UNIT IV. DETERMINATION OF SPECIES OF ORlOlN Species of Origin-Okier Methods

disagreement among various authorities over the certainty If the results of the examination of unknown bloodstains which could be attached to the results of these deter- are to have very much probative value, the species of origin minations. The controversy was never actually resolved. It must be established. It may be necessary on occasion to simply became irrelevant shortly after 1900, when the im- carry out a determination of species of origin on a body fluid munological methods were discovered and quickly put into or secretion other than blood, but since the question arises use in medico-legal practice. much less frequently with body fluids than with blood, less For the most part, the technique involved the recon- attention has been paid to the problem. If immunological stitution of the erythrocytes from bloodstains, and exam- tests are to be used, the principle remains the same, of ination of a fairly large number of cells microscopically. course, regardless of which fluid is under examination. Most Careful measurements were performed on the size of the current methods in common use for determining species of cells, and these compared with values for the known sizes of origin are immunological ones. The basis for these methods red cells from many different species. Most vertebrate red was established in the last few years of the 19th Century, and cells are enucleate, and it was usually not diicult to diag- the techniques actually devised and put into practice in nose invertebrate blood. But, particularly among the mam- 1901. Prior to that time, medicolegal investigatorswere very mals, a number of species have red cells which are quite concerned about the problem, but the techniques were not similar in size. The efforts which were made, and the dis- very satisfactory. agreements which existed, must likewise be considered in the context of the accuracy of the methods which were then 15.2 Chemlcrl Methods available. There was never complete agreement that species In 1829, J.-P. Barnel introduced a method for deter- could be correctly diagnosed using microscopical meas- mining the species of origin of bloodstains. Blood or blood- urements, or micrometry. A good many authorities came to stains would emit a completely characteristic odor, he said, believe that a proper examination could establish only that when treated with an excess of concentrated sulfuric acid. blood was of mammalian origin. He claimed, in addition, that human blood could be dif- The notion that red cell size might be exploited for the ferentiated with regard to sex of origin based upon the pun- determination of species of origin can be traced to some of gency of the odor. Odawas apparently convinced of the the earliest discussions of medico-legal blood examinations usefulness of this approach, for in 1835 the technique was in France. In 1821, Prevost and Dumas had established that used in a homicide case in which he served as the medico- the red blood cells of various species differed in size. In 1827, legal expert along with Barruel and Chevallier (see sec- Dulong is said to have remarked at a meeting of the Royal tion 3). although no results could be obtained with the Academy of Medicine that the size of red cells is a very exhibits submitted. In 1842, Mandl noted that the method characteristic feature, even when the blood is old and dried, was unreliable, and not suited for use in legal medicine. and that this property could be used to help decide the Schmidt (1848) evaluated Barruel's technique, and said that species of origin of the blood. Orfila (1827), however, was it was not reliable. In 1853, Tardieu et al. re-examined the not convinced, having camed out a number of experiments technique in connection with a case they were working on, to test the possibility. Orfila's reputation being what it was, and concluded, after a series of blind trial tests on stains of the matter does not appear to have come up again for several lcnown origin, that the test was not reliable. years. In 1848, Casanti reported that the blood of different mam- In 1842, Mandl published an extensive paper on the use of mals, including human beings, formed a characteristic resi- microscopy in medico-legal investigations, and strongly ad- due when dried and treated with concentrated phosphoric vocated the measurement of the red cells as a means of acid. He said, too, that menstrual blood could be dif- determining species origin. There was no diiculty in dis- ferentiated from circulating blood in this fashion. The tinguishing the nucleate red cells of birds, reptiles, and so method does not appear to have enjoyed any popularity, forth, from the enucleate ones of mammals. Mandl said, doubtless because of its unreliability. however, that one could not discriminate among the red cells of mammals by this method. Schmidt (1848) briefly dis- 15.8 Mlcromeblc Methods cussed the micrometric method in his book, and indicated Micrometry, the measurement of red cells as precisely as that he thought it was more promising than the methods possible in smears made from fresh blood as well as in which had been in use up to that time. Robin and Salmon reconstituted stains, came into use as a method for species readily distinguished human from duck bloodstains in a case determination of the 19th Century. There was considerable in 1857. The bloodstains at issue, they reported, had all the Solu~ebookin Forensic Semlogy, Imnology, and Biochemistty microscopic characteristics of human blood and could not within the experimental error of the measurements, that the have come from a duck as the defendant was claiming. Flem- interspecific variations were sometimes exceeded by intra- ing (1861) discussed the technique, but did not think it was specific variation, and that the cells underwent irregular very reliable. Roussin wrote a paper on the subject in 1865, changes in size and shape upon drying and reconstitution. advocating the method strongly. He introduced his solution Richardson and other adherents of the method did not fully for the preservation of red cells from bloodstains (see sec- accept these arguments. He thought that red cell size varied tion 5.3) in this paper, but he did not think that the blood of only very little within a species, and that the reconstituted mammals could be differentiated by the measurements. The cells were exactly the same size as those which had originally successful use of the method in two cases was reported. In dried out. 1869, Richardson in Philadelphia, whoebecame one of the In 1882, Vibert carried out a number of studies on the foremost advocates of the method, published a paper saying technique, proposing two solutions for the conservation of that one could, by careful measurement of the red cells, red cell size and shape as well. He did not think that human distinguish readily between human, sheep and pig blood. He blood could be differentiated from mammalian blood, how- extended his studies to other species, noting several years ever. Masson reviewed the material on this matter exten- later the importance of using high power objectives in carry- sively in 1885 in a series of papers. He conductd a number ing out the measurements, so as to obtain sufficiently high of experiments as well. A number of the solutions which had magnification to be able to distinguish relatively small been proposed were tested both with fresh blood and with differences (Richardson, 1874a. 1874b). He thought that bloodstains, Vibert's solution being recommended for the there was no difficulty in distinguishing human blood from former, Virchow's for the latter. A number of experiments the blood of pig, ox, deer, cat, horse, sheep and goat, even in were done to try and find out what factors cause the changes stains that were five years old. Later, he examined blood in red cells upon drying. Masson thought that the faster the from a number of people of different ethnic backgrounds, blood dried out, the better the cells were conserved in terms and said that there were no significant differences in the size of shape and size. He thought that unknown stains could be of the red cells among the different races of humankind assigned to one of several categories of animals, arranged (Richardson, 1877). Woodward (1875) took exception to according to the size of the red cells, but he did not think a Richardson's claims, noting that the cells of the dog were in specific diagnosis was possible with a bloodstain. Formad fact very similar in size to those of humans. He said that he (1888) in his book reviewed the entire subject extensively did not believe there was any method available for the and well. Most of the opinions which had been expressed on unequivocal diagnosis of human blood. Tidy (1 882), while the accuracy of the method were covered, and the references noting with interest Richardson's work, said that in giving given. Formad himself was persuaded that Richardson's testimony to a Court about the species of origin of a blood- conclusions were valid in many respects. He thought that stain, ". .. it is better, in the present state of science, at once there was no difficulty in distinguishing human blood from to confess our inability to give a definite reply." Hemphill that of domestic animals in stains, assuming that a large (1875) noted that he thought it possible to diagnose mam- enough number of cells could be examined and measured malian blood in stains, but not to establish which particular (500 was recommended), and omitting the guinea pig from species was involved. consideration. Human blood could not be distinguished from In 1875, Gulliver published one of the most extensive every animal blood, and he noted that the probative value of single investigations of the sizes and shapes of the red cells testimony presented by an expert who had conducted such of vertebrates. Dozens of species of animals were included in examinations relied in great part on the way in which the the studies, and extensive tables prepared to summarize the questions were asked of him. He thought it would be mis- findings. These data became known as "Gulliver's Tables", leading for the Court to gain the impression that human and were widely quoted and sometimes reproduced in the blood could not be distinguished from any animal bloods by medico-legal literature for 25 years. The relationships he the micrometric method. Ewe11 (1892a) measured 4000 red found in the sizes of red cells of various species were sum- cells, and found variations within the same species. He also marized in a figure in his paper, which is reproduced as said that different observers would get different values, and Fig. 15.1. that the variation was large enough to make one doubt the In 1873, a committee appointed by the Societt de published figures. He did not think red cells in stains could Medecine Ugale in France reported that it thought the be restored to their in vivo proportions. He thought that the organization should adopt the stance that it was possible for micrometric method could be useful in diagnosing blood as a qualified expert to decide by micrometric methods whether being of mammalian origin, but not more. or not a bloodstain was of human origin (Mialhe et al., In 1892, Jones reported on a murder case in Ascension 1873). "I1 [referring to the expert] mesurera les globules et Parish, Louisiana, in which he had examined bloodstain ev- pourra ainsi affirrner s'il s'agit ou non de sang humain," they idence. The body of a man named Simeon Cascio had been wrote. discovered September 22, 1892. Suspicion fell on the vic- The major objections voiced by opponents of the accuracy tim's brother-in-law, one Joseph Polito who had been seen of the methods were that the differences in size between with the victim shortly before the murder, and who had some many mammalian species cells were very small, and fell of the victim's possessions with him at the time of his arrest. Dr. Jones examined stains on the blade and handle of a knife, Axtell (1895) reported in detail on a murder case in Den- on a pocket book, on the collar of the victim's shirt and ver, Colorado, in which he had been able to determine by several stains on paper money bills. The stains on the knife micrometry that the stains on the suspect's clothing were and pocket book, he said in his report, "presented the char- mammalian in origin, and consistent with human blood. The acteristics of human blood." The stains on the currency were defendant had claimed that the stains were from the blood identified as being blood, and he said that the stains on the of a rabbit, but Axtell was able to exclude several different shirt collar were "due to human blood." Ewell(1892b) took species of rabbits as being the source of the bloodstains on very great exception to Jones' conclusion that he had deter- the basis of differences in red cell sizes. The defendant in the mined the stains on the collar to be human blood. "In view case entered a plea of guilty before Axtell had the oppor- of the consensus of opinion among microscopists who have tunity to present his findings before a Court. In 1892, Bell examined this question, that in the present state of science it reviewed the micrometric method in some detail, and is impossible to identify human blood, as such, it would be presented the veiws of most authorities. To illustrate the interesting to know how Dr. Jones reached a conclusion at kinds of tables that were employed, Bell's table of compara- variance with the almost unbroken current of authority," he tive measurements is reproduced as Table 15.1. Corin wrote. Jenne (1896) agreed with the view that human and (190 1) gave a brief review of micrometric methods. Several animal blood in stains could not be distinguished by micro- papers by 19th Century French authors, which go into the metric methods. subject in considerable detail, have been included in the Translations (Unit IX). Soundwok in F o Serology,~ Imm~ologv,and Biochemisttry

QUADRUMANA. CHEIROPTERA.

I S 6 s . ' 7 a *

IS n II I. I& PACHY DERMATA.

R* 81 .I

i;.G*j(yy &/I ML.N!U u RED BLOOD-CORPUSCLES.

Figure 15.1 Relationships of red cell sizes of many species (after Gulliver, 1875) Species of Origin-OIder Methods

All the objdue red b1ood-d- done to one md the asme de,which ir at tbe footof the drawing. The whole hgth of the alempreclenb %f an Englishinch, and sseh one of it. tea diririons of an inch. w dm & pn m. Only corpurdw of the ave.p eke.* quite regular d m am~ gircm ; and the ue a11 to the me,or nearly the mme, d-to wit, about 800 Lets=. Vrm~arr~~X&XATA. Hm. 25. Rpirue indious. 1.Corpurrlel' fht. 26. Eiyue caballus. 2 meme 27. I)m~tybrtorquatus. 3. Membranow bue of the me, 28. EY~"'F'en*s- afbr removal by rater of the Buminantin. aolouring-matter. 9. l'ngttltu jaranicus. Quadrununto. -90.Tngultr meminnr. . 31. lhgulur stanleyanun. 4. sitroglodyk 32. Cewua nemorirugua 6. Atdm ate?. 33. aplsaauarda. 6. bmur mgmudlllicr 34. Cnpm hima CLn'mptrra 35. h urm 7. cponyeteris COllariA 96. Camelo~r&littgirt&. 8. Vqer$!io neub. S7. Auchnr~aricugna. 9. VeeprtJIo p1pl8treU~. 98.39. AuclieniaAuchmnia glump~. Fcte. 40. Cumelus dro~t~abriue. 10. Sores tetragonurue. 41. Camelue bsetrianw. 11. Unue hbidue. 42. hueruericanw (sea page 488) 12. Busuis .stub Rudmticr. 13. Ccmole dvolvdua 43. Rydrochm capybara. 14. Triahec~ram~~~. 44. cutor fiber. 15. Csnir dingo: 45. &iurun cineroue. 16. Mustda zed. Mue m&w 17. Felis tigrir 46. 18. Paradorum Wi. Edmtata. 19. Pamd~~un~bondar. 47. Myrrnecophrga jubata, Cdm. 48. Bndypua dichtylua 49. hypus rillonus. 2Q. B.182~bpa 21. Jhlphinus globimp. Maraupialin. 22 Delpbinus phocl~m. 60. Phlompwuiiibat. H Poeiy~. Gl. ypdprymnus setonue. 23. Elephu indicua Monot+emafa. 5&.EIuuocaw indicua GZ. Echidna lrgrtrix. VE~~BBATAPYREX.QSATA. Aara 59. Columkr rufim 53. Rtwthio camelllr. 430. ~okrmbmigmtorin. 54. nemnm, ndo round and cle- GI. I)olichonyx o~zivorus. of c&ur by -tar. 02. BIHY)~rbinocartn. 55. Van@ deetmdur. 63. Psittaous aug;llcll ue. Xi, kiue excebitor. 64. Pl~anianussuperbus. 57. Bubo rirghbnu. Fi. Yclwanun tm~mtnl~ 58. Syrnea nycbe. 66. Trwliilw, ap.

