2 WILLCOX THE BIOLOGICAL TEST FOR BLOOD The Biological Test for Blood. BY SIR WILLIAM WILLCOX K.C.I.E. C.B. C.M.G. M.D. F.R.C.P., (Read at the Meeting November 2 1927.) UNTIL 1901 the examination of blood stains depended upon macroscopic chemical, spectroscopic and microscopic tests. By means of the microscopic examination of blood stains which had been carefully prepared by fixation and staining with a special stain such as 4aema-toxylin and eosin the species of animal from which the blood had been derived could not be stated with any certainty. Mammalian blood could be differentiated from that of birds fishes reptiles and amphibia since the red corpuscles of the latter four groups are nucleated, oval and larger in size whereas mammalian red corpuscles are non-nucleated.The red corpuscles of mammalian animals differ somewhat in size but the differences are so slight that one cannot with certainty differentiate the blood of man from that of other mammals by measurement of the diameter of the red corpuscles. It may be mentioned that the red blood corpuscles of the camel tribe are oval in shape and non-nucleated. About 1900 and onwards great attention was paid by research workers to the possibility of the use of biological tests for human blood. These tests depend on the production of anti-bodies in a living animal when foreign substances of protein nature of biological origin are injected into the living animal. It is interesting to consider the type of research which led up to the discovery of the Biological Tests for Blood.In 1890 Behring discovered that antitoxic substances were developed in animals following the injections of doses of diphtheria toxin and as a result of this work diphtheria antitoxin which has proved of so great therapeutic value, was discovered. Ehrlich in 1891 produced antitoxic substances in animals, which were capable of neutralising the poisons ricin and abrin which are present in castor oil seeds and jequirity seeds respectively. Experiments with snake venom whereby antitoxic substances were produced were made by Sewall in 1887 Calmette in 1894 and Fraser in 1895. Then Ehrlich propounded his beautiful theoretical explanation of the phenomenon occurring in the production of anti-bodies which is still at the present time of great value and wide application.About 1896 it was discovered by Durham and Widal that the injection or infection with specific bacteria caused the development in the body of anti-bodies which caused agglutination of these bacteria. On this work was founded the Agglutination Test for Typhoid Fever which bears the name of Widal and which has for many years been of universal use as a diagnostic test. It has applications in many other diseases such as the para-typhoid group Malta fever etc. In 1898 Bordet found that haemolytic bodies were produced in the sera of animals when foreign red blood corpuscles were injected into their circulation. F.I.C. D.P.H. (Medical Adviser to the Home Office) WILLCOX THE BIOLOGICAL TEST FOR BLOOD 3 It was found that these bodies “Haemolysins” were specific in nature.In 1900 Deutch proposed a haemolytic test for human blood. This depended on the removal of some of the red blood corpuscles from a suspected stain by means of carbolised normal saline. The suspension of the corpuscles of the suspected stain was treated with an antiserum which had been prepared by the injection of human red blood corpuscles into a rabbit and which contained haemolysins destructive to human red corpuscles. If the corpuscles in the suspension of the suspected stain were haemolysed it was thought that this was proof that the blood was human. This test has never come into use for forensic purposes owing to the many fallacies to which it is liable; for example the age of the stain and the atmospheric conditions to which it has been exposed may cause disintegration of the red corpuscles and solution of the haemoglobin quite apart from any specific action of the antiserum.THE PRECIPITIN TEST.-It was found that the injection of special proteins into animals (rabbits usually being chosen for this purpose) caused the develop-ment of anti-bodies in their blood serum. The serum of the animal after a suitable series of injections became very sensitive to the protein used for its immunisation and would cause the formation of a precipitate when mixed with even high dilutions of the protein in question. Thus Kraus in 1897 produced precipitins in the serum of animals submitted to injections of milk. Bordet in 1897 confirmed this work. Myers in 1900 produced specific precipitins from the injection of egg albumin and of serum albumin.Uhlenhuth about 1900, did a great deal of work on the precipitin test and was the first to apply it for medico-legal purposes in 1901. Nuttall and his collaborators Graham Smith and Sanger were working at Cambridge at this time on the biological tests for various species of blood. In 1904 Nuttall published his masterly treatise on Blood Immunity and Blood Relationship. As a result of the above work on the precipitin tests and of the improvement of technique a