Sourcebook in Forensic Serology, Immunology, and Biochemistry, Study notes of Biochemistry

Download Sourcebook in Forensic Serology, Immunology, and Biochemistry and more Study notes Biochemistry in PDF only on Docsity! UNIT II IDENTIFICATION OF BLOOD whether certain of them came from the same individual, assuming it could be established that they were stains of human blood. The e m established that a number of the stains were bloodstains using Orfila's chemical tests as criteria. They could not shed any light on the questions of species of origin, nor of common origin, nor on the question of the time i n t d which had elapsed since the deposition of the stains. Around this time, some authorities were considering mi- croscopical examination of bloodstains as a means of identification of blood in medimlegal inquiries. In some minds, microscopical results were more certain, and were preferable to the chemical methods. This subject is discussed in section 5.3. It was also recognized that carefully con- ducted microscopical observations of blood cells from blood- stains could, under some circumstances, help in diagnosing the species of origin. The blood of animals having nucleate red cells could, at least, be distinguished from mammalian blood. Some authorities, aware that the red cells of dserent species differed in size, thought that carefully conducted microscopical measurements of the red cells could, in some cases, serve as a basis for species determination, even among mammalian species. This subject is discussed in section 15. Blood Identitiwbn-Hiiory O d a (1 827b) looked into microscopical methods for blood identification and species determination. He was unable to obtain satisfactory results, and concluded that the chemical methods were much more reliable. Around 1836, Prof. Per- soz at Strasbourg introduced the use of hypochlorous acid as a reagent for discriminating bloodstains from other red stains, especially older ones which were not very soluble in water. Oriila evaluated this technique quite thoroughly in 1845,and found it unsatisfactory if used alone, but admitted that it might be useful as an auxiliary method in certain types of cases. A number of the early papers on the subjects discussed in this section may be read in their entirety in the translations (Unit IX). There is not much doubt that the carlitst scientifically systematic attempts to employ physical and chemical methods to the medico-legal examination of blood and body fluids are attributable to the French scientists in the early years of the 19th century. Some of the earlier papers on the identification of seminal fluid, other body fluids, menstrual blood, and so forth, are discussed in appro. priate sections and some have been included in the trans- lations as well (Unit IX). Blood Iden-n- Tenr SECTION 4. CRYSTAL TESTS 4.1 Struetura wrd [email protected] Porphyrlns dHematin Compounds The discussion of crystal tests and of spectrophotometric and spoctrotluorimetric methods which follows will involve many terms which refer to porphyrin and hematin com- pounds. The history and development of nomenclature of these materials is somewhat complex and can lead to consid- erable confusion. Interested readers are referred to the spe- cialized works by Lemberg and Legge (1949), Falk (1963) and Marks (1969). Most standard biochemistry texts also carry a discussion of the subject (e.g. White et al.. 1973; Pritham, 1968; Mahler and Cordes, 197 1 ).The present dis- cussion is designed primarily to provide an outline of the nomenclature, and give some indication of the different meanings that may be associated with various terminology. Potpbyrins are derived from the cyclic ring compound porphin, a structure with four pyrrole-like rings linked by methylene bridges. Represented in Figure 4.1 in two dif- ferent ways, the structures (a) and (b) are identical, neither is "correct" or "incorrect", and neither is preferable for any particular reason. Both are encountered in the literature. The porphyrins which occur in nature are all compounds in which side chains are substituted for some, or all, of the hydrogens at positions 1 through 8 in Figure 4.1. It is convenient in giving structural representations of these com- pounds to use a "shorthand" notation for the porphyrin nucleus, omitting structural detail, but allowing the dierent side chains to be shown in their correct positions. Figure 4.2 (a) and (b) shows the shorthand representations correspond- ing to the structures in Figure 4.1 (a) and (b), respectively. Introducing side chains into the molecule gives rise to a large number of d i i ren t structural isomers. If, for example, the eight numbered hydrogen atoms are substituted with four methyl- and four ethyl- groups, four isomers are possi- ble. Thesearc shown in Figure 4.3 according to both "short- hand" conventions, corresponding to Figure 4.2 (a) and (b), respectively. Only compounds deriving from structures I and 111, Figure 4.3, are found in nature, those from I11 being more important.In representing the isomers according to the shorthand notation, it is usual to number the positions as has been done in Figure 4.2. Not all authors number the posi- tions in the same way, however, and while any sequentially numbered system is interconvertible to any other by rotation in the plane of the paper, it eliminates confusion, in my opinion, if the numbering convention is clearly stated. Some of the different, naturally occurring porphyrins are indi- cated in Table 4.1. Only the type I and type I11 isomers (Figure 4.3) are given. If the number of types of substituent side chains is increased, then clearly the number of possible structural isomers increases as well. In the case of protu- porphyrin, for example, with three substituent types, there are Uteen structural isomers. All the porphyrins derived from hemoglobin and naturally occurring hematin com- pounds are of protoporphyrin type 111, Table 4.1. The mole- cule is more commonly referred to as protoporphyrin iX, since it was the ninth in a series of isomers listed by H. Fischer (White et al., 1973). Protoporphyrin IX is shown in shorthand notation in Figure 4.4. Porphyrins possess the ability to combine with many met- als, the most important biologically active molecules being those in which the porphyrin is combined with Fe or Mg. These compounds are collectively referred to as metal- loporphyrins. The hematin compounds, which are the only metalloporphyrhs of major interest to this discussion, are all iron-protoporphyrin compounds. The iron atom in an iron- protoporphyxin complex is coordinated to the four pynole nitrogen atoms in a planar configuration ( E i4.3, replac- ing the two dissociable hydrogens of the porphyrin nucleus. The detailed properties, including the nature of the coor- dinate binding and thennodynamic stabii, or iron and other metal po~~hyrinswere discussed by Phillips(1%3). The iron complexes readily add two additional ligands, which coordinate to the metal forming an octahedralstructure and in which the metal is then hexacoordinated. The valence of the iron atom is specified by the prefixes ferro- (for Fez+) and fern- (for Fe3+) in naming the various compounds. Heme isferroprotoporphyrin Heme is spelled haem in some countries, the spelling variation carrying over to other terms derived from the word heme, e.g. herno- globin/haemoglobin, hematin/haematin, hemochromogen/ haemochromogen, etc. Ferriprotoporphyrin,obtained as the chloride, is called hemin chloride, or hematin chloride. Ferriprotoporphyrin hydroxide is simply call hematin. The use of the term hemin is restricted by some authors to fer- riprotoporphyrin halides, especially the chloride, (Lemberg Ib) Figure 4.1 Porphin .