Pisr.5. Pemr cmmur, one corpu~~Iclying d.t, tho other nu edge. Tina vulgarin. Wmo fontidin. &ox luciua Clynnotus elPlchinrr. Sqcurlus oaurtlii~. Amtaomtea brmrcflialis. Lspidwiren WUI-

Figure 15.1 Explanation of the red cell size diagram on the facing page from the original article. Sourcebook in Foensic Serology, Immunology, rmdBiochemistry Specier of Origin-Immunologicul and Serological

SECTION 16. IMMUNOLOGlCAL TESTS WITH BLOODSTAINS pension as a mass. Uhlenhuth (191 1) attributed this discov- ery to Gruber and Durham in 1896. In 1897, Kraus made 16.1.1 Development of the precipitin test and its the important observation that bacterial antibodies have the medicolegal application property of precipitating cell free bacterial extracts. This The development of a specific immunological test for de- paper marked the first description of precipitin antibodies, termination of the species of origin of bloodstains was a which would shortly play an important role in immuno- major event in the evolution of legal medicine. It provided logical species differentiation. It was soon realized that bac- for the first time a technique for unequivocally establishing terial substances were not the only ones to which animals species of origin in stains. All authorities soon came to agree would elaborate antibodies. Bordet (1898) found that guinea on the specificity and certainty provided by the new method, pigs produce a potent red cell agglutinating serum against Wood (1902a) having said to the Massachusetts Medico- rabbit red blood cells when immunized with defibrinated Legal Society that one could finally testify absolutely as to rabbit blood. Similarly, rabbits which had received several the species of origin of bloodstains, rather than having to say injections of defibrinated hen blood yielded a serum which that the results were "consistent with" the stain having orig- actively agglutinated (agglutinin) and lysed (hemolysin) hen inated from a particular species. Precipitin reactions are red cells. In addition, it was found that the antiserum con- discussed more generally in section 1.3.4.2. Recognition of tained a specific precipitin for hen serum. This discovery was precipitating antibodies, and their application to forensic made, Bordet said, by Tschistovitch at the Pasteur Institute. investigations, must be regarded as natural outcomes of the In 1899, Tschistovitch found that rabbits elaborated a pre- rapid and ever-widening development that was .occurring in cipitin antibody to eel serum with which they had been immunology in the last years of the 19th century, and which injected. The precipitates were soluble in solutions of strong continued into the present one. In the years 1901 to 1920, base. To Tschistovitch, therefore, belongs the credit for the five Nobel prizes for Physiology and Medicine were awarded recognition of the fact that animals will form precipitating in immunology: in 1901 to Emil A. von Behring "for his antibodies to the serum of an animal of another species. work on serum therapy, especially its application against Bordet (1898) could show that milk proteins were antigenic diphtheria, by which he has opened a new road in the domain in rabbits, giving rise to precipitins for the casein. The serum of medical science and thereby placed in the hands of the containing the antibodies was referred to as "lactoserum". physician a victorious weapon against illness and deaths"; in Fisch (1900) in this country confirmed Bordet's observations 1905 to Robert Koch "for his investigations and discoveries on the antigenicity of casein. Wassermann and SchUtze in relation to tuberculosis"; in 1908 to Elie Metchnikoff and (1900) extended these studies to show that the precipitin test Paul Ehrlich "in recognition of their work on immunity"; in could be used to differentiate the milk of different species of 1913 to Charles R. Richet "in recognition of his work on animals immunologically. In 1900, Myers showed that crys- anaphylaxis"; and in 1919 to Jules Bordet "for hi discov- talline hen egg albumin was antigenic in rabbits, giving rise eries relating to immunity." to an antiserum which cross reacted to some extent with Immunology had gotten its start as a proper scientific field duck ovalbumin solutions. The antiserum was entirely of inquiry because of concern with infectious and highly specific for the ovalbumins, however, and did not react with contagious diseases, epidemics of which affected large seg- a number of other animal protein solutions. Independently, ments of the population at times. The recognition, and ulti- Dr. Paul Uhlenhuth, whose name came to be more or less mate acceptance, of the notion that these diseases were synonymous with the precipitin test in its forensic applica- caused by specific microorganisms provided a basis for de- tions, published a paper in 1900 in which he said that he had tailed investigations into the subject of immunity and its successfully differentiated the egg albumin from several spe- mechanism. cies of birds using precipitating antisera prepared in rabbits. Behring and Warnecke (1892) made the important dis- In the course of these experiments, he found that rabbits covery that animals injected with diphtheria toxin elabo- immunized with chicken blood elaborated precipitating anti- rated specific antitoxins. PfeXer (1894) discovered that if sera for the chicken serum, and this antiserum did not react animals were treated with bacteria, specific antisubstances with the sera of horse, donkey, cow, sheep or pigeon. form in the serum of the injected animal. If this serum and Further, the antisera prepared against hen blood showed the bacteria are mixed together and injected into the peri- only a slight cross reaction, and this after a long time, toneal cavity of a healthy animal, the bodies of the bacilli are with hen ovalbumin, demonstrating that immunological degraded. If bacteria are mixed with the antiserum in vitro, specificity could extend not only to the proteins of different the bacteria are agglutinated, and will settle out of the sus- species, but also to proteins from different sources within the Sourrebook in Forensic Serology, Immunology, and Biochemhttry same species. As Uhlenhuth remarked about these experi- urine, and it also reacted with human semen and with pul- ments some years later in the Harben Lecture (1911), monary mucus from tuberculosis patients to some extent. ". . .. the foundation was laid for the forensic method of The antiserum, therefore, detected human albumin, and was distinguishing between diferent specimens of blood, for the not necessarily specific for blood (Uhlenhuth, 1901e; Was- serum of rabbits treated with the blood of man or of any sermann and SchUtze, 1903). Uhlenhuth noted that anti- other animal gave a precipitate only with blood solutions of serum preparation required considerable experience, and the corresponding species to that whose blood had previously that there was variability in the rabbits used to produce it. been injected to the rabbit." He said that the use of high quality, carefully tested antisera The stage having thus been set, 1901 saw the publication was of paramount importance in forensic investigations, and of a number of papers which specifically addressed the im- suggested that antiserum production be carried out in a few munological differentiation of human from animal bloods specialized institutes. using the precipitin test, and recommended that the method Nuttall (1901a) confirmed the specificity and value of the be adopted in medico-legal examinations of blood stains. precipitin test. He noted further that the serum used for Uhlenhuth (1901a) reported in the February 7 issue of immunization needn't be fresh in order to elicit antibodies. Deutsche Medizinische Wochenschriftthat he had been able He showed also that the reaction worked well in dried blood- to distinguish cattle blood from horse, donkey, pig, ram, dog, stains and in putrefied blood. The investigations on the phy- cat, deer, hare, guinea pig, rat, mouse, rabbit, chicken, logenetic relationships among various animal species, using goose, turkey, pigeon and human bloods using a rabbit im- immunological cross reactivity as a basis, were begun at this mune anti-cow precipitin serum. Similarly, human blood time (Nuttall and Dinkelspiel, 1901a and 1901b), and it was could be differentiated from the others using serum from noted that anti-human serum cross reacted with the blood of rabbits which had been immunized with human blood. Nor- several species of monkeys. Antisera could also be prepared mal rabbit serum did not react with saline solutions of any against human pleural fluid, and this serum cross reacted to of the bloods. Further, it could be shown that saline extracts some extent with human tears and nasal mucus. Medico- of human bloodstains dried on pieces of wood could be dis- legal application of the precipitin method was strongly tinguished from horse and cattle bloodstains treated simi- recommended. Ziemke (1901a and 1901b) confirmed the larly. The stains were a month old when tested. A summary value of the technique, noting that species could be diag- of this work appeared in a later issue of Muenchener Me- nosed in bloodstains up to six years old as well as on a variety dizinische Wochenschrift (Uhlenhuth, 1901b). Indepen- of substrata. Frenkel (1901) and Stern (1901) likewise dently and almost simultaneously, Wasserman and Schiitze showed that the precipitin test was fully applicable to blood- (1901) arrived at the same conclusions regarding the species stains, and both noted the cross reactions with monkey specificity of the reaction and its applicability to dried blood- bloods. The term "antiserum" appears to have been intro- stains, this paper appearing in the February 18 issue of duced at this time simultaneously and independently by Berliner Klinische Wochenschrift. They noted that serum Nuttall (1901 a) and by Stern (190 1) to apply to the serum could be used as immunizing antigen in order to obtain containing the antibodies (usually, immune rabbit serum). antisera lacking antibodies to the cells (the agglutinins and Corin (1901) noted in his review that the precipitin antisera hemolysins). Uhlenhuth (1901~)reported the technique in were more specific if the serum globulins were isolated and detail in the medico-legal literature in May, noting that used for immunization. species differentiation was possible in 3 month old blood- The bulk of evidence thus indicated that the precipitin test stains, in blood frozen for 2 weeks in the snow, and even in was both Wicwithin certain Limits and fully applicable putrefied blood, provided a completely clear extract could be to forensic samples, and the use of the test rapidly became obtained for testing. A number of badly putrefied samples widespread. Many authorities confirmed its value and noted reacted readily with the antiserum, and human menstrual its limitations. Kratter (1902) dissented for a time from the blood in a urine specimen could be detected easily. Human current of opinion, saying that he had been unable to confirm urine containing albumin reacted with the antiserum as well a number of Uhlenhuth's observations on specificity, re- (Uhlenhuth, 1901d). This observation was also made by liability and medico-legal applicability of the test. Uh- Dieudonn6 (1901). Further studies on the precipitin test lenhuth (1902) responded to Kratter's article, giving further indicated that bloodstained materials from criminal cases, detailed experimental justification for the test's reliability made available from past cases by the judicial authorities, and specificity in the diagnosis of species in bloodstains on could be reliably diagnosed in every case. Results could even objects involved in criminal cases. Stonesco (1902) said that be obtained with bloodstains that had been washed (Uh- because of the known cross reactions of the antisera, it would lenhuth, 1901e). It was noted that cross-reactions occurred be better to use the term "probably" in giving evidence on between more closely related species, e.g. anti-ram reacted the species of origin of bloodstains in which a positive test to some extent with goat and cattle blood. Uhlenhuth recog- had been obtained for human blood. Schulze (1903) more or nized that the relationship between immunological cross re- less agreed with this view, saying that the Court should be activity in the precipitin test and species relatedness was a made aware of the test's limitations as well as of its great matter of fundamental biological importance. Anti-human value. Whitney (1902 & 1903) described successful results serum had been shown to react with albumin-containing with the method, and Wood (1902b and 1903) gave an Spec* of Origin-Zmmunologid und Serological account of the technique he used, and said that he had been ment of the game laws. Uhlenhuth (1901f) had shown able to identify human blood on an alleged murder weapon earlier that the method was applicable to the differentia- (a stone) in a case from New Hampshire. Ewing (1903) and tion of meats from different species of animals. Gay dis- Mallett (1903a and 1903b) both said they thought anti- tinguished between precipitins against sera (seroprecipitins) human sera prepared in chickens were more specific, and and those against meat protein (musculoprecipitins), and gave fewer cross reactions than rabbit antisera. Mallett attributed this distinction to Vallee and Nicholas. In the case thought there might be interracial immunological dif- in question, a man in Massachusetts was accused of illegally ferences which could be detected by means of the precipitin killing a deer, based on the finding of an animal heart in his test, this based on a few experiments with serum from Cau- possession by the police. The suspect claimed that the heart casians and Negroes. Bruck (1907) was able to obtain was from a cow. Dr. Gay prepared antisera to extracts of differences in the intensity of reactivity with the sera of authentic beef and deer heart muscle as well as to extracts people from different races using the complement fixation of the suspected heart. It could be shown that the heart technique (section 16.6.1), and thought that the method found in the man's possession was in fact that of a deer. might be useful in this respect. Marshall and Teague (1908), Clarke (1914) reported a similar case in California, in which in extensive experiments using both the precipitin and com- he identified deer meat in a poaching case, which went to a plement fixation techniques, also observed that there were jury trial. In 1917, Hunt and Mills reported on a case in slight differences in reactivity of the sera of persons from which the precipitin test failed to react with several blood- difTerent races with antisera prepared against the serum of a stained objects in a criminal case. Two anti-human sera were member of a particular race. But these differences were so used, one prepared by them and the other prepared abroad. slight that they did not agree with Bruck that racial origin These bloodstains were later shown to be of human origin by could be diagnosed by immunological techniques. the complement fixation technique. They cautioned, there- Some authorities around this time began using the term fore, that a negative precipitin test does not necessarily ex- "humanized serum" to apply to the antiserum. Thus, rabbit clude the presence of homologous antigen (blood) in a stain. immune anti-human serum was referred to as "humanized" In 1909, Uhlenhuth and Weidanz published a monograph rabbit serum. DeLisle (1905) prepared antihuman sera in which reviewed the subject of forensic immunology to the horses, and recommended this large animal as a source be- time, and provided very detailed experimental procedures cause of the large quantity of antisera which could be ob- and methods for every aspect of the precipitin technique. tained. It is now known that horse and a few other animal This book was a standard reference for a number of years. species often produce precipitin antisera which have some- In 1911, Uhlenhuth delivered the Harben lecture in Eng- what different characteristics from most rabbit antisera. The land, recounting many of the experiments that had led to the horse antisera are characterized by a narrower zone of development of the precipitin test, and giving his own equivalence between antigen and antibody, that is, the recollections. This paper was the only one of his many con- antigen-antibody complex is soluble in either excess antigen tributions which appeared in English. or excess antibody, and the range over which precipitation In 1914, Hektoen discussed the production of antisera to will occur is narrower than with rabbit antisera (Maurer, human and animal sera, finding that a single injection of 1971). This matter is discussed in more detail in sec- 30 mQ immunizing serum was about as effective as three tion 1.3.4.2. In 1903, Hitchens reviewed the highlights in the successive ones of 5, 10 and 15 d,respectively. In 19 17, development of immunology, emphasizing the value of pre- Hektoen carried out experiments to determine whether anti- cipitin antisera in medico-legal work. Ewing and Strauss body production in rabbits was enhanced if animals were (1903) gave an extensive review of precipitins and their used which had previously been employed for antibody pro- medico-legal applications. duction (not necessarily with the same antigen). There were An extremely comprehensive series of studies on the appli- no differences between such animals and immunologically cation of the test to forensic materials was reported by virgin rabbits, and Hektoen suggested that antisera were Graham-Smith and Sanger in 1903. Many of the test's pa- therefore best prepared in the latter. Ewing (1903) and Mal- rameters were explored in detail, and studies were conducted lott (1903a and 1903b) had recommended the production of on blood stains on a large number of different materials antisera in fowl. Sutherland (1914) preferred to prepare his under diierent conditions. A few chemicals exerted a delete- antisera in domestic fowl, but these antisera exhibited some rious effect on the antiserum, and some stains on leather peculiar characteristics. Sutherland and Mitra (1914) noted substrata were not able to be diagnosed. Very high titer that too rapid thawing of frozen antisera caused it to exhibit antisera could be produced using relatively small quantities interspecificcross reactions. These could sometimes be elim- of immunizing sera, of the order of 3 to 5 d at each of three inated by allowing the serum to sit in the dark for a time, or to four injections. Many workers had employed considerably by dilution with normal nonimmune fowl serum. Hektoen larger quantities of immunizing serum. (1918a) found that fowl were more reliable, liberal produc- Robertson (1906) recounted his experiences with the test, ers of antisera than were rabbits. Good, high titer antisera and noted that he got positive tests in stains that were 9 years could be obtained in most cases in 10- 12 days after a single old. In 1908, Gay reported what was probably the fust appli- intraperitoneal injection of 20 mll defibrinated blood or of cation of the precipitin test in this country in the enforce- serum. He noticed some of the same peculiarities which Sourcebook in Fo& Serology, Immunology, and Biochemistry