test of great value and reliability has been devised whereby it may be stated with certainty the species of animal from which a blood stain has been derived. Dr. Roche Lynch deals fully with the technique of this test (infra) and can speak with the knowledge derived from great practical experience.The only contribution I would like to make to this part of the subject is to emphasise the great importance of working with perfectly clear solutions of the suspected stain. A turbid solution of a few drops of the stain which has been teased and soaked in normal saline can be readily clarified by placing it in a small glass pipette the commencement of the narrowed part of which has been packed with a few shreds of torn filter paper. If a rubber teat is now placed on the open wide end of the pipette compression of it readily drives through the turbid liquid and clarifies it so that it is ready for the application of the test 4 WILLCOX THE BIOLOGICAL TEST FOR BLOOD Dr. John Glaister jnr. read before the Medico-Legal Society last year an excellent paper dealing with the technical details of the precipitin test in its application for medico-legal purposes.The precipitin test has been used by the Scientific Analysts for the Home Office since 1908 and I could quote numerous cases in which it has been successfully applied. In 1905 experiments were made by the late Sir Thomas Stevenson on the medico-legal application of the test. I t was not until 1908 that the test was used as a routine test in the examination of blood stains for forensic purposes by the Home Office. By this time much had been learnt as regards the preparation of very sensitive antisera and it was found that the reliability of the test was beyond dispute. The Stafordshire Case.-A blue serge jacket was sent to me from Staffordshire in 1912.I t had on the sleeves some stains which gave the chemical test for blood the microscopic test for mammalian blood and the precipitin test for horse blood. Inside the jacket pocket was a handkerchief with stains upon it of mammalian blood and the application of the precipitin test showed these stains to be human blood. On examination of the coat with a lens several short bay hairs were discovered and also two or three long black hairs. These hairs had the characteristics of horses’ hair. The coat belonged to a man who was accused of horse maiming his victim being a bay mare with black points. At the time of arrest the accused had a recently cut finger. The scientific findings entirely confirmed the charge against the accused. The test is of great value for forensic purposes and has a universal application.A modification of the precipitin test promises to be of great value in food analysis, e.g. in the determination of the kind of meat entering into the composition of potted meats sausages etc. Anti-sera containing precipitins specific for the muscle juices of different animals can be prepared and made use of for testing the specific nature of meat stuffs. The late Dr. W. A. Schmidt in 1908 published a valuable paper in the Cairo Scienti$c Journal on this subject. Before leaving the subject of the Biological Tests for Blood I should like to express the opinion that these should only be carried out by experts familiar with all the technique of the processes and who have themselves prepared the anti-sera which they use in their tests.Anti-sera for the precipitin test are on the market, but they are difficult to obtain. Their sensibility is often very variable and on this account it is best for an expert to work as far as possible with anti-sera which he has himself prepared and of which he knows the exact limit of sensibility. It depends on the prevention of haemolysis of the red blood corpuscles of an animal which has been sensitised to the inactivated serum of a particular animal the complement being derived from the solution of the suspected stain and being fixed by an anti-human blood serum. This test is stated to be a hundred times more delicate than the precipitin test. I t has not come into general use for forensic purposes because the COMPLEMENT FIXATION TEsT.-Bordet introduced this test LYNCH TECHNIQUE OF THE PRECIPITIN TEST AND ITS FORENSIC VALUE 5 haemolysis of red blood corpuscles is susceptible of so many additional factors, and it has been felt that the test is not sufficiently safe for forensic deductions to be made from it. The test may be used as a confirmatory test to the precipitin test. BLOOD GROUPING TEsTs.-The blood of human beings varies in certain properties and individuals belong to one of four groups as regards the agglutinating properties of their serum and corpuscles. The particular group to which an individual belongs is permanent for his lifetime. Dr. Martley speaks with experience of the application of this test. The Blood Grouping Test may be applied in cases of alleged paternity. It is thus seen that the Biological Tests for Blood enable the forensic expert to say from what species of animal a given blood stain is derived and if the blood is human he may state to what human blood group the stain belongs