%u#book in Fonmic Semlogy, Zmmunology, andBiochemirZry chromogens, and the terms hemichrome and porahematin have been applied to ferrihemochromes. Table 4.2, a modi- 19 + fication of Table I, Chapter V, of Lemberg and Legge (1949), gives a comparison of some of the diierent nomenclatures. 4 Another important consideration, which is not really a matter of nomenclature, but which may be worthy of brief 6 discussion here, is that of the interconvertibility of the vari- 6 5 ous hemoglobin derivatives. Both the crystal and spectral (a) (b) tests for the presence of blood in stains rely on these con- versions. There are, in addition, a number of methods de- Figure 4.2 Porphin - Shorthand Notation signed to determine the age of bloodstains which rely on the Figure 4.3 Structural isomers of Etioporphyrin - Equivalent Representations M - methyl and Legge, 1949; Phiips, 1963; White et al., 1973). Marks (1973) seems to be suggesting that the term hemin be re- served for femprotoporphyrin halide crystals (see Teich- mann, 1853). and that it should not be used in place of the term hematin Ferroprotoporphyrin may be called ferro- heme, as ferriprotoporphyrin may be called ferriheme (or ferrylheme). In compounds in which the Hth and sixth liganding molecules are nitrogenous bases, the term hemo- chromes is often applied. The names ferrohemochrome and ferrihemochrome may be used to specify the valence of the iron atom. Ferrohemochromes have long been called hemo- E - ethyl hemoglobin-methemoglobin interconversion. These are dG- cussed in a later section. The structure of hemoglobin will not be discussed here, but in a later section dealing with the determination of genetically-determined hemoglobin variants. Suffice it to say that native human hemoglobin is a tetrameric molecule, consisting of two a and two @ polypeptide chains, having one heme per peptide chain, or four in the intact molecule, and a molecular weight of about. 68,000. The iron atom is di- valent in hemoglobin. Oxidation of the iron atom to the ferric state gives rise to methemoglobin (hemiglobin; fer- Table 4.2. Comparison of Nomenclature of Hematin Compounds Coordinating Ligands In iron protoporphyrin IX Charge on over-all coordinate complex Valence of Fe Old Names Nomenclature of Pauling Et Coryell (1936) and Guzman Barron (1937) Nomenclature of Clark (1939). Clark et al. 11940) and Drabkin 11938 and 1942a) General Nomenclature four pyrrole N 0 2 reduced hematin: heme ferroheme ferroporphyrin heme four pyrrole N. two additional N of nitrogenous base 0 2 hemochromogens; reduced hemochromogens; reduced hematin ferrous or ferro- hemochromogens base 1e.g. dipyridine- . nicotine-) ferroporphy rin hemochromes four pyrrole N. water and OH- 3 hematin: hydroxyhemin; oxyhemin ferrlheme hydroxide ferriporphyrin hydroxide hematin four pyrrole N yes * 3 hemlns. e.g. chlorhemin, bromhemin ferriheme chloride, bromide etc. ferriporphyrin chloride, bromide etc. hemlns (CI, Br, etc.) four pyrrole N. two additional N of nitrogenous base yes* 3 parahematlns: oxidized hemochromogens ferri- or ferric hemochromogens base (e.g. dipyridlne-. nicotine-, etc.) ferriprotoporphyrln hemichromes * Charge depends on pH. Soamebook in Formdc Serology, Imntunology, and Biochem&irtry hematoporphyrin metHb-CN, + denatd globin + denatd Figure 4.6 Interrelationships Among Hemoglobin Derivatives Abbreviations used: Hb= hemoglobin HbOl= oxyhemoglobin Hb CO = carboxyhemoglobin metHb= methemoglobin metHb-CN =cyanornethemoglobin metHb-OH= hydroxymethemoglobin denatn= denaturation renatn= renaturation denatd =denatured [H] =reducing agent [0]= oxidizing agent oxidn=oxidation redn= reduction H* =acid OH' =base conc =concentrated Table 4.3 Some Modifications of the Hematin Crystal Test (modified from Lewin and Rosenstein, 1895) Blood Preparation Acetic Acid Other Acid NaCl Other Salt Heat or Reference Temperature I°C) dried, much oxalic, tartaric - - 25 - 62.5 Teichmann 11853) citric lactic dried, fluid slight excess - only i f none - cold or 40 - 60 BUchner and in blood Simon 11858) dried to fill space - yes - heat in flame Virchow. 1857 under covar slip - yes - no - gentle heat or spon- Morache. 1881 taneous evaporation dried few drops - no - heat until Janert, 1875 bubbles appear dried, fluid Ves - few drops salt sofn - heat in H,O bath Brilcke, 1857 dried yes oxalic. tartaric yes i f blood NaBr, KBr - Bikfalvi, 1886 i n alcohol CI free NH,Br. Nal KI sediment prepared by Ves - Ves BaCI,. SrCI, - Teichmann, 1856 copper sulfate pptn KC1 LiCI, CaCI,, and extraction w i th NH,Cf MnCt,. alcoholic sulfuric acid SnCI,, FeCI, MgCI, dilute solutions of blood NH,CI Struve. 1880 pigment, add ammonia. tannic acid, then acidify with acetic acid. get hematin tannate precipitate alcoholic extract of dried yes - yes CaCI. - Gwosdew, 1886 precipitate by sodium carbonate pptn of defibrinated blood s~~ncebook Semlogv, Immunologv, and Biochem&yin FO& 1909; Puppe, 1922) as reductants were described as well, being logical in view of HUfner's earlier observations (1 899). Nitrogenous bases other than pyridine will participate in hemochromogen crystals formation. Cevidalli (1905) suc- cessfully employed piperidine solutions, and Lochte (1910) noted that piperidine or picoline could be substituted for the pyridine. The most comprehensive study of hemochromogen in the early literature is almost surely that of Dilling, published in 1910 in both German and English. This volume records the results of Dilling's extensive experiments carried out in Prof. R. Kobert's laboratory. Hemochromogen crystals were pre- pared from blood, hematin and other derivatives of hemo- globin utilizing pyridine, piperidine and a number of other nitrogenous compounds, with ammonium sulfide, hydrazine hydrate, and ammonium sulfide in NaOH as reductants In each case the microscopic and spectral characteristics of the crystals obtained were described, as well as any problems