Sutherland and Mitra had earlier observed, and in another first recommended by Hauser in 1904. Schoenherr (1952) paper (l918b) suggested that it would probably be best to gave a technique for the precipitin test in Pasteur pipettes, use rabbits for making antisera. Hektoen and McNally dis- The ring test is still in use in many laboratories (see, for cussed the precipitin test in detail, including the preparation example, Boyd, 1946 and Hunt et al., 1960). The test can be and evaluation of antisera in their review of medico-legal carried out on a microscope slide and the precipitate ob- examination of blood stains in 1923. Fujiwara (1922a) said served in the microscope under dark field illumination that heat-coagulated serum elicited antibody production as (Marx, 1920). well as did fresh serum. Schmidt (192 1), working with egg The first immunodiffusion experiment was done by Bech- albumin, had shown that antibodies obtained with denatured hold in 1905, although his primary interest was not in protein could react with the denatured protein as well as antigen-antibody reactions. He allowed goat serum to with the native molecules. Blumenthal (1927) reviewed in diffuse into a gelatin medium into which had been incorpo- detail the methods for making antisera, and gave what he rated rabbit antiserum to the goat serum. The experiment regarded as the best protocol for immunization. Proom was done to obtain a precipitate different from those ob- (1943) was able to obtain potent antisera in over 80% of the tained when inorganic chemicals were allowed to diffuse rabbits used with alum-precipitated antigen. Over the years, together in gels and form precipitates. One can, for example, many investigators have looked into the preparation of anti- put a drop of Na2Cr207onto a gel into which has been sera for the precipitin test. Many of these studies are dis- incorporated AgNOS, and a series of concentric rings of cussed in some detail in Schleyer's (1962) review. AgCr207precipitate will form. These are called "Liesegang At present, many laboratories obtain antisera from com- rings", and Bechhold attributed their discovery to R. E. mercial sources. Schleyer (1962) said that antisera should Liesegang in 1898. No notice appears to have been taken of meet certain criteria before being employed in medico-legal Bechhold's experiments by the immunologists of the time. In work. They should be sterile, free from turbidity, of high 1920, Nicolle et al. devised a single immunodiffusion tech- potency and species-specific. By high potency was meant nique for the identification of bacteria. Bacteria were grown that the antiserum should have a minimum titer of 1:104 to in agar medium containing specific antisera for particular 1:2 X 10' against homologous antigen, and quite obviously bacterial species. If the bacterial cell wall antigens were the antiserum must not cross react with the sera of other homologous to the antiserum, a halo formed in the gel animals to be exlcuded in the tests at the dilutions at which around the colony. None of the early workers properly ap the test is performed. Otto and Somogyi (1974) said that preciated the meaning and potential applicability of multiple most of the cross reactions of antihuman sera with non- precipitin band formations, which were occasionally ob- human animal sera were accounted for by antibodies to IgG served, but thought they were similar to the Liesegang phe- and to a-globulins. nomenon. The possibility that they might represent more It is worthy of mention that the use of adjuvants in immu- than one antigen-antibody system, present in the same test nizing animals to obtain antisera has permitted better yields system, was overlooked. of higher titer antisera in many cases. Since the work of In 1946, Oudin published his first paper on immuno- Freund (see, for example, Freund and McDermott, 1942), diffusion. This work represented the beginning of the devel- many immunologists have employed adjuvants in the prepa- opment of the technique as a powerful immunological tool. ration of antisera. Herbert (1973) discussed the use of Antiserum containing gels were overlayed with homologous water-in-oil adjuvants, giving a review of the technique as antigen solutions and the behavior of the precipitin band well as some of the experimental details. which formed in the gel was studied. The precipitin band appears to diffuse through the gel in proportion to the initial 16.1.2 More recent deveiopments-gel methods concentration of antigen, its diffusion coefficient, and in- Another important development has been the evolution of versely to the antibody concentration. Multiple antigens methods which use gel media for detection of the antigen- cause the formation of multiple bands, which migrate inde- antibody reaction. Before gel media were introduced, the pendently. In succeeding years Oudin developed the method precipitin test was carried out in tubes. Often, antiserum, further and devised a theoretical basis for the observations which is more dense than the usual aqueous (e.g. saline) (Oudin, 1947, 1948 and 1949). A review of the principles antigen solutions, was placed into a tube and antigen solu- underlying Oudin's technique and immunodiffusion gener- tion carefully layered over the top so as to form an interface ally may be found in Oudin (1971). Aladjem (1 97 1) and at which the precipitate could then form. Alternatively, the Oudin and Williams (197 1). antigen solution could be placed in the tube first, and the Oudin's technique is called single irnmunodiffusion. It is antiserum solution layered underneath it by means of a cap- discussed in connection with the principles of immuno- illary pipette. This procedure is sometimes called a "ring diffusion in section 2.2. test". The earlier workers all employed this sort of method, The late 1940's witnessed the development of techniques in many cases using what would be considered by present for double diffusion analysis of antigen-antibody reactions in standards excessive quantities of reagents, on the order of gels by Elek in England and Ouchterlony in Sweden. The one to several mQ. Material can be conserved by carrying out method was developed primarily for assessing the diphtheria the test in small capillaries. The use of capillary tubes was bacillus toxin reaction with homologous antitoxin. Elek pub- Species of Origin-Immunological and Serological lished his preliminary findings in 1948, a more detailed samples and controls to be run simultaneously in a short account following in 1949. Ouchterlony published his first period of time, using only a few pIl of material for each test. account in 1948 as well (Ouchterlony, 1948a and 1948b). In this method, small wells about 1.5 mm in diameter are The theoretical basis was laid down in a series of papers in punched in an agar gel about 5 mm apart. The stain extract the following year (Ouchterlony, 1949a, 1949b and 1949~). (antigen) is placed in the cathodic well of a neighboring pair, Elek's interest in the subject did not continue, but Ouchter- and the antiserum in the anodic one. The y-globulin anti- lony has continued to work in the field, one of the best single bodies migrate cathodically because of electroendosmosis, references being his monograph, published in 1968. This while the other serum proteins migrate anodically. The net work covers not only the agar gel method, but discusses some result is a precipitin reaction occurring about midway be- of the other support media such as cellulose acetate mem- tween the paired wells. The test was perfonped in veronal branes. Detailed descriptions of various methods are given, buffers, pH 8.6, containing calcium lactate. Carny reported as well as the underlying theoretical basis for the variations a similar technique in 197 1. Grunbaum (1972) noted that which have been described. Crowle's book also gives a very crossed over electrophoresis could be done on cellulose ace- complete treatment of the subject,(Crowle, 1973). Oakley tate membranes as well. Although it is usual practice to and Fulthorpe (1953) developed the double diffusion tech- prepare antisera against whole human or animal sera, Tran nique for use in tubes, with special reference to its applica- Van Ky et al. (1968) recommended the use of antisera pre- tion to the analysis of bacterial toxins. Double diffusion is pared specifically against the y-globulin fraction. discussed in section 2.2.2. Lincoln (1975) reported on a very interesting series of Muller et al. (1950) applied Oudin's technique to medico- cases involving alleged witchcraft, illustrating how the legal practice. Gum acacia was tried as a medium but was events were unraveled by determining the species of origin of abandoned in favor of agar and agar-gelatin mixtures (Mul- the articles involved. ler & Michaux, 1952). In 1958, Muller et al. noted the applicability of the double diffusion methods. Species deter- mination could be carried out using a microdiffusion tech- 16.13 Effects of some external intluences nique in agar originally devised by Hartmann and Toillez in The age of a stain alone apparently does not prevent a 1957 (Muller et al., 1959). Mansi (1958) devised a positive precipitin test. Haseeb (1972) got a positive test on microimmunodi!Tusion technique which was used and rec- a 12year old stain kept at room temperature. Lioli (1971) ommended by Fiori (1963). Gajos and Brzecka (1968) said examined the "flesh and blood" from the eucharistic miracle that threads from bloodstained fabrics could be incorporated that is said to have occurred at Lanciano, Italy, in the 7th directly into the gel for species determination, thus avoiding Century A.D. The blood, which was about 1200 years old at not only the time required for extraction, but the concomi- the time of examination, gave a positive precipitin test with tant loss of material. Maresch and Wehrschntz (1963) de- anti-human serum. Smith and Glaister (1939) reported a scribed a precipitin test for species determination carried out positive precipitin test on extracts from mummified tissue on microscope slides in 2 mm thick agar gels. Feinberg many thousands of years old. It has been known for a long (1961) devised a "microspot" double diffusion test, which time, however, that old blood stains do not easily yield serum could be carried out on microscope slide cover slips. Katsura proteins to mild, aqueous extracting solvents, like water or (1976) utilized this method to diagnose the species of origin saline. Dorrill and Whitehead (1979) said that 5% (v/v) of very small amounts of blood using anti-human Hb anti- 0.880 ammonia was a much more effective extraction me sera (see section 7.1 ). The precipitin lines were visualized dium for older stains than water for purposes of extracting using a phase contrast microscope, and even stains 48 years serum protein for species determination in older stains. old were said to react within 3 hrs at 30". Feinberg (1962) Muller et al. ( 1966) showed that a positive precipitin test published a modification of his spot test for use on cellulose could be obtained on stained garments which had been dry acetate membranes. El-Guindi (1972) reported successful cleaned. There are circumstances, however, under which the results with an ordinary macroimmunodiffusion procedure precipitin test may fail. Since antisera are almost universally in agar gels. He used several arrangements of sample and prepared against serum proteins, serum protein must be antiserum wells. present in, and extractable from, the stain if a reaction is to In 1959, Bussard suggested taking advantage of the elec- be expected. There is not a necessary relationship between troendosmotic properties of agar in carrying out immu- the amount of hemoglobin and the amount of serum protein noelectrophoretic analysis of antigen-antibody reactions. A in a stain extract. One cannot judge how much serum protein system could be devised in which the antigen and the anti- may be present on the basis of the amount of pigment body migrated toward one another, a precipitate forming at present, as was pointed out by Okamoto in 1902. With fairly the point of their interaction. This technique, he called elec- freshly dried stains there is a strong likelihood that consid- trosyneresis. It has also been called electroprecipitation and erable blood pigment will be extracted, but sometimes with- crossed over electrophoresis or crossing over electrophoresis out sufficient serum protein to give a positive precipitin test, (see section 2.4.2). Culliiord (1964 and 1967) devised a particularly if extraction time is short (Mueller, 1934). crossed over electrophoretic technique for species determi- 24 hour extraction times are recommended in such cases. nation in forensic casework which allowed a large number of Mezger et al. (1933) noted that with a blood stain on wood, Sowrebook in Forendc hlogy, i~mmunoiogv,and Biochemirtv the dissolved superficial layers of stain did not give a precip Lee and DeForest (1976) devised an interesting variation itin test. It was necessary to extract the wood substratum, of the precipitin test, applicable to stains which contain the serum protein having soaked into the wood more deeply species-specific antigens, but which do not readily yield them than the red portion of the stain. Olbrycht (1950) showed up to extraction media. They could show that a substantial that extram of dried human blood mixed with earth can fail fraction of the species-specific serum protein (saline- to give a positive precipitin test with anti-human serum, pre- extractable protein) was lost upon steeping bloodstained sumably because of the absorptive properties of the soil. In cloth in water or detergent solutions. The loss of protein all cases in which the precipitin test failed, the extract did not positively correlated with the temperature of the washing contain detectable protein. The same results were obtained water from 25' to 100". This result was not in accord with with dried blood mixed with aluminum oxide or finely pulver- that of Smith and Glaister (1939), who observed that hotter ized iron ore. Some bloodstained earth samples did yield water extracted less protein from stained fabric than room precipitin-positive extram, as did bloodstained limestone and temperature water, and thought that the hotter water tended bloodmined sand. A few chemicals caused nompecSc to fix the stain, and prevent protein loss. Losses were greater precipitation of the antiserum, and of normal control serum in Lae and DeForest's experiments if a cold water rinse step as well. These included aluminum and iron chlorates, was included following the washing step. The results indi- aluminum chromate, tannic acid and an extract of spruce cated that only a very weak precipitin test at best could be bark. Some organic solvents, dilute acids and bases, and obtained from bloodstains exposed to washing water at peroxide cause non-spedlc precipitation of the antiserum temperatures of 75' or hotter, without agitation. (Vollmer, 1949). It was thought that some species-specific protein might still Exposure of bloodstains to lengthy steeping in cold water be present in the stains, however, notwithstanding the fact does not prevent their giving a positive precipitin test. Older that it could not be extracted with saline in quantities stains yield less protein to the steeping fluid than do fresher sufficient to give a precipitin test. An inhibition test was ones, and older bloodstains retained more serum protein in therefore devised in which the washed stained material was 50" water than in cold water (Smith and Glaister, 1939). incubated with relatively low titer antihuman serum (1:16) Detergents do interfere with the precipitin test. Some de- for 24 hr at 4". Aliquots of the antiserum were then removed tergents, even in dilutions up to 1:104, will give a flocculent and tested in an Ouchterlony double diffusion system with precipitate with antiserum as well as with normal serum fresh human serum to see whether the stained material had (Burger, 1956; Klose, 1961). Burger (1956) thought these substantially reduced the titer of the antiserum. Positive reactions were caused by alkyl- and arylsulfonates or sul- results were obtained, and it could be shown that washed fates in the detergents. Water rinsing does not entirely control bloodstains made from cow, pig, horse, sheep, duck, remove these chemicals, but rinsing stained fabric with a chicken, rabbit, dog and cat bloods had no inhibitory effect solution of methanolethanol-amyl alcohol-trichloroethylene on the antihuman serum. It was noted that this procedure prior to saline adraaion does rid the substratum of the in- could be applied equally well in situations in which the terfering substances, and allows the test to be performed. bloodstain contained a soluble substance which interfered Burger noted that these observations underscored the impor- with the ordinary precipitin test. The interfering substance tance of running cloth controls. Schoenherr (1957) noted that could be washed out, and the stain then subjected to the detergent residue on glass can cause precipitation of a test inhibition procedure. serum, a point well worth noting if the test is done in Itoh (1979) devised a somewhat similar kind of inhibition glassware that is washed and re&. test for species determination using anti-human serum. He Exposure of bloodstains to heat causes the precipitin reac- said that the procedure was applicable to bloodstains and to tion to become weaker, and reaction time longer. There is an muscle tissue. Here, anti-human serum of appropriate titer upper limit of exposure, beyond which the reaction will be was incubated with the material to be tested. The absorbed negative. The limit is a function of both the temperature and antiserum was then titrated with latex particles, coated with urposure. the time of Smith and Glaister (1939) could still serum protein antigens (see also in section 16.5). get a positive test on stains exposed to 1W or 150' for 15 min, or to 200" for 2 min. Exposure to heat also tends to At various times, there has been interest among immu- 6x the stains and render the protein increasingly insoluble. nologists in the preparation and characteristics of antisera This phenomenon was observed very early by Katayama against heatdenatured serum proteins, particularly the (1888) and Hammer1 (1892) with regard to hematin com- .comparison of reactions of such antisera with native and pounds. Stains which have been ironed may be extremely denatured antigen (see, for example, Schmidt, 1908). These insoluble (Schech, 1930). Much longer extraction times are experiments were of significance in advancing immu- required for stainsthat have been exposed to heat than under nological knowledge, but not of much practical significance ordinary circumstances (Schech, 1930; Schleyer, 1948 and for medico-legal investigations (Schleyer, 1962). It is clear 1962). It is sometimes necessary to use 0.1N NaOH for that moderate exposure to heating, e.g., 10 min at 55-70', extraction in these cases since saline will not extract the causes denaturation in serum proteins leading to changes in proteins even after extended periods of steeping (Schleyer, the shape of the molecules and giving rise to aggregation, but 1948). such proteins retain their antigenicity (Peeters et al., 1970). 16.1.4 Tests with anti-human hemoglobin antisera the color of the extract spot compared with the colors of the Preparation and use of anti-human hemoglobin sera has "standard" spots. Lynch (1928) mentioned three other been discussed in section 7.1. These antisera can be used for methods for estimating the protein concentration in stain the determination of species of origin as well as for the extracts. The so-called "foam test" is based on the fact that identification of the presence of blood. Anti-hemoglobin sera 1:1000 dilutions of serum in saline, which have air bubbled are generally not as potent as anti-serum sera, because he- through them, will form bubbles at the surface which persist moglobin is not as antigenic as most of the serum proteins. for about 10 min while in greater dilutions of serum, the The same considerations apply to the use of anti-human bubbles disappear almost at once. Nitric acid precipitates hemoglobin as do to the use of anti-human serum reagents. protein, and extract can be layered over a nitric acid solution Assuming species specificity has been established for an anti- to see whether an opalescent precipitate forms at the inter- human Hb senun, however, its reaction with a bloodstain ex- face. In 1:1000 dilutions of serum, a faint precipitate will be tran may be regarded both as an indication of the presence observed, while in greater dilutions, no precipitate will form. of hemoglobin (presence of blood) and a diagnosisof human A similar test is based on the protein precipitating power of origin. sulfosalicylic acid. The opalescence caused by addition of '/, of a volume of 50% sulfosalicylic acid to bloodstain extract 16.1.5 The antigen-antibody reaction. Optimization of may be compared to that in a series of standards prepared reactant concentrations from dilutions of whole serum. Schleyer (1962) discussed No complete review of the theory of antigen-antibody the sulfosalicylic acid test in his review. It is usually said that reactions would be appropriate here. A good deal of work a 1:1000 dilution of whole serum or its equivalent in a stain has been done on the subject and some discussion is given in extract is optimal for carrying out the precipitin test. section 1.3.4.2. Munoz and Becker (1950) and Becker et al. It may also be mentioned that the pH of stain extracts (1951) gave a detailed treatment of the reaction as it takes should be in the neighborhood of neutrality for best results place in Oudin's single diffusion system. The precipitin reac- with precipitin reactions, and to diminish the possibility of tion in solution was discussed by Kendall (1942). The effect pseudoreactions. Normally, there will be no problem be- of salt concentration was studied in detail (Aladjem and cause saline is usually used for extraction, but if 0.1N Lieberman (1952) and Lieberman and Aladjem (1952). A NaOH or ammonia were required as an extraction medium, very good review by many specialists may be found in Chap- some neutralization would be necessary. ters 13 and 14 of Williams and Chase (197 1). With some variations, depending upon the system under 16.2 Antlhuman Olobulln Serum study, antigen-antibody reactions have certain character- istics in common. It has been known for a long time that, if lnhlbltfon one titrates a fixed quantity of antigen with decreasing Antihuman globulin serum (AHG serum, Coombs' amounts of antibody, the antigen-antibody reaction does not serum) is an antiserum prepared against human serum glob- occur at extreme excesses of either component. In many ulin. Since the antibodies of serum are in the globulin precipitin systems, for example, no precipitation is observed fraction, AHG serum contains antibodies to the other anti- at the excess antigen or the excess antibody extremes of the bodies, a fact whose significance will become clear in the titration series. It is important, therefore, to obtain some discussion below, and see section 1.3.4.1. idea of where the optimum precipitating concentrations lie In 1949, Wiener et a!. suggested a new serological test for in order to insure that the conditions chosen will not be the determination of human serum globulin. The serum characterized by an excess of either component. In carrying globulin is human specific, and the application of the method out the precipitin test for determination of species, it is to species determination is based on this property. There are common practice to make dilutions of the serum, or of the certain kinds of antibodies to the Rh, (D)receptor of human stain extract. The antiserum is often employed undiluted or red cells which do not agglutinate Rh+ cells in saline. These at a partiahconstant dilution. Sithere is considerable so-called "incomplete" or "blocking" antibodies do, how- variation in the concentration of proteins in bloodstains, as ever, combine with the antigenic receptor and sensitize the well as variation in the efficiency of extracting them, some Rh+ cells (see sections 1.3.4.1 and 22.4). The presence of attention has been paid to determining the protein concen- such antibodies on red cells can be detected by using anti- tration of stain extracts. The extract can then be diluted if human globulin serum. The AHG antibodies are capable of necessary to optimize the protein concentration prior to per- combining with the anti-Rh antibody which is, in turn, forming the test. Allison and Morton (1953) devised a sim- bound to the red cell. If AHG serum is added to red cells ple test for approximating the amount of protein in a stain with incomplete antibody bound to their Rh,, (D) receptor, extract. Drops of progressively doubly diluted blood solu- agglutination will occur. Cells with incomplete antibodies tions were placed onto a filter paper and dried. A dried drop attached to their receptors are said to be "sensitized. This of stain extract was treated similarly, and the concentration principle is illustrated in Fig. 16.1. A bloodstain contains the of protein estimated by eye on the basis of color. If a more human serum globulin proteins, and, if incubated with AHG accurate estimate is wanted, the dried spots could be stained serum, will bind the antibodies and reduce the titer of the with a protein stain, the paper background destained, and AHG serum. An inhibition test can therefore be devised, in 3 A + >- yAYfi 1 Red cells with A representing Rho (Dl receptor Incomplete anti;Rho (Dl Wash to remove % unbound antibody 9L I C

