BLOOD STAIN'S 97 ascertaining its source, otherwise pus, semen, etc., if present, will respond to the test and will "be interpreted as blood. After this preliminary pre- caution, it is necessary to see that all test tubes, pipettes and other glass- ware articles employed in performing the test are scrupulously clean. The next important item is to prepare an extract of the stained material by soak- ing it in a small quantity of 0.85 per cent saline solution. The addition of potassium cyanide or any other chemical for dissolving old stains is not desirable and is deprecated nowadays. The extract must be perfectly clear and bright and may be filtered or centrifuged if necessary. It should then be diluted with normal saline to make up a dilution of 1 in 1,000. The anti- serum is not diluted and 2 drops of it are gently added to three-fourths of a cubic centimetre of the diluted stain extract in a small tapering test tube held in a slanting position. The antiserum slowly settles down at the bot- tom, and at the junction of the two fluids a white ring with well-defined borders appears in the case of a positive reaction. The ring is situated mostly in the antiserum and not in the extract. In the case of a negative reaction no ring appears. A positive reaction should begin in 10 minutes and be read in 20 minutes. Several controls are put up to guard against all possible errors. The following controls are the most important: — (a) The normal serum control of the extract, i.e. the saline extract put up with the normal serum from the same species of the animal which has yielded the antiserum; it should give a negative reaction. (b) The positive control, and (c) The negative controls, which have already been mentioned. The results are all qualitative and expressed as positive or negative. " All doubtful reactions are read as negative for the purpose of medico-legal work. The negative results of old, faint and insoluble stains are reported as " disintegrated " with a view to avoid favouring the accused unduly by reporting that no human blood was found on the exhibit. Exhibits stained with blood of good quality but not giving the reaction of human blood, how- ever, are reported as " not stained with human blood " or as " stained with the blood of a ruminant animal/bird as the case may be.23 Limitations of the Test.—Very small stains do not give a satisfactory reaction and as such they are reported as "too small for identification of their source ". Blood stains which have been washed or mixed with mer- curic chloride solution (1 in 1,000), potassium permanganate, copper sul- phate, iron sulphate, calcium chloride, zinc chloride, sodium bisulphite, alcohol, formalin, acids and alkalies will not respond partially or completely to this test. Owing to some unknown reasons, the reaction in some cases may be such as " no opinion can be given as to origin "^for medico-legal purposes. A well-equipped laboratory and long expe^^tl^ in this kind of work are essential for giving a decisive opinion. Hende one who is not con- versant with the technique of the test and has nc^ got sufficient experience in this branch of the serological work is not just^led to undertake this work for giving an expert opinion. *j> Blood Grouping.—This is based on a jP^al phenomenon of agglutina- tion of the red blood corpuscles on comingjfKto contact with the blood serum derived from another individual of the/same species. Investigations into this phenomenon led Landsteiner, Decastelo and Sturli, Ottenberg, Jansky and Moss to divide all human beings, without regard to race, sex or state of health, into four groups according to the behaviour of their sera and red blood corpuscles. It is universally admitted that the red blood corpuscles contain two distinct agglutinable substances (agglutinogens), called iso-hsemagglutino- 23. The Imperial Serologists* Ann. Rep., 1937-38.