encountered in the course of applying a particular technique. As nitrogenous bases, Dilling tested pyridine, piperidine, nicotine, methylpiperidinc, ethylpiperidine, coniin (2-pro- pylpiperidine), conhydrine (2-(a-hydroxypropyl) piper- idine), and its isomer pseudoconhydrine, 2-methyl- and 3-methyl-pyridine (a- and 8-picoline, respectively), a-di- methylpyridine (a-lutidine), as well as trimethylpyridine (collidine)' and tetramethylpyridine (panoline), the two last mentioned probably consisting of mixtures of the isomers. Pyridine and piperidine were found to be the most satis- factory of all these compounds for crystal formation from blood. In addition to presenting his results, Dilling gave a good, comprehensive review of the pre- 19 10 literature. Somewhat less comprehensive reviews have been given by Kalmus (19 10) and by Kurbitz (19 10). A great many authorities since 1912 have preferred to prepare hemochromogen crystals using the reagents de- scribed by Takayama in that year (Akaishi, 1956; BNnig, 1957; Gonzales et al., 1954; Greaves, 1932; Hunt et al., 1960; Kerr and Mason, 1926; Kirk, 1953; Lopez-Gomez, 1953; Mahler, 1923; Olbrycht, 1950; Rentoul and Smith, 1973; Thomas, 1937; Ziemke, 1924). Takayama's name has come to be used to describe not only the reagent he devised, but alsothe procedure and the hemochromogen crystals thus obtained, in much the same way as did Teichmann's name in the case of hematin halide crystals. The original paper in 19 12 is cited in several different ways in the literature3 and some difiiculty was encountered in locating it. The paper, writfen in Japanese, appeared in Kokka Igakkai Zasshi. Curiously, Takayama's paper was published in 1912 in Japan, but it does not appear to be mentioned in the Euro- pean literature until Strassmann's paper appeared in 1922. It would be of interest to know how the information got from Japan to Germany. Takayama was in Germany around the turn of the century, but before 1912 (see in Unit IX, Trans- lations). We had some correspondence on this point with Prof. Dr. Hiroshi Hirose of Kyushu University in Fukuoka, Japan. After some extensive searching in the early literature, Prof. Hirose discovered the paper by Strassmann (1922), and kindly shared the fruits of his search with us. His letter to me, in which the historical details are given and fully documented, has now been published (Hirose, 1979). Strass- mann learned of the Takayama test from Prof. Fujiwara who was a student of Takayama's, and who was in Europe from 1920-1923. Takayama had noted that Reagent I re- quired that the preparation be warmed for best results, while Reagent I1 did not require warming. Perhaps for this reason, many authorities preferred the second reagent, even in Japan where the test was widely used before its introduction in Europe. As mentioned in the footnote, the citation of the original paper was not correct in the German and English literature. Neither we nor Prof. Hirose could h d any jour- nals with the titles given in the incorrect citations. Ta- kayama proposed two solutions, I1 being a sort of improved version of I. Solution I: Solution 11: 10% dextrose 5 mQ Saturated dextrose solution 3 mQ 10% NaOH 10mf! 10% NaOH 3 d pyridinc 10-20 mP pyridiie 3mQ water 65-75 mP water 7 mQ The crystals, obtained by treating a small amount of blood or stain fragment with these solutions are shallow rhom- boids, salmon-pink in color. Gentle heating was needed with the first reagent, but not with the second. As mentioned previously, Mahler's (1923) extensive study of blood crystals included hemochromogen crystals as well as hematin crystals. He compared a number of different methods and solutions for obtaining the crystals from a large number of different types of dried blood specimens. Kerr and Mason (1926) discussed the test in some detail as well, concluding, as had Mahler, that Takayama solution I1 was the most reliable reagent for obtaining hemochromogen crystals. The speed with which the crystals appear depends on temperature, being almost immediate if the material is heated, somewhat slower at room temperature (1 to 6 min- utes), and slower yet if in the cold (e.g. if the reagent has been kept in the refrigerator). The reagent is stable for 1-2 months, crystals taking somewhat longer to form with older reagent. Greaves (1932) recommended the use of Takayama solution I1 for hemochromogen crystals as the method of choice for blood identification. He preferred not to heat the material, and suggested waiting up to several hours if neccs- sary before deciding that the result is negative. Puppe (1922) also noted that good results were obtained without heating. Oustinoff (1930) suggested that gum arabic be included in the reagent, 1 part to 3 parts pyridine. There are a number of advantages to the hemochromogen ' 2,4,6-trimethylpyridine is now called y-collidiie, while u-collidine is ethyl-2-methylcollidine and fl-collidine is 3-ethyl-2-methyl-cofidine. * && (lqW & (lg2.) the -= had appeared m the Japanese Journal of Toxicology. Ziernke (1924) and dted as having appearrd in Japao. mm fiu Staatsatznhde, this having pmbabfyhem taenfrom S- (1922). test, as compared with the hematin test. A technical advan- tage is that heating is not required to obtain results within a reasonable amount of time; and even if one does prefer to apply heat, the test is not subject to being ruined by over- heating. The test also yields positive results under some of the circumstances where the Teichmann test fails. Thus, Mahler (1923) obtained a positive Takayama test in cases of various 22 year old bloodstains on linen and of stains on rusty knives up to 23 years old, which failed to give Teich- mann crystals. Similarly, Kerr and Mason (1926) showed that stains on linen and glass which had been heated to 1 SO0 for 30 minutes, stains (relatively fresh) washed in hot water, and stains on rusty metal surfaces up to 45 years old, all yielded hemochromogen crystals but did not give hematin crystals by the Sutherland technique. According to Kirk (1953), both the specificity and sensi- tivity of the hemochromogen test are about the same as those of the hematin crystal test. Ken and Mason (1926) and Greaves (1932) say that the hemochromogen test never failed in their hands in the known presence of blood. Mahler (1923) got some failures, but with solutions other than the Takayama 11. The proof value of a positive hemochromogen test, with spectroscopic confirmation of the identity of the product, is not widely disputed. A negative crystal test, how- ever, should not necessarily be interpreted as meaning that blood is absent (Kirk, 1953; Olbrycht, 1950). That blood- stains which have been exposed to heat, or are old or weathered, become increasingly insoluble has been known for a long time (Katayama, 1888; Hammerl, 1892). In cases