representing the antigenic determinant Agglutination Antihuman globulin on the -globulin anti-Rho ID) antibodies antibody molecule

Figure 16.1 Scheme of Agglutination of Sensitized Rh+ Cells by Antihuman Globulin Serum Species of Origin-Immunological and Serologkal which bloodstained material is incubated with AHG serum, reagents in order to select the optimal concentrations for and the serum then tested to see whether it will agglutinate actually performing the test. Schleyer (1962) discussed this sensitized Rh+ test cells. If no agglutination occurs, the technique in his review. Grobellar et 41. (1970a) said that bloodstained material has removed the antibodies from the they selected their antisera for the test based on the criteria AHG serum, and it is, therefore, of human origin. If agglu- established by Proom (1943) for precipitin sera, namely that tination does occur, the stain did not reduce the combining human serum should react at dilutions of 1:8000 while other power of the AHG serum and it is, therefore, not of human animal sera should not react at dilutions of 150 or greater. origin. The test for human origin is illustrated schematically It is important to include proper controls in this test, includ- in Fig. 16.2. In practice, the test is slightly more involved, in ing the usual cloth control, and controls on the various re- that appropriate dilutions of the AGH serum must be chosen agents used in the test. Cramp (1959) said that he routinely so that the reduction in titer brought about by the serum included a positive human bloodstain control and two nega- globulin in a human bloodstain can be detected by the test tive animal bloodstain controls as well. Hunt et al. (1960) system. Wiener et al, utilized a doubling dilution titration showed that the antihuman globulin inhibition test was pos- technique, and could quantitate the serum globulin in the sible with a 1 cmZpiece of cloth containing no more than test sample. Urine, semen, cerebrospinal fluid, saliva, and 0.1 pjl of blood. Saliva and tears caused reduction as well if the sera of ox, horse, rabbit and Rhesus monkey did not they were not too dilute. They recommended an AHG serum inhibit the AHG reaction, and it was suggested that the which, when diluted 1:400, gave a titer of 1:8 against R&+ method might find a medico-legal application. (D+)cells strongly sensitized with anti-D (anti-RhJ. Anderson (1952) reported that the method was more Klose (1962) indicated that the presence of detergents in specific than the precipitin test, and could be applied to the test samples will interfere with the test, unless a separation detection of any animal serum, provided specific anti- procedure is employed to get the globulin away from the globulin serum was available. The work was extended in surfactant material. The procedure using paper chro- 1954, and he could show that the technique was applicable matography, described in section 7.2, was found to be satis- to bloodstains. Human blood could not be distinguished factory, and the detergent material could be located by its from chimpanzee blood, but could be differentiated from all fluorescence under UV light. other animal bloods tested. Allison and Morton (1953) Patzelt er al. (1977) said that the test could be done using confirmed Anderson's (1952) findings. They showed that the human y-globulin-coated latex particles. These are available test worked well with bloodstains on different substrata. It commercially for use in testing for Rheumatoid Arthritis also gave positive results with alkalidenatured serum pro- Factor in serum. Rheumatoid Arthritis Factor behaves as an tein, although the inhibitory effect was reduced, and a posi- antibody to human y-globulin. Patzelt et al. said that false tive test for human blood was obtained on cadaveric material positive reactions could be gotten with the blood of higher from a mummy 5000 years old. mammals, and false negative results with sera from people The value of the test was soon confirmed by a number of with high concentrations of Rheumatoid Arthritis Factor. other laboratories. Several different techniques were pro- The false negatives were not seen, however, in bloodstain posed, some calling for titration, while others arranged the tests. dilutions of the reagents in such a way that a one-step "all- or-none" procedure could be followed. Vacher et al. (1955) gave their method and said that semen, saliva, urine, me- conium and fecal matter did not give inhibition. A variety of natural and synthetic textile materials were found not to In 195 1, Boyden observed that certain preparations of interfere. Ruffit and Ducos (1956) published their method inulin could render red cells able to absorb proteins onto for carrying out the test, and Dbobert et al. (1957) enlarged their surfaces from solution. Inulin is a storage polysac- upon their earlier (Vacher et a[., 1955) studies. charide in certain plants (e.g. the Jerusalem artichoke) and Jungwirth (1956) in Germany, Dell'Erba (1957) in Italy is a polymer analogous to starch except that it is made up of and Liberska and Smigielska (1958) in Poland all confirmed fructose units instead of glucose units. Boyden found that the specificity and sensitivity of the method. Duws (1958a) preparations of inulin which rendered the cells able to absorb described the techniques for the AHG serum inhibition test, protein could, at high dilution, agglutinate the cells. Inulin as well as for the precipitin test and the passive hemag- preparations not having the property did not agglutinate the glutination test (see Section 16.3) in detail. He noted that if cells. It had been known since the work of Reiner and all three tests were performed on a sample, interpretation is Fischer (1929) that dilute tannic acid solution could aggluti- simple if all results agree, but that it becomes very difficult nate red cells, and it was decided to test tannic acid solution if different tests give conflicting results. Cramp (1959) de- for their ability to render red cells protein-absorbing. It was scribed a relatively simple version of the test which he said found that sheep red blood cells treated with 1:20,000 dilu- had been found to be quite satisfactory in 170 cases in New tions of tannic acid readily absorbed proteins from saline South Wales. Mosinger et al. (1960) described a two dimen- solutions. The cells could then be washed, and an antiserum sional titration protocol for the AHG serum and the anti- homologous to the absorbed proteins would readily cause Rh, serum which, it was said, should be carried out with the agglutination of the cells. Red cells which have been treated Bloodstained fragments with representing the Antihuman globulin Remove aliquot Add Rh+ cells human globulln antigenic antlbodles of antiserum sensitized with Result receptor incomplete anti-Rh

