematocrit values are not included in the recent manual "The Physiology of Fishes" (Brown, 1957). Hematological examinations of fish are usually made either in the course of research or used as a tool for quality control in fish culture or management. Selection of methods of hemato- logical examination for research purposes depends on the nature of the investigation and must be left to the individual worker. In order to be used gen- erally as quality control tools, the hematological methods selected must be simple, rapid and as free as possible from procedural errors. Hem- atological examination may be used to detect some types of malnutrition (Tunison^^, 1939), chron- ic diseases, or disturbances caused by unfavorable environment or pollution (MjCay, 1929). Red cell counts and estimation of hemoglobin have been the procedures used most frequently in the past. Hematocrits were used rarely, because the early hematocrit methods required large quan- tities of blood which seldom were obtainable from fish of the size raised in hatcheries. A detailed review of reference material pertaining to hema- tology of fishes can be found in papers by Yokoy- ama (1947) and Katz (1949). Critique of the significance and of the limitations of red cell counts, hemoglobin and hematocrit determination can be found in recent- ly published manuals of hematology or clinical pathology, as for example the well known books by Wintrobe (1958)and Wells (1956). TTie purpose of this investigation was to determine the practicability of large-scale micro - hematocrit measurements in fish culture and to establish values which may serve as normal for trout until more extensive data are collected. Determination of normal values for fish is difficult. Changes in concentrations of dis- solved oxygen rapidly affect hematological values in fish (Hall £tal_., 1926; Adrianov, 1936; Phillips, 1947; Dombrowski, 1953, and others). For this reason hematological standards for fishes are likely to have wide amplitudes. Only a very thorough examination of fishes under different concjitions may ultimately result in establishing significant standards within normal amplitudes. Our 'observations are a contribution to this aim . While a microhematocrit method was described by Guest and Siler as early as 1934, equipment became commercially available only recently (McGovern et al. 1955). Since then the microhematocrit method has gained general ac- ceptance in clinical laboratories. Procedure described by Guest and Siler requires one drop of blood or 20 to 40,ul (microliters or cubic milli- meters). In the ultra -microhematocrit method of Strumia et al. (1954) only 5 to 10, ul of blood are used; thus hematocrits can be performed with fish from which even less than one drop of blood is obtained. METHOIDS The technique we used is essentially that recommended by McGovern et al. (1955). Trout used for this work were hatchery stock kept in water of 12° to 14°C. (54° to58°F.). They were picked at random, several at a time, and kept in a small tank supplied with fresh and freely flowing water of the same temperature until all were ex- amined. Immediately prior to examination each fish was completely anesthetized by exposure for about one minute in a water solution (approx. 1: 2000) of tricaine methanesulfonate (MS 222) . After wrapping in paper toweling to blot the surface and to cover the vent in order to prevent contamina- tion of the sample, the caudal peduncle was rapidly cut off with sharp scissors. Blood which welled from the dorsal vessels was collected in heparin- ized capillaries 75 mm long and one end closed either in flame or with modeling clay. Capillaries were centrifuged in an International Microcapillary Centrifuge Model MB for 5 minutes and hematocrit determined by means of a plastic reader. (The supernatant plasma is used for other tests) . The usefulness of the hematocrit deter- mination depends upon its speed, accuracy and close relationship to red cell counts and hemoglobin concentration . In order to perform all three tests simultaneously, blood was collected in a spot test plate depression containing a trace of dried anti- coagulant. The blood was continuously stirred until all needed pipettes and capillaries were filled. Red cell counts were made in the usual manner and hemoglobin was determined with a hemoglobino- meter (Spencer #HB meter No . 1000) . Fish selected for these observations were fingerlings and yearlings of eastern brook trout, brown trout (S . trutta) and rainbow trout (S . gaird- neri) fed Cortland diet No. 6 mixed with beef liver and spleen . For the purpose of comparison, a separate series of hematocrits were run simultaneously in 75 and 32 mm heparinized and plain capillaries. In another series plain capillaries were filled with untreated blood which was permitted to co- agulate completely. At the same time blood treated with anticoagulants was also introduced into similar untreated capillaries and both were centrifuged in the same manner and at the same time. Hemoglobin values obtained with the hemo- globinometer were checked with a Bausch and Lomb "Spectronic 20" spectrophotometer. The presentation of quantitative data in tables including mean values, mean deviations, variances or standard deviations and frequency distributions may seem unnecessary to some readers, but may be of value to others who want to compare statistically their own data with those presented here. RESULTS Data presented in this report are based on examination of about 300 trout. Red cell counts and estimation of hemoglobin, in addition to micro- hematocrit, were made with 64 of the trout examined. In table 1 data are presented on micro - hematocrits. In most cases to or three hemato- crits were determined for each trout. This was done to determine the reproducibility of the hema- tocrit method when applied to trout blood. The frequency distribution of these replicated hema- tocrits shows that in 80 to 90 percent of the cases the difference between the replicates is + 1 unit or less. This is a very high degree of reproduc- ibility. The mean hematocrit values for all three species of trout were between 40 and 50, with an average about 45 . The frequency distribution of hematocrit values seemed to become less regular with increasing age. It seems likely that this was caused by gonad development in yearling trout, and/or high incidence of mycosis -like granuloma (Wood et al., 1955) observed in yearling eastern brook trout. The relationship between the hematocrits, red cell counts and hemoglobin as determined in the same trout are presented in table 2 and figure 1 . From this it can be seen that as in other vertebrates, there is a correlation between these values. Hematocrit readings in apparently normal trout differ only slightly from average hematocrit levels in humans and most other vertebrates. It is believed, therefore, that figures obtained for trout at the Leetown hatchery may be tentatively considered "normal" for trout. How representa- tive these figures are for the same species of trout of the same age but with different environ- mental conditions remains to be demonstrated. Comparative data on microhematocrit values from rainbow trout blood pre -treated with anticoagulant with that taken directly into two dif- ferent brands of heparinized capillaries are presented in table 3. Pre-treated samples gave values 18 percent lower than those taken in heparin- ized capillary tubes . When fish blood was collected directly into capillaries treated with anticoagulants and such capillaries were kept in vertical position, there was no visible sedimentation of blood cells. How- ever, if blood was treated with the same antico- agulants before introducing into capillaries, blood cell sedimentation proceeded at about the same rate as with similarly treated human blood. Also when untreated and pretreated blood was kept in capillaries for some time and then blown out on a porcelain plate, the pretreated blood was uniformly liquid while blood collected directly into treated capillaries had a gelatinous consistency. Blood collected into plain capillaries was coagulated and the clot was well separated from the clear serum . Samples of fresh human blood were run at the same time. Blood collected in treated capil- laries did not clot. Cell sedimentation was rapid and hematocrit values, regardless of the type of handling, were identical within the range of ex- perimental error. These observations show that even in treated capillaries some coagulation of trout blood occurs and hematocrit values are somewhat higher than for pretreated blood. Data are presented in table 4 on hematocrits run with trout blood in 75 mm and 32 mm capillar- ies (Strumia, 1954) . 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