such as these, solubility can be a problem in itself; if it is not possible to solubilize any hemoglobin or hemoglobin deriva- tives, it will obviously not be possible to obtain a positive crystal test. Comparisons of sensitivity present some difficulty because various different authors express sensitivities in different ways. It is not always possible to convert one set of units or reference frame to another. This problem is encountered in many of the identification and serological and biochemical tests used in this field. As to hemochromogen crystal tests, Greaves (1932) men- tions only that the fragment of stain or stained material should be very small, only just large enough to be seen and manipulated onto a slide. Antoniotti and Murino (1956) noted that the test is still positive with 1 pf!of blood or about 0.1 mg hemoglobin, while Hunt et al. (1960) could obtain crystals from a stain fragment containing only 0.2 pll of blood. Akaiishi (1965) stated that a positive test could be obtained from a stain made from a 1:30 dilution of whole blood, but did not state how much stain was taken for the test. Miller (1969) thought that the Takayama test was considerably more sensitive than the Teichmann test. He could obtain Takayama crystals from 5 p i of a 1: 1000 dilu- tion of whole blood, provided that the material was dis- pensed onto a slide in 10 separate 0.5 pll aliquots in order to keep the area occupied by the .test material as small as possible. Not long after Kerr and Mason (1926) published their article on the Takayama test, Dilling (1926), in a letter to the Editor of the British Medical Journal, suggested that the hemochromogen test should not supplant the hematin test, but rather be used as to supplement it. He brought up two other points: (1) Kerr and Mason had said that the only discussion of hemochromogen tests in English prior to their paper was Sutherland's discussion, and Dilling correctly pointed out that he had published an extensive study of the subject in 1910; and (2) he believed that the purpose of the sugar in the Takayama reagent was to decrease the solubility of hemochromogen, and not, as Ken and Mason had sug- gested, to serve as a reductant. Kerr (1926a) replied to the letter, saying that he agreed with Dilling's interpretation of the mechanism of action of the sugar. He further said that there was no prior account of the Takayama method in English, and that he still believed it to be much superior to the hematin test for medico-legal work. Recently, Blake and Dillon (1973) have investigated the question of false positive hemochromogen crystal tests. They correctly note that the question has received virtually no attention in the medico-legal literature. Of particular inter- est was the issue of whether other iron-protoporphyrin con- taining substances, such as the enzymes catalase and peroxidase, would give misleading false positive crystal test reactions. Three presumptive tests were also carried out on the material, including the benzidine and phenolphthalin catalytic tests (sections 6.3 and 6.4) and the luminol test (section 6.7). A number of microorganisms were tested, since these are known to be particularly rich in catalase and peroxidase activities. Pure samples of both the enzymes gave Takayama crystals, those formed with catalase S ing virtu- ally indistinguishable from the crystals obtained with blood. These materials also gave positive results with all three pre- sumptive tests. The effectiveness with which different bacte- ria reacted with benzidine, in a two stage test, was directly related to the catak content of the cells. One drop (about 0.05 mP) of a suspension of Citrobacter having a cell concen- tration of 400 x 106/mPgave a positive benzidine reaction, and several microbid suspensions which had been dried as spots on filter paper gave positive benzidine reactions after 2 months. Heating a number of different bacteria at 100"for 30 mi-or at 150" for 15 mi.did not abolish the benzidine reaction. Although pure catake and peroddase could give a positive crystaltest, none of the bacteria tested contained suf- ficient concentrations of these enzymes under the test wndi- tions to give a false positive Takayama test. Blake and Dillon cautioned that great care should be used in the interpretation of catalytic and crystal tests, and in c o m b i o n s of them, since it is not only possiile, but even likely in some situations, that case material will be contaminated with bacteria. Blood Ident~YWion-Spectral mrd MknxcopW SECTION 8. SPECTRAL AND MICROSCOPICAL METHODS 5.1 Spectroscopic and Spectrophotometrlc Methods Spectroscopic and/or spectrometric analysis of hemo- globin and its various derivatives is considered to be among the best methods for the certain identification of blood in stains (BNnig, 1957; Derobert and Hausser, 1938; Ewell, 1887; Gonzales et al.. 1954; Gordon et al.. 1953; Lopez- Gomez, 1953; Mueller, 1975; Olbrycht, 1950; Rentoul and Smith, 1973; Simonin, 1935; Sutherland, 1907; Walcher, 1939; Ziemke, 1924). The methods are not technically diflicult in practice, but as with so many of the methods and tests, a good deal of care should be exercised in the inter- pretation of results. Most of the older workers employed hand spectroscopes or microspectroscopes or both, since spectrophotometers were not widely available until rela- tively recently. Some authorities have said that the identity of hemochromogen crystals (section 4.2.4) should be con- h e d microspectroscopically. While the spectral methods might be thought to have the singular advantage of being nondestructive, they really do not if the tests are carried out properly. I t does not suace in the opinion of most experts to determine the spectrum of an extract of a stain, and infer from that alone the existence of blood in the stain. Even in cases where one can be reasonably certain that the material being examined is pure, the identity of a substance should never be inferred solely on the basis of the observation of its absorption spectrum. Lemberg and Legge (1949) state this concept especially cogently: While it is certainly true that under identical conditions the same substance cannot have different absorption spectra, spectra which are apparently identical are insufficient evidence for chemical identity. . . . . . . . It isalwaysnecessary to demonstrate identical altemtions in spectra when chemical reactions are per- formed, before identity of two substances can be con- sidered in any degree certain. This caveat must be considered especially relevant to medico-legal identifications, not only since there are a large number of porphyrin compounds in nature, many of which have common spectral features, but also since it can almost never be assumed that stain evidence is uncontaminated. Most of the spectral