Agglutination

or non-human bloodstained fragment

Figure 16.2 Scheme of Testing Bloodstain for Human Origin by the Antihuman Globulin Inhibition Method Speck of Origin-Zmnolo~and Semlo&d with dilute solutions of tannic acid to render them protein- "agglutinated" to the fiber rather than being agglutinated to absorbing are referred to as "tanned cells". one another. Coombs et al. (1952) used a somewhat diierent technique A test for human species origin of a bloodstain was devised to achieve essentially the same result, namely to devise an using this principle, and the technique called the "mixed agglutination method for the assay of soluble antigens. antiglobulin reaction" by its originators. A bloodstained Using benzidine tetramnium chloride as a conjugating thread, which, if human blood is present, contains human agent, egg albumin and chicken globulin proteins could be globulin, is incubated with AHG serum. The test material is conjugated to incomplete Rh antibody molecules without then washed to remove excess, unbound antibodies. Test affectingthe latters' ability to combine with the Rh receptor cells are Rh positive and have been sensitized with an incom- on the red cell. Red cells could then be sensitized with anti- plete anti-Rh antibody. The mixed agglutination (mixed an- Rh antibodies to which had been conjugated either oval- tiglobulin) reaction occurs if any AHG has been bound to bumin or chicken globulin protein. Antisera to either of the fiber, i.e., if the fiber contined a human bloodstain. The these proteins would then bring about agglutination of the principle is illustrated in Fig. 16.3. Hara et al. (1969) em- cells having the homologous antigen present. ployed the method, although they did not use AHG senun. Ducos (1956) put the tanned red cell technique to use in An anti-human serum mum, which had been absorbed with medico-legal species determination. Using a modified monkey serum,was used. The test cells were tanned erythro- tanned red cell preparation method described by Dausset cytes, to which had been absorbed human serum proteins. et al. (1955), he incubated bloodstain extracts with the They said the technique was more sensitive than the precip tanned red cells. The cells were then washed, and tested for itin test either in tubes or in agar gels. They referred to the agglutination with AB serum, a series of blood group anti- technique as the "double combination method", a term bodies, and AHG serum. Of greatest interest was the result which the Japanese workers have usually preferred to obtained with AHG serum. Only cells which had been ex- "mixed agglutination" (see section 1.3.4.1 ). posed, after tanning, to human bloodstain extract, and which had therefore absorbed human globulin, were agglutinated by the AHG serum. The reaction was very specilic and Ducos noted that it had the advantage over the AHG- 16.5.1 Sensitized colloidon particks inhibition method (section 16.2) that agglutination in this In 1925, Freund established the groundwork for particle case represented a positive test, whiie a positive test in the agglutination techniques in his physico-chemical studies on former case was represented by the absence of agglutination. the agglutination of tubercle bacilli, and of protein-lipid Ducos extended these studies (1958a, 1958b) showing that, extracts from these cells. The cells and the lipoprotein par- whiie this technique was somewhat less sensitive than the ticles derived from them are maintained in a dispersion by a precipitin and AHG-inhibition tests, it was more specific. In surface potential difference. When the potential is reduced cases of exposing tanned red cells to human bloodstain ex- below a threshold value, the dispersion is destabilized and tracts in doubting dilutions, and testing with AHG serum, the particles aggregate. All sera have the effect of reducing agglutination could be observed at dilutions of 1:5000. In the potential, but if the particles are coated with an antigen, 1960, Ducos again described the method, suggesting that in specific antiserum reduces it more effectively to a sub- medico-legal cases, several of the techniques should be used threshold value, and aggregation occurs. Jones (1927) in order to be more certain about the species of origin. Hara applied this principle to the agglutination of colloidon par- et d. (1%9) incubated stain extract with anti-human senun, ticles, to which had been absorbed ovalbumin, by a specific washed the samples, then eluted the bound antiiy, which was using anti-albumin serum. In 1940, Cannon and Marshall de- detected tanned red cells to which had been ab- scribed a technique for the accurate determination of the sorbed human serum protein. titer of precipitin antisera using sensitized colloidon par- ticles. Antigen was absorbed onto the particles, which were then washed to rid the suspension of unabsorbed antigen. 16.4 MlxdAntlglobulln Tochnlquo The antisera could then be titrated with accuracy using these In 1963, Styles et al. described a mixed agglutination "indicator" particles. technique for species determination based on the species- Giaccone (1958) applied this principle to the deter- sptcific serum globulin present in bloodstains. The principle mination of species of origin of bloodstains. Bloodstain ex- underlying mixed agglutination is as follows: a complete tracts were incubated with washed colloidon suspension for antibody to an antigen contained in the bloodstained fibril is 12-24 hrs at 4". The particles were then thoroughly washed allowed to react with the receptor in the stain, and the excess and resuspended in saline. A small aliquot of the particles antibodyis then washed away. Red cells containing the same was incubated with an equal volume of antiserum, the mix- antigen are then incubated with the "sensitized" stain fiber, ture centrifuged, shaken lightly, and read for agglutination and will be bound by the remaining combining site of the either macroscopically or microscopically. Positive, specific antibody, resulting in the cells arranging themselves in an reactions were obtained with undiluted and with 1: 10 diluted orderly fashion along the fiber. The test cells are in effect bloodstain extracts. Concentrated control sera incubated Sourcebook in Forensic Stmlogv, Zmmnology, and Biochemistry with the colloidon mixture instead of bloodstain extract of 1: 1600) being given by milk. It was found, however, that sometimes caused spontaneous agglutination. Spontaneous milk agglutinated the unsensitized latex particles as well. agglutination also sometimes occurred when stain extract Whitehead and Brech (1974) indicated that the latex par- was added, but did not survive the washing steps. ticle technique could be applied to an extract from a single Schleyer (1962) reported that he had conducted experi- thread, which could then be employed for blood grouping by ments on this method in collaboration with Hilgermann in the elution technique since the species-specific antigens are 1960. The method was found to be specific and useful, and water soluble whereas the blood group antigens are not. no spontaneous agglutination was observed in the course of these studies. Human serum in dilutions of 1:8000 gave positive, though weak reactions, whereas the precipitin test 16.6 Other Immunological Test8 could not be obtained at dilutions of human serum greater 16.6.1 Complement fixation than 1:4000. Complement (or alexin) is a multi-component system of 16.5.2 Sensitized latex particles serum proteins, and participates in the action of lytic anti- In 1956, Singer and Plotz applied the particle aggluti- bodies (see section 1.3.5). Antibody must be present on a nation principle to the serological diagnosis of rheumatoid cell, bound to its receptor, before complement can combine arthritis, using sensitized latex particles. Robbins er al. with that cell causing lysis. In most antigen-antibody (1962) showed that latex particles coated with human cho- reactions, the resulting complex will combine with com- rionic gonadotropin (see section 8.2.1) were agglutinated by plement if it is present. Such combination is known as com- a specific anti-HCG serum, and suggested that the technique plement fixation. For purposes of the present discussion, might well lend itself to the development of a pregnancy test. complement fixation can serve as an indicator that an In 1973, Cayzer and Whitehead applied the sensitized latex antigen-antibody reaction has occurred (see section 1.3.5.3). particle technique to medico-legal species diagnosis. The Since complement participates in the lysis of red cells by latex particles were sensitized with immunoglobulins, iso- specific hemolysins, and is in fact required for hemolysis, lated from a sheep immune anti-human serum, in glycine hemolytic systems are convenient tools for the detection of buffered saline at pH 8.2. Protein concentrations of between the presence or absence of complement. All sera contain I and 2 mg/mrP with equal volumes of 2% latex suspensions complement, but the amount varies even among members of were routinely employed for sensitization, but these condi- the same species. Guinea pig serum is a common source of tions varied depending upon the particular globulin prepara- complement in complement fixation assays. If an antigen tion. Dilutions of serum or of stain extract were mixed with and its antibody are allowed to react in the presence of the sensitized latex particles on a glass plate, the mixture complement, some complement will be taken up, or fixed. rocked gently for 2 min, and agglutination read macro- The amount of complement added to the system can be scopically against a dark background. The test was positive arranged so that all of it is fixed. If the solution is now tested with human serum dilutions as high as 1: 10,000. Cross reac- with a hemolytic system, its hemolytic power will be found tions with the sera of closely related primates were positive to have been lessened or abolished. A common hemolytic at similar dilutions. All other animal sera tested gave nega- system used in these assays consists of sheep red blood cells tive results at serum dilutions greater than 1:100, and in which have been sensitized with an anti-sheep red cell hemo- some cases at dilutions greater than 1:40. Extracts of 2 year lysin prepared in rabbits. If such test cells are incubated with old human and primate bloodstains gave strongly positive a test solution, and hemolysis occurs, complement was still reactions while extracts from stains of turkey, rabbit, ham- present in the test solution indicating that it has not been ster, rat, cat, mouse, dog, sheep, horse, cow, pig and donkey fixed, i.e. that the antigen-antibody reaction which was being bloods gave negative reactions. Neat human saliva gave a tested has not taken place. Conversely, if no hemolysis oc- strongly positive reaction but the reaction diminished rap- curs, complement has been fixed and the antigen-antibody idly at saliva dilutions greater than 1: 100. Neat human reaction may be inferred to have occurred. The complement semen gave a weak positive reaction. These studies were in the serum containing the hemolysin is inactivated by heat- extended to animal bloodstains (Whitehead et al., 1974) and ing prior to sensitizing the cells. latex particles were sensitized with the immunoglobulins of It may be noted that a number of the early studies on antisera against deer, cow, sheep, dog, cat, horse, mouse, hen complement, and its ability to be "fixed" in antigen-antibody and guinea pig as well as human sera. Some cross reactions reactions, were carried out by the Nobel laureate immu- were seen in the experiments with sera, but with the blood- nologist Jules Bordet and his collaborators. Bordet showed ' stain extracts, the only cross reaction was a weak reaction of in 1898 that an anti-rabbit erythrocyte hemolysin, prepared cow bloodstain extract with anti-deer particles. A few of the in guinea pigs, would first clump and then dissolve (hemo- anti-animal particles reacted with human rheumatoid sera, lyze) rabbit red cells. He could show further that both anti- but not at dilutions greater than 1:500. A number of other body and complement were required for the lysis to take substances were tested for non-specific reactions with the place, and that complement, but not antibody, was inac- anti-human particles. Soap solutions and fabric softener tivated by heating for about 30 min at 60'. Since it was not gave weak reactions, the only strong reaction (to a dilution clear at the time that one and the same antibody molecule Sourcebook in Fore& Serology, Immunologv, and Biochemisiry could bring about a variety of different effects when the overlooked the fact that sweat may contain serum pro- antigen-antibody reaction occurred (such as agglutination, teins which can react with an anti-human serum serum lysis, fixation of complement, etc.), antibodies were given (Sutherland, 1907). Ehrnrooth (1906) got inhibition of descriptive names, based upon their action in a particular hemolysis with 1:80,000 dilute human serum in a system system (e.g. agglutinin, precipitin, lysin, etc.). Antibodies which used rabbit serum as the source of complement and a which "sensitized" cells so that they could subsequently be goat red cell-rabbit immune anti-goat erythrocyte antiserum lysed in the presence of complement were called "sensi- as the detection device. Muir and Martin (1906) showed bilisatrices" by the French workers and "amboceptors" by that complement fixation occurred with 10 n!2 human serum the German workers. Gengou (1902) established that com- incubated with 50 $2 of a potent rabbit anti-human serum. plement could be fixed in precipitin reactions as well as in 1pQof human serum was required to obtain a definite precip lytic ones. This fact, he demonstrated using rabbit antiserum itin test, indicating that the complement fixation test was to cow milk proteins and to ovalbumin, as well as in several about 100 times more sensitive than the precipitin test. They other precipitating antigen-antibody systems. These studies suggested, as had Friedberger (1906), that only very potent were enlarged upon by the work of Gay (1905a and 1905b) antisera, which could bring about complement fixation with and of Bordet and Gay (1906). 10 nQ human serum, should be used in medico-legal The complement fixation test for the medico-legal diagno- investigations. sis of species of orgin was introduced by Neisser and Sachs Uhlenhuth (1906a) tested a number of articles in his col- (1905 and 1906a). They were prompted to carry out their lection by complement fixation and obtained a number of experiments by the studies of Moreschi (1905) on the anti- false positive reactions. Some materials contained sub- complementary properties of certain sera. Neisser and Sachs stances which could bring about the non-specific fixation of used a hemolytic system consisting of rabbit immune anti-ox complement. A number of other materials, such as cotton- erythrocyte serum, known to be hemolytic for sheep red wool, pasteboard and gauze, which could be substrata for cells, and the sheep red cells were sensitized with this anti- bloodstains, were negative. He got a complement fixation serum. Fresh guinea pig serum served as the source of com- test in a case (Uhlenhuth, 1906a) from a bloodstain on a plement. The antiserum had had its complement inactivated. sack, when the precipitin test was negative and where exam- 1 mQof a 5% suspension of sheep cells was readily hemolyzed ination of the bloodstain showed quite unequivocally that it in the presence of 5 pL antiserum and 50 pL guinea pig was of avian origin. He was inclined not to put too much serum. Anti-human serum serum, prepared in rabbits, did confidence in the test, therefore, and did not think it should not interfere with the hemolytic reaction. However, if 0.1 pl be trusted as a check on the precipitin test (Uhlenhuth, human serum or 1 pl monkey serum and 0.1 pl anti-human 1906b). Neisser and Sachs replied (1906b) that adventitious serum was incubated with the complement, and the indicator substances could sometimes bring about non-specific com- system then added, no hemolysis occurred. Rat, pig, goat, plement fixation, and that a boiled bloodstain extract control rabbit, horse and ox sera had no such inhibiting effect on the should be run, since boiling would destroy the specific reac- hemolytic reaction. Saline extracts of 3 month old human tion but not the non-specific one. Another means of detecting bloodstains, diluted in some cases to the extent that a precip such non-specific reactions is the running of a control in itin test could not be obtained, inhibited hemolysis in the which stain extract is incubated with saline or non-immune system just as had human serum. Saline extracts of similarly serum (Sutherland, 1907). Schiitze (1906) found the com- aged bloodstains from sheep, fowl, rabbit, guinea pig, ox and plement fixation test to be specific and more sensitive than horse did not inhibit hemolysis. They recommended that the the precipitin test. Graetz (1910 and 1912) carried out a test be employed as a control on the basis of the fact that a number of studies on the method. Sutherland (1907) dis- hemolytic reaction is easier to read than a weak precipitin cussed the test, and his own experiments on its applicability. reaction, that hemolytic sera are easier to obtain than pre- Stockis (1910) reviewed this method along with the other cipitin sera, and that opalescence of the antiserum, which is major tests for species determination. Complement fixation a problem in a precipitin tube test, presented no difficulties was reviewed in detail by Pfeiffer in 1938. In 1914, in this test. Sutherland mentioned that the test was not done routinely in In another series of experiments Neisser and Sachs India because sufficiently potent antisera could not be ob- showed that with normal rabbit serum as the source of com- tained. Olbrycht (1950) noted that the test is employed only plement, hemolysis was inhibited by as little as 10 nP fresh infrequently in medico-legal practice, because of the experi- human serum incubated with 10 pIl anti-human serum. They ence required to carry it out properly, the controls that are said that an antiserum which would inhibit hemolysis with necessary, and the fact that the precipitin test is usually 0.1 p5! fresh human serum, i.e. a very high titer antiserum, more convenient and simpler. In the older literature, com- should be used in carrying out forensic tests. Friedberger plement fixation is sometimes referred to as "complement (1906) conducted a number of experiments on the tech- deviation". nique, and thought that its sensitivity might be a drawback. He obtained a "human" reaction with a saline extract of a 16.6.2 Anaphylaxis (Hyperseusitivity) stain of human perspiration and chicken blood. He was using The principle of anaphylaxis as a means of detecting a very high titer antiserum, however, and appears to have antigen-antibody reactions was discussed briefly in sec- Speciesof Origin-Immunological and Serological tion 10.4.2, and more generally in section 1.3.6. Anaphylaxis and that dessicated proteinaceous matter worked equally as as an imrnunologicai device for the medico-legal diagnosis of well in inducing hypersensitivity as the fresh substances. species of origin was explored by the earlier workers, but the Thomsen (1909) could show that bloodstain extracts were technique is too time-consuming and cumbersome to be of completely suitable as sensitizing antigens. The reaction was any real value in practice, when simpler, equally effective not completely specific, however, in that some monkey sera alternatives are available. The anaphylaxis test is also some- could cause the reaction in animals sensitized with human times called the hypersensitivity test (herempfindlich- serum. Pfeiffer (1909) showed that guinea pigs which have keitsprobe). been hypersensitized to an antigen show a marked decrease A number of workers appear to have suggested that the in body temperature upon administration of the shocking anaphylactic test be applied to medico-legal problems injection, even if the other symptoms of anaphylaxis are not around the same time, and independently of one another. present. This temperature change, he believed, should be the Thomsen wrote a full paper on the subject in 1909, which sole criterion for judging the presence of the reaction in an appeared in the March 25 issue of the Zeitschrift fur animal in medico-legal work (Pfeiffer, 1910). Uhlenhuth Immunitatsforschung und Experintentelle Therapie. Short- and Haendel (in Uhlenhuth and Weidanz, 1909) showed ly thereafter, Sleeswijk (1909) published a paper on serum that even old material could cause hypersensitivity in test hypersensitivity. On the page preceding the title page of this animals. A fourteen year old, decomposed bloodstain, which article, he made a point of noting that he had discussed the would not give a precipitin reaction, gave a positive anaphy- forensic applicability of the technique at an Academy pro- laxis test. Sutherland (19 10) pointed out that, were it possi- ceeding in Amsterdam on March 27,1909, and that his work ble to hypersensitize the test animals in advance and have had been done independently. Pfeiffer's paper appeared in them sitting around at the ready, and then simply test blood- 19 10, and had been delivered at a September, 1909, meeting stain extracts as they were received in the laboratory, some of the German medico-legal organization. Uhlenhuth took of the objections to the test's inconvenience would be an- credit for having been the first to note the medico-legal swered. Unfortunately, the shocking dose must be quite applicability of the anaphylaxis method (Uhlenhuth and large, and there is seldom enough bloodstain to produce a Weidanz, 1909). This claim is in fact true. Although he did sufficiently large amount of extract for the purpose. Since not publish a full paper on the subject at the time, he did the sensitizing dose can be very minute, the bloodstain ex- make the point clearly in a discussion which took place at the tract must normally be used in this way, necessitating the 10 Berliner Militararztliche Gesellschaft on December 14, to 14 day waiting period. Minet and Leclerq (191 1) con- 1908 (see in Dtsch. Militaeraerztl. Z. 38, Vereinsbeilage, ducted extensive experiments on the technique with blood- pp. 3-4, 1909). stained materials. Stains on a variety of substrates were In principle, the test is simple, but time consuming. An tested, and a number of chemicals, including ammonia, po- animal, usually a guinea pig since this animal tends to ex- tassium permanganate and phenol, were mixed with blood- hibit relatively consistent and characteristic symptoms of stains before testing. In all cases, the reaction took place. anaphylactic shock, is injected with sensitizing antigen. Ex- Stains 12 and 20 years old gave results that were indistin- tremely small doses are required to bring about the sub- guishable from fresh blood, while a 28 year old stain gave a sequent hypersensitivity, which becomes evident after about somewhat less intense reaction. They recommended that 10 to 14 days. A second, usually much larger dose of homol- Pfeifier's temperature decrease criterion be used in deter- ogous antigen will then bring about anaphylactic shock in mining the presence or absence of the reaction. The tech- most of the animals. They normally go into convulsions, nique was recommended for medico-legal investigations, and while displaying a characteristic set of symptoms, and die particularly in situations where the precipitin and com- fairly soon. Some sensitized animals do not show the charac- plement fixation tests would not give a result. Pfeiffer (1938) teristic shock reaction, however. Sera to be used for sensi- discussed the method in some detail in his review, and con- tization must be heated to 55-60" for 30 min so that it does cluded that the technique should be used where other, sim- not cause a reaction in the animals. And the animals must be pler ones would not suffice. Olbrycht (1950) noted in his given injections which are as close to their body temperature review that the method was prohibitively cumbersome, and as possible, or the symptoms very similar to anaphylaxis also said that the judgment of the presence of the anaphy- may result and mislead the observer (Sutherland, 1910). lactic reaction was too subjective, even if the body tem- Rosenau and Anderson (1907, 1908 and 1909) conducted perature decrease is used as the criterion. extensive experiments on the anaphylaxis, hypersuscep- tibility phenomenon. Among other things, they could show that a variety of proteinaceous substances would cause ana- Hemolysins are antibodies which bring about the destruc- phylaxis in guinea pigs, that maternal hypersensitivity was tive lysis of the red cells which contain the homologous transferred to the fetus whether sensitization had occurred antigen. In 1869, Creite observed that if an animal received prior to or after conception, that the active principle in horse a transfusion of foreign blood, it developed hemoglobinuria. serum causing sensitization was inactivated by heating to He also noted that the animal's red cells were dissolved 100" for 1 hr, that exceedingly small doses (0.1 p11) of sensi- (lysed) by foreign serum, but he apparently did not appre- tizing antigen were required if administered intracranially, ciate the relationship between the two observations. Landois Sourcebook in Forensic Serology, Immunology, and Biochemistry