methods, therefore, involve the prepara- tion of various hemoglobin derivatives, followed by verifi- cation that these have actually been obtained by measuring the spectra. This subject, along with the crystal tests and other chemical tests, has been excellently reviewed by Fiori (1962). I am indebted to this work for source material as well as for lucid explanations of many aspects of the various tests. Since there is quite a large number of different hemo- globin derivatives that may be used in identification pro- cedures, and a fairly extensive literature on their absorption spectra, it seemed most profitable to present a cross section of this information in tabular form (Lemberg and Legge, 1949; Fiori, 1962). When there are multiple absorption max- ima, the bands are sometimes called I, 11,111, etc., in going from longer to shorter wavelengths; another convention is to refer to the major visible bands as a and 8, the a-band being at longer wavelength. A very intense band in the region of 400 nm, characteristic of all conjugated tetrapyrrole struc- tures, is known as the "Soret band". Table 5.1 gives the absorption maxima reported by different workers for a num- ber of hemoglobin derivatives. A composite representation of the spectra of some of the derivatives, as presented by Lem- berg and Legge (1949), is given in Figure 5.1. The absorp tion bands for a number of derivatives, as seen in the spectroscope, are given in Fig. 5.2, as originally shown in the review of Hektoen and McNally (1923). Representations such as that in Figure 5.2 are still seen in textbooks of legal medicine. Among the earliest papers on the spectral properties of the "coloring matter of blood" was that of Hoppe (1862). He observed and reported the absorption bands of hemoglobin and several of its derivatives in the visible range of the spec- trum, and suggested that the spectral method be employed for the forensic identification of blood. More elaborate stud- ies were done by Stokes (1864) who recognized the dif- ference between Hb and Hb02, described the spectral properties of hematin and, for the first time, of hemo- chromogen. Sorby (1865) independently studied the spectra of a number of hemoglobin derivatives and advocated the spectral method for the identification of bloodstains. In 1868, Herepath discussed the techniques in some detail. He was able, using the microspectroscope, to identify blood on the wooden handle of a hatchet which had lain exposed in the country for several weeks. In this case (Reg. v. Robert Coe, Swansea Assizes, 1866), the amount of blood remaining on the handle was very small. The technique was stated to be sufficiently sensitive to detect ". . .. less than one thou- sandth of a grain of dried blood, the colouring matter of which had been dissolved out by a drop and a half of distilled water." Bell (1892) noted that Dr. Richardson of Philadel- phia had said he was able to detect the blood on an ax handle equivalent to '/,,grain of blood in a case that he had had. Sorby (1 870) described his own technique, and stated that it was equally applicable to medico-legal blood identification and to the clinical identification of blood in urine. These applications were based on his earlier studies of the spectra of the various derivatives (Sorby, 1865), in which he had first Soumbook in Forensic Ser010gy.Immunology, and Biochem&v 1 2 3 4 5 IOXYHEMOGLOBIN (Varying Concentrations) HEMOGLOBIN 6 CO-HEMOGLOBIN 7 8 IALKALINE HEMATlN (Dilute and concentrated) 9 HEMOCHROMOGEN 10 METHEMOGLOBIN 11 ACID HEMATIN 12 13 14 !O1!l 0 I I.I ' I "0 I I 1 I .o 1 AClD HEMATIN (in alcohol) AClD HEMATOPORPHYRIN ALKALINE HEMATOPORPHYRIN Figure 5.2 Absorption bands for hemoglobin and some derivatives. Wavelengths in nm for Fraunhofer lines: B 686.7, C 656.3,D 589.3, E 527.0, F 486.1, G 430.8. From: L. Hektoen and W. D. McNally Medicolegal Examination of Blood and Bloodstains in: F. Peterson, W. S. Haines & I?. W. Webster (eds.) Legal Medicine and Toxicology, 2nd ed., V . 11, 1923. Reprinted by permission of W. B. Saunders Co. recommended this technique for forensic examinations. Dr. Taylor recommended Sorby's technique for medicolegal cases, and wrote a sort of introduction to the 1870 paper (Taylor, 1870). The spectral methods were incorporated into the reper- toire of techniques for examining bloodstains fairly quickly. The earlier texts of Chapman (1892), Ewe11 (1887) and Reese (1891) carry discussions of them. Relatively fresh, reasonably well-preserved stains may still show hemoglobin spectra. As aging proceeds, the hemoglobin is converted into methemoglobin and later into hematin. The last-mentioned is exceedingly less water-soluble than hemoglobin, it being necessary with old stains to extract with acid, base or solvents. The extracts may then be examined for the appro- priate hemoglobin derivative. Reese (1891) was apparently recommending the preparation of alkaline methemoglobin in cases of stains, though present-day terminology for the derivatives was not then used. In the early English literature, as noted by Gamgee (1868), hemoglobin was called cmorine, after Stokes' suggestion (1864). Hb02 was called "scarlet" cruorine, while Hb was "purple" cruorine. The early workers regarded the Hb + O2eHb02 reaction as one of oxidation-reduction, and they seem to have had dBculty squaring this concept with their results using ox- idizing or reducing agents which were affecting the valence of iron. Sorby (1865) apparently suggested that the term "brown cruorine" be applied to methemoglobin. It was Hoppe-Seyler (1864) who introduced the present-day name "hemoglobin". "Um Verwechselungen zu vermeiden, nenne ich den Blutfarbstoff HBmoglobulin oder H'ioglobin," he wrote. [In order to avoid confusion, I call the blood pigment hernoglobulin or hemoglobin]. All hematin compounds can be transformed into hemo- chromogens by additon of base, reducing agent and a nitrogenous compound (Lemberg and Legge, 1949). The derivative, characterized spectrally by a sharp a-band in the 550-560 rm region, is therefore one of the most useful in diagnosing bloodstains. As discussed previously in section 4.2.4, heme will combine with a variety of nitrogenous bases to form hemochromogens. Bloodstain extract may be treated with strong alkali and ammonium sulfide to form hemo- chromogen (Sutherland, 1907). Simonin (1935) suggested extracting the bloodstain with 10 mN HCl, and treatment of the extract with KOH and hydrosulfite. The characteristic band at 560 nm is detectable by this method at 1500 to 1:1000 dilutions of whole blood. Smith and Simpson (1956) and Glaister and Rentoul (1957) recommended a reagent made up by shaking 2 g Na2S$04 in 5 mQ 10% NaOH or KOH and adding 1 mP alcohol. This solution must be freshly prepared before each use. Pyridine hemochromogen may be obtained using Takayama's solution (Smith and Simpson, 1956; Fiori, 1962), as discussed in Section 4.2.4. Riegler (1904) used a solution of hydrazine sulfate in alcoholic NaOH. A drop of hydrazine sulfate in 20% KOH, intro- duced under the cover slip on a slide containing the sample, was recommended by Hesselink (1 93 1). Olbrycht (1950) dissolved the stain directly in pyridine, then added hydrazine Blood IdenaYiation-Spectral and Miiroscopical sulfate as reducing agent. Meixner (1927) recommended KOH in glycerol as a good medium for extracting older stains. Hankin (1906), who was concerned with stains that underwent rapid putrefaction when damp, because of the tropical climate, recommended treatment with boiling water, followed by addition of ammonium sulfide on a slide. Cyanide hemochromogen may be prepared as well (Hankin, 1906; Fiori, 1962) if KCN solution is used to dissolve the bloodstain. The use of the hematoporphyrin spectrum for bloodstain identification was first suggested by Struve in 1880, but not until Kratter published the results of his experiments in 1892 did the method begin to enjoy more general use. Hema- toporphyrin is formed from bloodstains upon treatment with concentrated HISO,. The iron atom is removed by this treat- ment and the vinyl residues of the porphyrin nucleus are oxidized to hydroxyethyl groups (see Table 4.1). This mate- rial was originally called "iron-free hematin". The treat- ment with sulfuric acid can cause charring of the fabric, and carbonized fabric fragments interfere with spectral exam- ination. Ipsen (1899, 1900) thought that the treated mate- rial could be washed in order to get rid of such particles, and more acid added. But Ziernke (1901) found that this pro- cedure did not work well. He advocated 24 hr sulfuric acid treatment of the stain, glass wool filtration and neutral- ization to form a precipitate. The precipitate is then washed, dried and an ammonia-alcohol extract of it examined spec- trally. Takayama (1905) allowed the acid to act on the stain for 5-7 days, after which the material was heated, diluted to about three times its volume with water, filtered through glass wool, and examined. The hematoporphyrin procedure may well be the least desirable of all spectral tests, if proof of the presence of blood is wanted. There are probably more naturally occurring sub- stances with spectra similar to hematoporphyrin than to the other derivatives. When bloodstains are very old, however, or when they have been exposed to high temperatures for ex- tended periods, they become very difficult to dissolve, and often fail to give hematin or hemochromogen. In these cases, hematoporphyrin preparation may be the only recourse. Fiori (1962) notes that strong acids should be avoided for spectral determination, and that hematoporphyrin is, there- fore, best prepared by the milder method of Dotzauer and Keding (1955). The stain is dissolved in 0.1N HC1, with heating if necessary. After 15 mhiutes, concentrated thio- glycollic acid is added and the material heated to boiling for 1 minute. Hematoporphyrin formation is complete in about a half hour. Olbrycht (1950) has pointed out that bloodstain extracts can easily be contaminated with substances from the substratum which may then interfere with the identifi- cation tests. The use of concentrated sulfuric acid would only tend to aggravate such contamination problems because the acid is such a strong solvent. This consideration strengthens Fiori's contention that strong acids should be avoided. Many conditions to which bloodstained material can be exposed affect the spectral tests (as, of course, they affect any of the identification or grouping tests). Weathering, Sourcebook in Forensic Serology, Zmmunology, md Biochemktty submersion in water, exposure to sunlight, heat, washing, or rust can all cause great difficulty. Rust formation is acceler- ated on bloodstained ferrous metal surfaces (Buhtz, 1933), and rust contamination often precludes the identification of blood. Scheller (1937) studied the spectral identification of bloodstains on various substrates after exposure to air and weathering in different humidities. Damp environments cause more rapid deterioration of the stain than moderate ones, and accelerate the formation of rust on bloodstained ferrous metal surfaces and of ZnC03 on bloodstained gal- vanized sheet metal surfaces. Green plants are particularly difficult contairninants of samples to be subjected to spectral tests because of the chlorophyll, which, being itself a mag- nesium porphyrin derivative, shares spectral characteristics with hematin compounds and which may be convertible under some circumstances to similar derivatives (Mayer, 1933). A bloodstain on a green leaf may therefore present special problems in this regard. Hirose (1966a, 1966b) stud- ied the effects of soil on bloodstained fabrics buried in earth. Hemoglobin leeches out of such fabrics, the rate being di- rectly proportional to soil moisture content, but independent of soil temperature. Hemoglobin solubility decreases with time in such samples as well. In the case of a bloodstained cotton cloth buried in soil of pH 5.3, Hirose found the order of efficiency of extraction of hemoglobin (from most efficient to least efficient) to be as follows: 20'30 pyridine 1% NaOH > Veronal buffer, pH 9.2 > Veronal buffer, pH 7 >Veronal buffer, pH 5.3 >water. The hemochro- mogen spectrum could be obtained in some samples after 40 days in the soil. Olbrycht (1950) camed out extensive studies on spectrophotometric and spectrographic identi- fication of stains at various dilutions of blood. Micro- spectrometric determination can fail if stains on linen were made from blood diluted more than 1: 150; spectrographic tests in the UV region of the spectrum, however, can detect stains made from up to 1:400 dilute non-hemolyzed blood and 1:750 dilute hemolyzed blood, these being too dilute to give any evidence to the naked eye of there being a blood- stain on the cloth. Sunlight, metal oxides (especially rust) and admixture with fine, aluminum and iron containing soils were found to be the host deleterious conditions 01- brycht's studies. Haseeb (1972) reported spectral identi- fication of Hb and metHb in a 12 year old bloodstain kept on the laboratory bench in the Sudan. Hirose (1976) mixed blood with iron powder and allowed the mixture to age in order to mimic the aging of a bloodstain on an iron surface. The pyridine-hemochromogen spectrum test was carried out at 1, 1 1 and 51 days, with the results shown in Table 5.2. The 20% pyridine-1% NaOH extraction medium was found to be best one under these circumstances, and the higher pH veronal buffer more efficient than the lower pH one, just as he had found in the case of the bloodstainedcloth b&ed in soil (Hirose, 1966b). 