(1875) carried out extensive experiments on the transfusion substances other than blood. Carrara (1904) looked into the of blood from one animal to another. Transfusions between technique. He found that the agglutinins were inactivated by unrelated species were always accompanied by hemo- a few minutes exposure to 70'. He also said that saturated globinuria in the recipient animal, and Landois recognized borax solutions or Paccini's solution (see Section 5.3) could that this was the result of hemolysis brought about by the be used to extract agglutinins from bloodstains that had donor serum. In transfusions between closely related species, become insoluble. Martin (1905) carried out extensive ex- these results were not seen. In 1898, Belfanti and Carbone periments on the test with a number of different animal showed that artificial hemolysins could be produced by im- bloods, and concluded that it was not reliable and should not munization of an animal with the blood of another animal, be used in forensic practice. Sutherland (1907), having got- and that they were specific for the red cells of the animal ten agglutination of fresh human cells with an extract of whose blood had been used for immunization. Artificially human menstrual blood, and having failed to get it with a produced hemolysins are used in the complement fixation number of animal bloodstain extracts, agreed with Martin. assays described in sections 16.6.1 and 1.3.5.3. In 1901, Leers, in his 1910 monograph, described his own pro- Deutsch proposed the direct use of hemolytic antisera for the cedure for carrying out the test, but emphasized that it was diagnosis of species of origin of bloodstains. The method not to be regarded as a substitute for the precipitin test, and relied on the lysis of the red cells in the bloodstain by the that it was necessary to use proper controls. Most authorities antiserum. Hemolysins could be prepared against any de- agreed that a negative test was noninterpretable, because it sired animal or human red cell, and complete hemolysis was could not be known whether agglutination had failed to obtained within 24 hrs of incubation of homologous blood- occur because the blood was human, or because the aggluti- stain with antiserum. Normal serum, used as a control did nins had become inactive in a stain that was in fact of animal not bring about hemolysis. The difficulty with such a method origin. Baecchi (1910a and 1910b) took a look at the issue is in judging the degree of hemolysis which has occurred in of the presence of human isoagglutinins in the stains. Marx a dried bloodstain. In a red cell suspension, it is relatively and Ehrnrooth had originally suggested that the iso- easy to assess hemolysis, but in bloodstains, which may al- agglutinins of human blood deteriorate faster in bloodstains ready be partially hemolyzed, there are many problems in than do the heteroagglutinins in animal bloodstains. Fur- interpretation. Nuttall (1904) noted that he thought there ther, they suggested that the two could be distinguished on were many potential sources of error in this method, and that the basis of differences in agglutination behavior. Baecchi it would be better to use the more reliable precipitin test. The recognized the problem, and said that one should carry out subject was briefly reviewed by Sutherland (1907) and does the test with a number of examples of human red cells. If not appear to have been pursued. these were all agglutinated uniformly, he said, the proba- bility of the stain having contained heteroagglutinins, and 16.6.4 Agglutinins therefore, of being of animal origin, would be very much When the serum of an animal is mixed with the red cells increased. Lattes (1913) took up the problem of dis- of another, not too closely related animal, the cells are agglu- tinguishing between the isoagglutinins and the hetero- tinated. Sutherland (1907) attributed the initial observation agglutinins in this procedure, and appears to have been the of this phenomenon to Landois in 1890. In 1904, Marx and first investigator to have suggested a simple method for it Ehrnrooth attempted to devise a species test for bloodstains based on the principles governing isoagglutination in human based upon this principle. A saline extract of a nonhuman, blood (see section 19 for full discussion). His objection to mammalian bloodstain would cause fresh human cells to be Baecchi's approach was that it was "blind", in that no ac- agglutinated, while with an extract of a human bloodstain, count was taken of the blood group of the test cells ud. there was either no effect or else rouleaux formation oc- False positive results in the test, caused by the presence curred. They thought that the hagglutinins of human serum of human isoagglutinins in human bloodstains, could be did not persist in an active state in bloodstains lonpr than avoided, Lattes said, by the very simple expedient of using 1 month. In cases of bloodstains less than a month old, they group 0 test cells. Under these conditions, he noted, the test said that a drop of saline diluted human serum, obtained by could be of value provided it gave positive results. Negative allowing a saline diluted drop of human blood to settle for 24 results could not be interpreted. He felt that the test could be hrs, should be added to the text mixture. This addition would used to screen samples for a subsequent precipitin test, and cause the clumping by homologous isoagglutinins to become in addition, could serve to indicate possible human-animal more marked, while it would tend to break up the aggregates blood mixtures in the stain in cases where the agglutination formed by heterologous agglutinins. They said that the test test was positive and the precipitin test with anti-human would be a useful preliminary one, to be carried out prior to serum was also positive. doing the precipitin test. Pfeiffer (1904) obtained positive results with the test on bloodstains on wood and lien that 16.6.5 Serum-bemoglobi precipitation were 37 and 24 years old, respectively, and felt that it was a In 1967, Kimura published a series of papers on the pre- good preliminary test. De Domincis (1904) found that heat- cipitin reaction of human hemoglobin with human serum. ing bloodstains to 150' prevented the reaction, and he ob- The reaction was first observed in Ouchterlony gels, and was tained false positive reactions with a number of body fluid noted to be independent of the ABO group (1967a). A series Species of Origin-I~ologicaland Serological of experiments was conducted with a number of animal sera In 1974, Bhatia conducted a number of experiments on and hemoglobins as well as with human serum and Hb. The phytagglutinins from a number of plants with the blood of a only homologous reaction besides the human one which oc- number of different animals. Three lectins were specific for curred was that of monkey hemoglobin with monkey serum, guinea pig blood, while five others gave a combination of and it was weaker than the human reaction. Human serum reactions, the patterns of which were distinctive for cow, reacted weakly with monkey and rabbit hemoglobins chicken, sheep, rabbit, frog and rat bloods. Bloodstains were (1967b). It could be shown by immunoelectrophoresis that not studied in these experiments. hemoglobin was reacting with the albumin of the serum Tumosa (1976) looked at the reactions of the red cells of (1967c). A medico-legal application of the phenomenon was 37 different species with the "anti-A," lectin from Dolichos proposed (1967d) in which human serum was used as test biporus, and found that only those of the barasinga, chim- reagent, the bloodstain serving as the source of hemoglobin. panzee, rhinoceros and wallaroo reacted. It has been appre- Extracts of 6 months old human bloodstains gave the test ciated for some time that the ABH blood group receptors of with serum dilutions of up to 1: 16. human red cells are actually widely distributed surface In 1971, Hartmann and Oepen said that they had tested structures in many different living things. The matter is many combinations of sera and hemoglobin, and could not discussed in more detail in appropriate parts of section 19. In confirm Kimura's findings. They said that the precipitin re- this respect, Tumosa (1977) has found that a variety of actions between serum or albumin and hemoglobin were animal bloods are fully reactive with anti-A, anti-B and with nonspecific and could be avoided in imrnunodiffusion tests by anti-H. keeping the distance between wells less than 6 mm and the Chowdhuri et al. (1975) looked at the phytoprecipitin temperature lower than 20'. Human serum would react with activity in 50 diierent plant seed extracts against human a number of animal hemoglobins in their experiments, and and a variety of animal sera. 25 plant extracts showed phy- they said that the method was of no value in diagnosing toprecipitin activity, and many of these were nonspecific. species of origin. Two extracts were specific for horse serum, one for chicken serum, one for goat serum and another for dog serum. Four 16.6.6 Phytoprecipitin and phytagglutioation methods lectins reacted with human and monkey sera. That the seeds of certain plants contain agglutinins for red cells has been known for a long time. In 1888, Stillmark 16.6.7 Gamma-globulia deviation found that aqueous extracts of Ricinus communis were hem- In 1957, Ambrosi attempted to take advantage of the fact agglutinating (Gold and Balding, 1975). Plant extracts that antibodies are present in the y-globulin fraction of which agglutinate red cells are usually called lectins, or serum, proposing that a reduction in the y-globulin fraction phytagglutinins. More recently, the term "receptor specific of the antiserum after incubation with homologous antigen proteins" has been introduced, and includes agglutinins could serve as an indication that the antigen-antibody reac- from animal sources (protectins) as well. This subject is now tion had occurred. The quantities of y-globulin present quite complicated and its literature immense. More is said before and after the reaction were detected by paper electro- about lectins in section 19 (ABO system). Some plant ex- phoresis. Although this method is not, strictly speaking, tracts can also cause precipitation of animal sera. These are purely immunological, it is no less appropriately presented in called phytoprecipitins. this section than in a separate one dealing with electro- In 1963 Haferland reported that aqueous extracts of phoresis. Ambrosi said that a consistent reduction in Bryophyllum diagremontienum contained a phytoprecipitin y-globulin was observed when small aliquots of antisera specific for human serum. There was a weak reaction with were tested following incubation with 1:500 dilutions of ho- some monkey sera. In 1964, he showed that the precipitin mologous bloodstain extracts. No reduction was seen with detected human serum in bloodstains up to 5 years old on a heterologous antigen. The absolute value of the reduction number of substrata by the agar gel diffusion test. In one varied in the homologous case, but was never less than instance, the phytagglutinin reacted when anti-human 14.7%. Schleyer (1962) said that Ambrosi's paper electro- serum did not. Haferland recommended the phytoprecipitin phoresis experiments had not been carried out very rig- for medico-legal species diagnosis. orously, and that Hilgermann, working in his (Schleyer's) In 1963, Raszeja reported that the phytagglutinin from laboratory in 1960, had utterly failed to confirm Ambrosi's the mushroom Lacchana laccata Berk was specifically in- results. hibited by human serum, as well as by human saliva, semen and milk. The sera of 35 animals showed no inhibition. This 16.6.8 F'Iw)re!scentantibody technique principle was proposed as the basis of a forensic species In 1968, Gajos reported that he had conducted explora- diagnosis test, and an extract of a 36 year old human blood- tory experiments on the applicability of fluorescent-labelled stain showed the inhibition reaction. The lectin has an anti- antibody technique to medico-legal immunology. The results H specificity, and it was found in subsequent studies that were not very satisfactory, one of the problems being that "anthropomorphic" monkey (e.g. chimpanzee) sera also many fabrics contain fluorescent "brightening" substances cause inhibition, but that the sera of "zoomorphic" monkeys which have similar emission maxima. He believed further (e.g. Rhesus monkey) do not (Raszeja, 1966). work would be required to render the technique applicable to Sourcebook in Forensic Serology, Immunology, and Biochemistry forensic problems, such as that of species of origin deter- Welsh and Chapman (1910) indicated that they could mination. immunologically differentiate the ovalbumins of closely re- lated species by carefully determining the weight of the precipitates under carefully controlled conditions. Berkeley (1913) could not confirm Uhlenhuth's contention that Immunoelectrophoresiswas first described by Grabar and specific precipitins to human proteins could be prepared in Williams in 1953, who applied the technique to the sepa- monkeys. They were unable to raise precipitin antisera to ration and characterization of human serum proteins. Since humans in two species of monkeys, although it is now known that time, it has become a standard method of immu- that this can certainly be done. Fujiwara (1922b) said that nological investigation (see Grabar and Burtin, 1964 and human and monkey bloods could be differentiated by the section 2.4). An important development was Scheidegger's complement fixation test using anti-human serum absorbed (1955) introduction of a micro-immunoelectrophoretic with monkey blood. method, which was widely adopted and modified by many In more recent times, a good deal of work was done on the workers. Immunoelectrophoresis has not been widely em- problem by the South African forensic scientists, the differ- ployed as a means of species determination. Muller and entiation of human and primate bloods being a serious prob- Fontaine (1960) explored the possibility, but said that the lem in their practice. In 1952, Taylor published a detailed method was not inherently superior to other gel methods, paper on the subject, in which the standards of potency and and that the specificity of the test was a function of the specificity for antisera as stated by many authorities were specificity of the antiserum. Schleyer (1962) gave a detailed reviewed. In addition, the various techniques for attempting description of a method which he had devised in collabo- to render antisera specific for a single species were reviewed, ration with Schneider. He agreed with Muller and Fontaine and a number of experiments conducted. He had some suc- in noting that the specificity of the antiserum was the critical cess with anti-human sera prepared in baboons and vervet feature in determining the specificity of the test. monkeys, but none of the antisera could differentiate human blood from that of the chimpanzee, the most closely related primate. Shapiro (1954) flatly stated that Taylor's experi- 16.8 Cross Raactlons of Antlsera-The ments had conclusively shown that human blood could not Problam of Closely Related S~las be differentiated from that of the most closely related pri- It has been recognized since the earliest experiments with mates. Gradwohl (1956) tested anti-human serum with the precipitating antibodies that antisera to a particular protein sera of several monkey species and a small baboon species cross-reacted with closely related proteins. This fact was with negative results. The antiserum did react to chim- clear from the studies on the reactions of anti-hen ovalbumin panzee, gorilla and orangutan sera though. An anti- with duck ovalbumin (Myers, 1900, Uhlenhuth, 1900). Very chimpanzee reacted with gorilla, orangutan and human sera, soon after the precipitin test for medico-legal diagnosis of as did an anti-orangutan preparation. He did not think there species was proposed, it was clear that antisera to a particu- was any way to distinguish these species by means of the lar species cross reacted with the sera of closely related precipitin test. Vagnina (1955) discussed the problem of the species, as for example, anti-ram sera with goat and cow differentiation of a closely related species, and said that blood (Uhlenhuth, 1901e) and anti-human serum with mon- Dr. Boyden had told him that it was indeed possible to dis- key bloods (Nuttall and Dinkelspiel, 1901a and 1901b; tinguish closely related species by precipitin tests, but not Frenkel, 1901; Stern, 1901). The cross reactions of anti- using the methods employed by most medico-legal investi- human serum have been a matter of particular concern to gators. A photoelectric turbidity measuring system had to be medico-legal investigators ever since that time. In some employed, and antisera selected with very great care. countries, which have indigenous monkey and other primate Boyden and DeFalco (1943) and Boyden et al. (1947) de- species, and where these bloods may be present in exhibits scribed their technique in detail. An antigen was titrated to submitted for examination, the problem is a serious one. In its endpoint with very carefully selected antiserum, the tur- this country and in Europe, it is improbable that such cross bidity being measured in each dilution from the prozone, reacting primate bloods would be encountered. through the equivalence zone, and into the postzone with the A number of approaches have been taken to find ways of Libby photronreflectometer. Curves can then be constructed differentiating the bloods of closely related species, particu- in which tube number (in order of increasing antigen con- larly of humans and primates. Among these are selective centration) was plotted against turbidity units. The areas absorption of antisera with the cross-reacting antigens (Wei- under these curves, which vary both in area and in shape chardt, 1905), so-called cross-immunization ("kreunveise with homologous vs heterologous antigens, are proportional immunisierung") (Uhlenhuth, 1909, and various tech- to the summated turbidities in the titration series. The niques for carefully estimating the quantity of precipitate summed turbidity value is sometimes referred to as "relative obtained. By cross immunization is meant the preparation of area". This type of analysis is sensitive to very small antibodies to the proteins of a particular species by immu- differences in antigen structure, and could differentiate be- nization of a member of the closely-related, cross-reacting tween closely related antigens, including human and chirn- species. panzee serum. A representative set of results from Boyden Specks of Origin-Immunological mtd SeroIogical