5.2 Spectrofluorimetric Methods In 1916, Heller proposed the use of hematoporphyrin fluorescence, resulting from excitation by ultraviolet light, for the identification of bloodstains. Methods of preparing hematoporphyrin have been discussed in the previous section. Hematoporphyrin gives a red fluorescence when illuminated with 366 nm light (Fiori, 1962). Dotzauer and Keding (1955) studied this method in some detail, and their method for preparing hematoporphyrin is given above, in section 5.1. The fluorescence is dependent on the solution used, the substrate concentration, the pH and the tem- perature. Scheller (1973) suggested this method for identi- fying bloodstains on oxidized metals. Dotzauer and Kedding (1955) showed that bloodstains up to 10 years old, exposed to heat, sunlight or humidity can give positive results. Further, chlorophyll does not interfere under their conditions because the acid concentration is too low to bring about its con- version to hematoporphyrin. Ju-Hwa and Chu (1953) ob- tained hematoporphyrin and its characteristic fluorescence by adding a solution of 50 ml glacial acetic acid and 50 ml 0.1N HCl, containing 1 g hydrazine di-HC1, to very dilute blood solutions (1:5000 - 1:20,000). Direct application of the reagent to stains on fabrics or paper was unsuccessful, but treatment of saline extracts gave good results. Chlorophyll, however, gave positive results in this system as well. Schwerd (1977) recommended hematoporphyrin fluores- cence as a certain test for the presence of blood in stains. He preferred the sulfuric acid method of preparation to the HCl/thioglycoUic method of Dotzauer and Keding (1955). Amounts of blood in stains corresponding to 0.6 pg Hb could be detected, and Schwerd indicated that he thought the possibility of false positive results with this test, when prop erly carried out, had been overstressed. The same constraints concerning interpretation must, of course, apply to the spectrofluorimetric test for hematopor- phyrin as do to the spectrophotometric test for it. The ubiquitous biological occurrence of porphyrin compounds must be kept in mind. Bile, feces and meconium may give positive results in these tests because of porphyrin com- pounds contained in them (Fiori, 1962). 5.3.1 Blood identitication by microscopical techniques Microscopical tests have been applied to bloodstains for a number of different reasons, including: Identification of blood in stains Determination of species by noting whether red cells have nuclei or not and/or structure of white cells Examination of leucocytes for chromatin bodies for cyto- logical determination of sex of origin of the stain Examination for epithelial and other cells which might indicate the origin of the bloodstain from a particular organ or tissue Examination for the presence of pathological conditions, such as hematological diseases, presence of parasites, etc. The discussion presented in this section has primarily to do with microscopical methods as means of identifying blood. and aniline, and methylene blue. Neutrophil granules are stained purple-red, eosinophil granules, lighter red, and ba- sophil granules, a deep purple. The nuclei are stained dark blue and the cytoplasm light blue. It is said that the mono- cyte granules become peroxidase negative in bloodstains in about a month, about 8-12 months being required for neu- trophil granules to be negative, while eosinophil granules may be positive in up to 5-year old stains (Undritz and Hegg, 1959). Environmental influences can greatly influence these values. Undritz and Hegg also noted, as did Ziemke (1938), that tissue and organ cells could be histologically differenti- ated in blood stains, such as in cases on blood crusts on a knife which had been used in a stabbing, or where stains may contain nasal or epithelial cells. Le Breton did not share the same level of confidence in or enthusiasm for the micro- scopical methods (Fiori, 1%2). De Bernardi (1959) suggested paraffin embedding, thin sectioning and staining with hematoxyiin preparations con- taining alum for bloodstains in deeply absorbed in wood. DIubler (1899) had employed a similar procedure much earlier. If blood crusts are detached from rusty surfaces by the celloidin technique, the film may be fixed and soaked in a saturated solution of oxalic acid containing a bit of ura- nium nitrate, then exposed to light to dissolved the rust, prior to histological staining (Romanese, 1930). Some of the more recent texts in forensic medicine do not mention microscopical methods for identification of blood at all, e.g. Glaister's Medical Jurisprudence and Toxicology, 13th ed. (Rentoul and Smith, 1973). It should be kept in mind that these techniques require a comparatively large amount of material, and that a certain degree of skill with histological technique is doubtless required if good results are to be expected. In some instances, however, some work- ers prefer these methods. If whole (liquid) blood is encoun- tered, microscopical identification might well be the method of choice, since a smear could be quickly and easily prepared. Fiori (1%2) said emphatically, however, that microscopical methods should never be employed in plclce of other, more reliable procedures, such as spectral, chromatographic and immunological ones. Discussions of microscopical methods are, in any case, of more than purely historical interest, because of the newer methods for the cytological deter- mination of sex of bloodstains (Section 48). 5.3.2. Biological stains and dyes The histological stains employed in this work have known, and often differential, staining a f f ~ t y for blood cells. MOG of the stains commonly employed in histological work have been in use since the last century. Considerable infomation about the structure, properties and mode of action of biolog- ical stains is available (Clark, 1973; Conn, 1933; Gurr, 1960; Gurr, 1962; Lillie, 1977). In 1922 a Commission on Stan- dardization of Biological Stains was set up in this country to devise standards of uniformity for commercially available products and techniques, and to disseminate information. The handbook of biological stains, published under the Com- Blood Zdentz~iwn-Spectral and M~croscopical mission's auspices, has gone through nine editions (Lillie, 1977). Conn's History of Staining (1933) was authorized by the Commission, and a guide to recommended staining pro- cedures, now in its 3rd edition, is published as well (Clark, 1973). A brief discussion of the more commonly used stains is given here. Readers interested in histological stains should consult the above-cited specialized works on the subject. Hematoxylin was first employed as a stain in 1863, appar- ently without success. It was successfully employed two years later by B6hmer (Clark, 1933). Hematoxylin itself is a naturally occurring glycoside