(1958) is shown in Fig. 16.4. The turbidity determinations showed that the technique was applicable to bloodstain ex- were shown to correlate well with other methods of quanti- tracts, and that human and chimpanzee bloodstains could be tative determination of precipitin reactions, such as gravi- differentiated from one another (i.e., chimpanzee serum metry and total antibody nitrogen. Gravirnetry, the accurate could be shown to be non-identical with human) as had been measurement of the mass of the precipitate, suffers from the done in the case of the sera. disadvantage that combining valencies of antibodies may Lichter and Dray's studies (1964) indicated that anti- differ. Determination of total antibody nitrogen is accurate, human sera prepared in chimpanzees and in Rhesus mon- but more complicated than turbidimetry (Kwapinski, 1972). keys could distinguish a number of human-specific serum In 1955, Coetzee proposed an interesting, if complex tech- proteins. Gempel et al. (1960) could differentiate human nique which was capable of differentiating human from and monkey sera using an anti-human serum prepared in the chimpanzee serum. The method employs sensitized tanned monkey. The antiserum was apparently of very low potency, red cells as indicator cells (see section 16.3), and is based on however, and the reaction was very slow. Using anti-human the principle that homologous antigen should be able to and anti-monkey sera prepared in rabbits, and absorbed with annul its antiserum, while heterologous antigen (even if heterologous serum, it was possible to differentiate between closely related) should not be able to do so to the same human and monkey sera on the basis of the number and extent. Another way of putting it is that the failure of species structure of precipitin lines in Ouchterlony gels (see section X proteins to annul anti-species Y antiserum shows that X 2.2.2). Bloodstains were not studied in the course of these and Y are, in fact, different. The test demonstrates non- experiments. Sivaram et 41. (1975) indicated that monkey identity, although it could be used to establish identity if blood could be distinguished from human blood using a very enough experiments were carred out. The test was arranged similar sort of analysis of the Ouchterlony diffusion precip- as follows: serial dilutions of human serum in one set of tin bands, but employing a very potent anti-human globulin tubes, and serial dilutions of chimpanzee serum in another, Serum. were incubated with serial dilutions of anti-human globulin Grobellar et al. (1970b) utilized the AHG inhibition test (AHG) serum. After a time, tanned red cells, sensitized with (section 16.2) to differentiate between human and primate human serum globulin, were added to all the tubes, and sera. AHG serum was prepared in goats, and with selected agglutination read after a suitable interval. A set of results examples of the reagent, no inhibition was observed with is shown in Table 16.1. In a similar way, beef and horse meat primate sera at dilutions greater than 150 while human extracts could be readily distinguished. In 1958, Coetzee serum gave inhibition at dilutions of 1:1000. The test was

5 + 5 L 7 8-9 to 11 U 13It ANTIGEN DILUTION

Figure 16.4 Quantitative Precipitin Analysis by Reflectrometry. Reactions of an anti-human albumin serum with a series of primate sera. From "Comparative Serology: Aims, Methods and Results" by Alan Boyden, in: Seroiogical and Biochemicai Comparisons of Proteins, ed. by William H. Cole. Copyright @ 1958 by Rutgers, The State University. Reprinted by permission of Rutgers University Press. Table 16.1 Results of the Application of the Coetzee Method to Human and Chimpanzee Sera (after Coetzee, 1955)

Dilutions of anti-human globulin serum (reciprocal)

3 6 12 24 48 96 192 384 768 1536 3072 C

C C C C C C C - - - - -

+ C C C C C C - - - - -

16 + + C C C C C C - - - - Dilutions of - - - human1 32 + + C C C C C C - chimpanzee 64 - - - sera + + + C C C C C C (reciprocal) 128 - - + + + t C C C C C C - + + + t + + C C C C C - + + + + + + t C C C C - + + + t + + + + C C C + + t + + + t + + + + -

L Key to abbreviations in figure: + = agglutination with both human and chimpanzee sera; c = agglutination only with chimpanzee serum; - = no agglutination; C = control (normal rabbit serum). Species of Origin-lmmunologicoland SeroIog&/ recommended for medico-legal species diagnosis in South that a number of medico-legal authorities had reported their Afrjca. inability to differentiate closely related species by the precip Camy (1969) recommended immunoelectrophoresis as a itin test. The use of quantitative immunological reactions to means of discriminating between the bloods of closely re- establish taxonomic relationshi~swas discussed in the re- lated animals. view as well, and some data presknted. Readers interested in this subject are also directed to Leone (1964), which con- 16.9 Serum Protdn Structure and tains articles by many specialists on all the varied aspects of taxonomic serology and immunology. Phylogeny-Taxonomic Serology and Sensabaugh (1975) pointed out the intimate connection Immunology between immunological cross reactivity of those proteins Structural similarities in the proteins of different species, which have evolved rather slowly, and the medico-legal and their corresponding immunological cross reactivity are a differentiation of species of origin. Depending on the dis- reflection of phylogeny. This fact was recognized almost criminatory capability of the technique being employed, the simultaneously with the discovery of precipitins for blood phylogenetic relationships between related species impose proteins. The undisputed pioneer in this field was G. H. F. intrinsic limits on the ability to differentiate related bloods. Nuttall. In 1901, he was preparing antisera to scores of Medico-legal tests would be far more satisfactory in these animal bloods, and recognized the fundamental biological terms, he said, if monospecific antisera to the more rapidly importance of immunological cross reactivity as a delicate evolving proteins were employed, thereby taking advantage measure of protein structural similarities (Nuttall, 1901b). of greater differences in the structure of similar proteins, In the course of several years time, antisera were prepared even in very closely related species. by Nuttall and his collaborators against hundreds of animal In the full paper on the subject, Sensabaugh (1976) noted bloods. These were tested with homologous and heterologous that cross reactions between the proteins of closely related sera in a systematic fashion. The results were obviously com- species are an inevitable consequence of evolution. Cross plex, and took up dozens of tables. They cannot be readily reactivity can be turned to advantage in medico-legal in- summarized. All the material is collected in Nuttall's classic vestigations, because of the known relationship between de- work, Blood Immunity and Blood Relationship, which ap gree of cross reactivity and species relatedness. Proteins peared in 1904. The book additionally provided a detailed must differ from one another in sequence by about 40% of review of immunological investigations up to the time, and their residues in order to be completely non-cross reactive to included a chapter on medico-legal species differentiation by an antiserum to one or the other. In discriminating between the precipitin test. primates, anti-Ig gamma chain and anti-Ig kappa chain sera More recently, quantitative techniques have been em- were found to be much better than antisera to albumin or ployed in comparative immunology and serology. These transfemn. Sensabaugh noted further that diierent exam- were reviewed by Boyden (1958). The reflectometric pro- ples of commercial "anti-human" serum contained anti- cedure for assessing the quantity of precipitate formed in the bodies against different serum proteins, and that unless such precipitin reaction was discussed in the foregoing section. characteristics of an antiserum are known, it is difficult to Boyden said that with appropriate quantitative precipitin make the most productive use of a species test or indeed to technique and careful selection of antisera, there was no select the most suitable antiserum. reason for being unable to differentiate very closely related Bauer (1969, 1970a, 1970b and 1970c) has carried out species in forensic medicine, including human and chim- extensive studies of human and animal serum proteins using panzee blood. He said that the usual technique employed in immunological methods with the objective of determining medico-legal investigations, the use of antiserum against phylogenetic relationships among the species. Particular whole serum rather than a specific protein, and the use of a proteins could be placed into what he called "immunological single concentration of antigen and antibody for the test, was evolution groups" based upon their cross reactivity, a direct quite crude. He did not find particularly astonishing the fact measure of the speed with which they have evolved. Spech of Origin-Other Methods

SECTION 17. Other Methods for Specles Determination of Blood and Bloodstains, Body Flulds and Tissues 17.1 Dlfferentlal Denaturation of and Rengei in 1959 and 1960 devised a species of origin test Hemoglobln wlth Alkall based on fibrinolysis (Rengei and SzBllasy, 1959; Rengei, Although the absorption spectrum of hemoglobin in vari- 1%0, Szolliisy and Rengei, 1%0). A fibrin plate was pre- ous species does not differ greatly, the rate at which the pared using bovine fibrinogen in buffer in the presence of a molecule denatures in alkali is decidedly species-dependent. small amount of thrombin. This mixture was allowed to clot Kriiger observed this phenomenon in 1887. Magnanimi and the surface then dried in a 37" incubator. A small (1898) applied the principle to bloodstains, and thought it quantity of streptokinase was mixed with a buffer extract of had forensic applicability. Zeimke (1901~)confirmed many a bloodstain, and a drop of this mixture applied to the sur- of the previous fmdins, likewise recommending the method face of the fibrin plate. Streptokinase is an Micial activator for medico-legal examinations. Kriiger had another exten- of proactivator, and its addition to the serum in the blood- sive paper on the subject in 1925, although he had no interest stain sets in motion the fibrinolytic machinery. After a suit- in forensic blood investigations. Schleyer (1962) quoted the able incubation period, a marked lytic area develops in the Russian authors Blumenfeld and Krasovickaja as having fibrin plate if the extract was from a bloodstain of human said in 1955 that human blood could be readily distinguished origin. The authors claimed absolute specSk&y for the from a number of animal bloods by following the rate of method, as well as a sensitivity of 0.3 pg human serum pro- alkali denaturation of hemoglobin spectrophotometrically. tein. Positive results were obtained with bloodstains on Analogous results were obtained with bloodstain extracts. various substrata, including iron and glass, which were up to Their observations were generally in agreement with those of 8 months old, even ones which had been kept at 56'. It was the earlier investigators. Human hemoglobin denatured at noted that human milk and tears would give the fibrinolytic the fastest rate (1-2 min), followed by cat Hb (6-7 min) and reaction as well. In 1%2, Kumano utilized the technique, and dog Hb (12-14 min). Fowl, goat, ox and sheep hemoglobins said that it was sensitive and spedic. He got a positive result do not significantly denature in alkali after an hour. Schleyer with a 30 year old bloodstain, and on stains which had been conducted a number of experiments with this technique in exposed to 100" heat for an hour, or washed. Putrefied liquid 1961, and summarized his results in the 1962 review. It blood could not be diagnosed satisfactorily by this method. could be confirmed that human Hb denatured faster than Schleyer (1962) presented a description of a method that of any animal hemoglobin tested. He said, in addition, which he had found to work well. The method was found not that the pattern of denaturation might actually be more to be species-specific, however, if non-human sera were less characteristic of the species than the rate. Schleyer thought dilute than 1:100. At serum dilutions of 1: 1000, only human that the technique was primarily applicable to fresh blood or blood gave a positive reaction, and Schleyer noted that with to very fresh bloodstains. The alkali denaturation curves are this method, as with the precipitin test, it was important to characteristic for whole blood dilutions, but not for pure employ dilute (1 :1000) bloodstain extracts to insure speci- solutions of hemoglobin. Human hemoglobin is not dena- ficity. Mohri (1963) confirmed the results of previous work- turable by alkali at blood dilutions greater than 1:950. ers, and Morioka (1965) said that putrefaction destroyed the It must be noted that fetal hemoglobin (Hb F) is quite fibrinolytic activity of blood steadily over the course of a resistant to alkali denaturation. This fact was used as a basis month. Mikami er al. (1966) recommended the technique for differentiating Hb F from adult hemoglobin (Hb A) for species determination in small fibers of bloodstained ma- prior to the development of electrophoretic techniques (see terial, to which they said they could consecutively apply a section 8.3.1). The presence of Hb F in a bloodstain could catalytic test, the fibrin plate test, and a mixed agglutination cause a grave error if the alkali denaturation technique for technique for the determination of the blood group. Akaishi species differentiation were being applied. Hb F is further (1965) noted that the test was sensitive to a whole blood discussed in section 38. dilution of 1:25,600. Hirose (1976), in his studies of the effects of rust on dried blood (see section 5.1 and Table 5.2), 17.2 The Bbrln plate method said that the ~reci~itintest remained positive for 51 days The fibrinolytic system was discussed in section 8.1.2 (and after the mixkg oi the blood with the'iron only if pH 94 see Figure 8.1). The work of Mullertz and Lassen (1953) on buffer solution was used for extraction. The fibrin plate test the fibrinolytic system had indicated that human serum con- was barely positive after 51 days using pH 9.4 buffer as tains substantially larger amounts of proactivator than most extraction medium, and it became negative at 7 days if saline animal sera. On the strength of this observation, Szbll6sy was used. ~rcebookin Fore& Serology, Zmmunologv, and Biochembtry

17.8 Hemoglobin Separatlon by in section 37.12) in a series of vertebrate bloods. Herr (1979) Chromatographic and Electrophoretic showed that a number of differences existed in the red cell M.thods acid phosphatase (see in section 29) enzymes of several ani- mal species as compared with the human isozymes. Herr and Some efforts have been made to apply paper chromato- Konzak (1980) suggested that these ACP patterns, as well graphy or gel electrophoresis to the diagnosis of species by as those of red cell PGM and AK (see in sections 27 and 28), separation of the hemoglobins. Fine et al. (1956) got sepa- might be helpful in differentiating species of origin. ration of the hemoglobins from distantly related species us- ing gel electrophoresis, but the hemoglobins of most mam- 17.S Specks Dlagnosls In Other Body mals tended to migrate about the same distance. These ob- Fluids and In Tluues servations were largely confirmed by Depieds et al. (1960). Fiori (1957) tried paper chromatography of hemoglobins, It is not often necessary to carry out species tests on body and tested a large number of different solvent systems. He fluids other than blood. In theory, the immunological tests could not get satisfactory differentiation of human hemo- could be applied for the purpose to any body tluid in the globin from that of other species. Vidoni and Marenghi same way as they are used for blood, provided that specific (1957) tried to distinguish blood species on the basis of the antisera were available. The preparation and use of antisera patterns obtained by paper chromatography. Schleyer to semen has been discussed in a previous section (10.3), as (1962) said that he and Schneider were unable to reproduce has the preparation and use of antisera to saliva (1 1.4). these results. Santini (1960) applied circular paper chro- There do not seem to have been extensive studies on the matography to the separation of human and animal hemo- species-specificity of these antisera. Most of the concern was globins. He said that a specific, reproducible Rf of 0.72 could with cross reactions with other body fluids, since the objec- be obtained for human hemoglobin using this technique. tive of the immunological tests was primarily identification Berg (1967) said that the hemoglobin electrophoresis and of the body fluid. chromatographic methods for species determination were Popielski et al. (1963) said that the precipitin test could be unreliable, and that other techniques should be used. carried out on extracts of urine stains provided these were subjected to a protein concentrating- step- in advance. Pathol- ogically albuminous urine can be tested more easily. 17.4 Isoenzyme Patterns Tissue samples are sometimes encountered, and must be In 1962, Samico et al, reported that aqueous extracts of tested for species of origin. The species of muscle tissue may stains of human, cat, ox, sheep, goat, pig, turkey, chicken, be conveniently determined by the precipitin test, as has rat, monkey, possum and toad bloods could be differentiated been known since the early work of Uhlenhuth (1901f). on the basis of their esterase profiles following starch gel The subject of tissue antigens is complex, and beyond the electrophoresis and specific staining for esterase activity. scope of the present discussion. The species of origin of tis- Thick starch gels were employed for the separations, and sues can be determined based on their presence, however, if staining effected with a-naphthyl acetate or a-naphthyl bu- appropriate antisera and techniques are available. Milgrom tyrate and Fast Blue RR salt. The banding patterns were and Campbell (1970) reported on a case in which a piece of different with each animal tested, and the results were better intestine and a piece of mesentery, found in a sewer in Ni- with the butyrate ester. The single band from human blood- agara Falls, N.Y., were submitted for species determination. stains was due to cholinesterase activity. This band persisted It was known from previous work (Milgrom et al., 1964a and in bloodstains for 210 days if extraction was carried out for 1964b) that tissue contains species-specific antigens which 30 min with water, but if extraction were done at 37O for were called "BE antigens", because of their resistance to 24 hrs, the band persisted to 3 10 days. boiling in water, and their solubility in ethanol. A rather Madiwale et al. (1972a) tested blood samples from bul- complicated mixed agglutination technique, previously de- lock, buffalo, sheep, goat, rabbit and human sources for scribed by T6nder er al. (1964), for determination of these lactic dehydrogenase and malic dehydrogenase activity band antigens was employed in the case. A rabbit antibody to the patterns by polyacrylamide disc gel electrophoresis. The pat- tissue antigen is first attached to the tissue antigen. Test cells terns were reproducible, and different for the various ani- are then prepared by sensitizing sheep red cells with rabbit mals. Bloodstain extracts gave similar results (1972b), and anti-sheep erythrocyte antibodies. These cells are then the method was considered very sensitive, only about 1 rll of treated with a goat anti-rabbit y-globulin antibody, and the blood being required. agglutinates which form are broken up. The test cells then Some of the isoenzymes which are under the genetic con- have, in effect, one free end of the goat anti-rabbit y-globu- trol of polymorphic loci in human beings are found in certain . lin antibody which can combine with the rabbit antibody animal bloods. The various human isoenzymes are discussed attached to the tissue antigen, giving the mixed aggluti- in Unit VI. In cases where animal blood isoenzyme patterns nation reaction. The tissue submitted in the case was deter- are consistent, and differentiable from human patterns, elec- mined to be pig intestine using the technique. trophoretic enzyme determination might offer a method of It may be noted in conclusion that the entire subject of helping to identify the species of origin of bloods. Gallango species of origin determination was reviewed by Pfeiffer and Suinaga (1979) studied the red cell enzyme UMPK (see (1938), by Boyd (1946) and by Schleyer (1962). References for Unit IV

FOR UNIT IV Akaishi, S. 1965. Studies on the group specific double combination method. spezifischer prlcipitierender Antisera fllr den forensischen Blutnachweis. Jpn J.Leg.Med. 19:177-187 8' Dtsch. 2.Gesamte Gerichrl. Med. 10: 17-30 Aladjem. F. 1971. Diffusion theory for antigen-antibody reactions in gels, Bordet, J. 1898. Sur l'agglutination et la dissolution desglobules rouges par in. C. A. Williams and M. W. Chase (4s.) Merhods in Immunology and le serum d'animaux injectes de sang defibrine. Ann Insr. Pasteur. Paris Immunochemisrry, 3: 108-1 17, Academic Press, New York & London 12: 688-695 and see Bordet (1909) Aladjem, F. and M. Lieberman 1952. The antigen-antibody reaction I. The Bordet, J. 1909. Studies in Immunity, papers collected and translated into influence of sodium chloride concentration on the quantitative precipitin English by F. P. Gay, John Wiley & Sons, New York and Chapman & reaction. J. Immunol. 69: 117-130 Hall, Ltd., London Allison. A. C. and J. A. Morton. 1953. Species specificity in the inhibition Bordet, J. and F. P. Gay, 1906. Sur lcs relations des sensibilisatricesavec of antiglobulin sera. A technique for the identification of human and I'alexine. Ann. Insf. Pasteur. Paris U): 467-498 and see Bordet (1909) animal bloods. J. Clin Pathol. 6: 3 14-3 19 Boyd, W. C. 1946. Forensic immunology. J. Crim. Law CN'minoI. 36: Ambrosi, L. 1957. I1 metodo elettroforetico in emktologia forense. La 455-472 diignosi spccifica su macchia di sangue: vantaggi sul metodo siero- Boyden, A. 1958. Comparative serology: aims, methods and results, iti precipitante. G. Med. Leg. Injorrun Tossicol. 3: 203-21 1 W. H. Cole (ed.)Serological and Biochemical Comparisons of Proteins. Anderson, J. R. 1952. The agglutination of sensitized red cells by antibody Annual Conference on Protein M*abolism. Rutgen Univ., Bureau of to serum, with special references to non-specific reactions. Br. J. &xp. Biological Research, pp. 3-24, Rutgers Univ. Press, New Brunswick, Parhol. 33: 468-483 N.J. Anderson, J. R. 1954. Application of antiglobulin reaction to bloodstains in Boyden, A. and R. J. DeFalco. 1943. Report on the use of the photron- determination of animal species. Am J. Clin Parhol. W. 920-928 reflectometer in serological comparisons. Physiol. Zool. 16: 229-241 Axtell, E. R. 1895. The medicelegal examination of the red stains found on Boyden, A,, E. Bolton and D. Gemeroy. 1947. Precipitin testing with special the clothes of Charles Ford, with a plea for the use of the camera lucida reference to the photoelectric measurement of turbidity. J. Immunol. 57: in the microscopic examination of blood stains. J. Am. Med. Assoc. 25: 21 1-227 139-144 Boyden, S. V. 1951. The absorption of proteinson erythrocytes treated with Baecchi, B. 1910a. Sulla diagnosi individuale di sangue umano. Arch. tannic acid and subsequent hemagglutination by antiprotein sera. J. Exp. Anfropol. Crim. Psichiatr. Med. 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Cenrmlbl. Complexity of antigen-antibody systems as demonstrated by a serum agar Bakreriol. Parairenkd. Infektionskr. 1 Abt.. 0";. 99: 704-712 technique. J. Immunol. 65: 47-58 Mm,A. M. 1920. Neue Methoden zur Diffemzierung kleinster Blutspuren Myers, W. 1900. On immunity against proteids. Lancer 2: %la) mittels der Uhlenhuth'schen Serumpiddpitinreaktion und Unter- Neisser, M. and H. Ms. 1905. Ein Verfahren zum forensischen Nachweis suchungen iiber die Wiung von Kokto-Antisrmm auf gekochtes der Herkunft des Blutes. Berl. Klin. Wochensrhr. 42(44): 1388-1389 1 koaguliertes muskdeiweiss. wHe!iahrschr. Gerichrl. Med. Oeff. Sani- Neisser, M. and H. Sachs. I-. Die forensische Blutdifferenzierung durch taetnuer. w39: 149-176 9' antihimolyische Wikung. Berl. Klin. Wochenrrhr. 4Y3): 67-69 1 Man, H. and E. Ehmrooth. 1904. Eine einfache methode zur formsirhen Neisser, M. and H. Sachs. 1906b. Bcrmerkung zu der Arbeit von Prof. Unterscheidung von Menschen- und Siiugetierblut. Muench. Med. 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Dinkelspiel. 1901b. On the formation of anti- Milgrom, F. and W. A. Campbell. 1970. Identifaion of species origin of bodim in the blood following upon treatment with the sera of different tissue found in a sewer. J. Fomnsic Sci. 15: 78-85 animals, together with their use in legal medicine. J. Hyg. 1: 367-387 Milgrom, F., M. Tuggac, W. A. Campbell and E. Witebsky. 1%4a. Ther- Oakley, C. L. and A. J. Fulthorpe. 1953. Antigenic analysis by diffusion. J. mostable ethanol-insoluble antigens of brain. J. Immunol. 92: 82-90 Parhol. Bacreriol. 66: 49-60 Milgrom. F., 2. M. Tuggac and E. Witebsky. 1964b. Studies on species Okamoto. Y. 1902. Untersuchungcn iiber den forensisch-praktischen Werth specif-. J. Immunol. 93: 902-909 der Serumdiagnostischen Methode rue Unterscheidung von Menschen- Minet, J. and J. Leclercq. 1911. L'Anaphylaxie en medecine legale.- Son und Thierblut. Viene(johrschr. Gerichrl. Med. Oeff. San~taetw.24 application a la damnination de la nature humain ou animale du sang. 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Wochenschr. 32(31): 12441248 tion of their Source. Balliere, Tidal and Cox, London Uhlenhuth. P. 1906b. Komplrmmtablenkung und Bluteiweissdif- Suthaland, W. D. 1910. The applicabity to medico-legal practice in India fmnzierung. Wh.Med. Wochenschr. 32(51): 2072-2073 of the biochemical tesu for the origins of blood stains. SEientifi Memoirs Uhlenhuth, P. 191 1. The Harben Lectures. 191 1. On the biologd differen- by Offim of the Medical and WtcvyDepartments of the Government tiation of proteids by the pdpitin reaction, with special reference to the of India, New Ser., No. 39, Supt. GOM.Printing, Calcutta forensic examination of blood and meat. J. R. Inst. Public Health 19: Suthaland. W. D. 1914. Blood Stains. in: L. A. Waddell. Lyon's Medical 641-662 Jurirpmdence for India, 5th ed.. pp. 166-I&, Thacker, Spink & Co., Uhlmhuth, P. and 0. Weidanz. 1909. Pmktkhe Anleirung air A~ufiihnmg Calcutta des biologkhen Eiweirsdflferenziemn&~erfahrensmil bewnderer Suthaland. W. D. and G. C. Mitra. 1914. Misleading reactions obtained hiicksichtigrmg der forekhen Bht- und Fk&hunter~uchung, sowie with precipitating antiscra and how to avoid them. India J. Med. Res. 1: der Gewinnung priuipitierender Sm. Gustav Piher, Jma 707-710 Vacher, J., E. Sutton, L. bbert and J. Moullec. 1955. Une nouvdle Sziillbsy, E,and B. Rengey (Rengei). 1959. Spezifwher Nachweis von men- mahode pour h recherche de I'origin humaine da taches de sang. schilichm Blutspurm auf Gmnd des im Blute mthaltenen fibrinolytirhen L'lnhibition de I'antiglobuline. Ann. Med. Leg. Cnininol. 35: 29-33 Systems. Naturwisenschqften 46: 175-176 Vagnina, L. L. 1955. Medico-legal problems in blood grouping. J. Forem'c Swlliisy, E. and B. Rengei. 1%0. Identitlation of human blood on the basis Med. 2: 247-250 of its proteolytic mryme system and its application. J. Foremc Sci. 5: Vibert, Ch. 1882. De la possibilitt de distinguer le sang de I'homme de celui 331-337 damannifh (Etude medico-kgale). Arch. Physio. Norm. Pathol. 142 Tardieu, A., J.-J.-E. Barrue1 and J.-B. Chevallier. 1853. Expirimces sur ser. 9):48-58 1 I'odeur du sang. Ann. Hyg. Publique Med. Leg. * 413417 Vidoni. G. and I. Marcrighi. 1957. Sub diagnosi specifics di macchie di Taylor. J. E. D. 1952. The medico-legal precipitin tat for human blood. S. sangue a neno deb cromatografia su cam. Minento Medimleg. 7% qfr. Med. J. 26: 81-86 45-47 Tchiistovitch, Th. 1899. etudes sur I'immunisation conue le drum Vollmer, 0.1949. ifbet die Beeinflussung des Abkufs der Eiweissdiffmn- d'anguilles. Ann. I&. Pusfeur (Paris) W: 406-425 hng nach Uhknhuth durch beabsichtigte Behandlungen von Thornsen. 0. 1909. Ueber die Spaifat der Saumanaphylaxie und die Bluupuren zun~Zwecke da Unkenntlichmachung. Dtcrh. 2. Geurmte Moglihkeit hrcr Anwendung in der medikoforrnsischen Praxis zur Dif- Mhtl. Med. B 628437 famzkmg von Menschen- und Thiilut (m Blutfleckm, etc.) 2. Im- Wassermann, (A.) (and A. Schfitze). 1900. Neue Beitrage mr Kenntnis der munitae&fomh. Erp. Ther.. Orig. 1: 741-770 Eioffe mschiedener h4ikhMm. Dtcrh. Med. Wochenschr. 26, Tidy, C. M. 1882. Legal Medicine, Vol. 1, Wm. Wood and CQ.. New York Verrins-BciJage No. 29: 178-179 Tonder. 0..F. momand E. Witebsky. 1%4. Mixed agglutination with Wasurmann, A. and A. Schutze. 1901. Ueber dne mue forcnsische Meth- tisue sections. J. Etp. Med. 119: 265-273 ode zur Unterscheidung von Menschen- und Thierblut. Berl. Klin. WO- Tran tLan Ky, P., L. Lenoir and P. Muller. 1968. Utilisation d'une im- chenschr. rn:187-190 1 munoirum anti yG-globuba pour la recherche de l'origine hurnaine Wasmnann. A. and A. Schutze. 1903. Ueber die SpezifitSt dcr Eiwk da taches de sang. Med. Leg. Dommoge Corpor. 1: 392-395 priizipitierendm Sera und dmn Werthbemessung fiu die Praxis. Dtsch. Tumosa, C. S. 1976. The reaction of the DoGhm bifom lectin with non- Med. WWocnschr. 29(11): 192-195 250 Referencesfor Unit IV

Weichardt, W. 1905. Zur Frage des Nachweises individueller Woodward, J. J. 1875. On the similarity between the red blood-corpuscles Blutdifferenzierung. Vierteljahrschr. Gerichtl. Med. &B: Santuetswes. of man and those of certain other mammals, especially the dog; consid- 29(3F): 19-27 s2 ered in connection with the diagnosis of blood stains in criminal cases. Welsh, D. A. and H. G. Chapman. 1910. On the differentiation of proteins Mon. MirraPe. J. 0. 65-76 of chdy rrkted spsies by the prdpitin reaction. J. Hyg. 110: 177-184 Ziemke, E. 1901a. Zur Unterscheidung von Menschen- und Thierblut mit Whitehead. P. H. and A. Brech. 1974. A micro-technique involving species Hicines spezifischen Serums. Dtsch. Med. Wochenschr. 27(26): identification and ABO grouping on the same fragment of blood. J. 424-426 Forensic Sci. Soc. 14: 109-1 10 Ziemke, E. 1901b. Weitere Mittheilungen nber die Unterscheidung von Whitehead. P. H., A. Brech and I. Cayzer. 1974. The identification of the Menschen- und Thierblut mit Hilfe eines spezifischen Serums. Dtsch. species origin of bloodstains using sensitid later J. Forensic Sci. Soc. Mcd. Wochenschr. 27(42): 731 -733 14: 103-107 Ziemke, E. 1901c. Ueber die Ungleiche Resistenz des Blutfarbstoffes ver- Whitney. W. F. 1902. Notes on the production of the test serum in rabbits. schicdener Thiere gegen Alkalien und cine hierauf geflndete Methode Boston Med. Swg. J. 146(17): 429 zur Unterscheidung von Menschen- und Thierblut. Vierteljahrschr. Whitney, W. F. 1903. The serum test in rabbits. Med.-kg. J. (N.Y.) 21: Gerichtl. Med. Oeg Sanitaetswes. 22(3F): 77- 103 5 33-35 Wiener, A. S.. M. A. Hyman and L. Handman. 1949. A new serological test (inhiiin tat)for human serum globdin. Proc. Sm. &p. Bbl. Med. n: %-99 Bibllognphlo Not08 to Refennoes for Unit IV Williams, C. A. and M. W. Chw (eds.) 1971. Methods in Immunology and Immunochnnistry, v. 3, Academic Press, New York and London $'Japanese Journal of Legal Medicine (Jpn. J. Leg. Med.) see note 1 to Wood, E. S. 1902a [no title. Society Report]. Boston Med. Surg. J. References for Unit V 144(17): 440 5'VierteQahrschrifl fur Gerichtliche Medizin und ijffentliches Wood, E. S. 1902b. The serum tat for blood. Boston Mcd. Surg. J. Sanitatswesen (Vierteljahrschr. Gerichtl. Med. Oe# Sunitocrswes. ) see 146(17): 427-429 Note 7 to References for unit I1 Wood, E. S. 1903. The ~rmmtat for blood. Med.-Leg. J. (N.Y.) 21: jJForemic Serology News (Forensic Serol. News) sec Note 1 to 26-32 References for Unit V1