from logwood, and is not a dye. It is easily oxidized to the dye form, however, by ox- idizing agents or exposure to air. The oxidation product, which is the dye, has the unfortunate name hematein, and should not be confused with the hemoglobin derivative he- matin. Fig. 5.4 (a) and (b) shows the structures of hema- toxylin and hematein. Eosin, also called eosin Y, is a tetrabromofluorescein (Fig. 5.5), and was discovered in 1871. Giemsa stain, which came into use in 1902, is a mix- ture of methylene blue and its oxidation products, the azurs, in combination with eosin Y. May-Griinwald stain (1902) is a mixture of eosin Y and unoxidized methylene blue (Fig. 5.6), and is equivalent to Jenner stain (1899), after the latter of whom it should no doubt have been named. Wright's stain is the result of heating methylene blue in the presence of NaHC03, adding eosin, collecting the precipitate which forms and dissolving it in methanol. The so-called Feulgen reaction for staining of nuclei relies on the acid hydrolysis of the purine residues from DNA, and reaction of the liberated aldehyde groups with Schiff s fuchsin-sulfurous acid reagent to form red-purple complexes. Basic fuchsin is a mixture of pararosanilin and related compounds. Biological stains and dyes will come up again in this book in other contexts, for example in the visualization of sper- matozoa under the microscope (Section 10.2.1) and as cou- pling reagents for the visualization of the naphthols liberated when naphthyl phosphates are employed as substrates for acid phosphatase (Section 10.3.2). These dyes and stains have frequently had a number of names over the years. It is, thus, not always apparent to the uninitiated that two very different sounding names may refer to the same material. This problem has existed for a long time. There have been standardized lists of dyes and stains prepared over the years, but different lists have not been consistent with one another, nor has there always been consistency in the same list as it was revised and re-revised over time. Dye manufacturers and trade associations have usually designated products by number. There is a lengthy history to the various dye indexes (see Lillie, 1977), but for purposes of this book, suffice it to say that there is now a fairly widely accepted numbering system which is derived from the Colour Index in its most recent edition. Most stains and dyes are assigned a five digit "CI number". In Table 5.3 are listed a number of stains and dyes which come up in medico-legal biology. The preferred name is given, along with synonyms and CI number. Table 5.3 Biological Stains and Dyes Preferred Name Synonyms C.I. Number Chemical Name or Nature 2 Amido Black 10B Naphthol Blue Black; Naphthalene Black 10B 20470 Acld Diazo Dye Aniline Blue WS Water Blue I:Cotton Blue; China Blue 42755 Acid Triphenylmethane Dye Biebrich Scarlet Scarlet 38 of B of EC: Croceine Scarlet 26905 Diazo Dye Brilliant Indocyanin G Coomassie Brilliant Blue 6-250; Supranolcyanin G; 42655 Arylmethane Dye Carmine; Carminic Acid Cochineal 75470 Derivative of Anthraquinone Glycoside Crystal Violet Methyl Violet 108; Gentian Violet 42555 Hexamethylpararosanilin Eosin B, BMX Eosin BN, BA, BS, BW or DHV; Saffrosin; Eosin Scarlet 45400 Dinitro derivative of dibromofluorescein Eosin Eosin V or G; 45380 Tetrabromofluorescein Erythrosin Erythrosin R or G; Pyrosin J 45425 Diiodofluorescein Erythrosin B; N or JN; Pyrosin B 45430 Tetraiodofluorescein Fast Blue B Diazo Blue B; Dianisidine Blue; Fast Blue Salt BN; 37235 See Fig. 10.3 Naphthanil Blue B; Brentamine Fast Blue B Fast Blue RR Blue RR; NRR; Diazo Blue RR 37155 Diazonium Dye Fast Red AL Naphthanil Diazo Red AL; Red AL; ALS 37275 See Fig. 10.2 Fast Red RC Red RC; RCS; Red Salt I; Diazo Red RC; RS: 37120 See Fig. 10.4 Fast Red 4CA Hematoxylin; Hematein Logwood 75290 See Fig. 5.4 - Table 5.3 (cont'd) Preferred Name Synonyms C.I. Number Chemical-Name or Nature * Kernechtrot Nuclear Fast Red; Calcium Red 60760 Anthraquinone Derivative Malachite Green Solid Green, 0;Victoria Green, B; Malechlte Green BXN 42000 See Fig. 6.6 Methyl Blue Helvetia Blue; Cotton Blue; Soluble Blue; Ink Blue; Sky Blue 42780 Aminotriarylmethane Dye Methylene Blue Methylene Blue Chloride 52015 A Thiazin Dye Methykhiazolyldiphenyl Tetrazolium (BS8) MTT; Chelating Tetrazole MTT - 3-14.6-dimethylthiazolyl-21-2, 5- diphenyl tetrazolium bromide &-Naphthylamine Fast Garnet B 37265 See Fig. 10.1 Nitro Blue Tetrazoliurn lBS8) NBT; Nitro BT: Ditetrazollum Chloride - 3,3'(4,4'-di-o-anisylenel-2,2'-di(p- nitrophenylJ-bisl5.phenyl) Pararosanilln Magenta 0;Basic Fuchsin 42500 Trieminotriphenylmethane chloride Ponceau S Fast Ponceau 2B 27195 Polyazo Dye Procion Blue HB Cibachron Blue F.3GA 61211 Arninoanthraquinone Derivative Procion Brilliant Red M-2B Mikacion Brilliant Red 2BS 18158 A Monoazo Dye Pyronin Y Remazol Brilliant Blue R Pyronin G Remalan Brilliant Blue R; Ostazin Brilliant Blue VB; Primazin Brilliant Blue RL 45005 81200 Xanthene Derivative Arninoanthraquinone Derivatilie Rhodamine B Brilliant Pink B; Rhodamine 0 45170 Xanthene Derivative Soumebook inFore- Serology, Zmnologv, and Biochemkt~ Geelong, Australia, confirmed most of the results of earlier workers in 1867. He employed "omnized ether" for the test, and correctly surmised that it worked because of the per- oxides which formed when it was exposed to the air. In what may well have been the first reported use of the test in a medico-legal case, Dr. Day reported that he had detected blood on the trousers of a Chinese man suspected of a mur- der in a place called Scarsdale, Australia, on October 19, 1866. The trousers had been washed by the time they were taken as evidence, and the Government's forensic chemist could find no traces of blood on them by microscopical exam- ination. Day wrote of this case to Dr. Taylor in London, and even enclosed a portion of the trousers on which he said he had detected blood. Dr. Taylor re-examined the cloth, some months old by then, by means of the guaiacum test, and c o n b e d Day's findings (Taylor, 1868). He reported this result in a rather lengthy study of the guaiacum test, prompted apparently by Day's communication. Taylor also noted that he gave the bloodstained cloth from the trousers in Day's case to Mr. Sorby, who was unable to confirm the presence of blood using his microspectroscopic method (see section 5.1). Taylor regarded the test as a useful one, to be used in conjunction with microscopical and microspec- troscopic methods. Negative results were conclusive in his view, while a positive result ". . . enables a chemist to speak with reasonable certainty to the presence of blood . . .". The "false positive" results caused by oxidants which blued guai- acum in the absence of peroxide were easily eliminated by applying the guaiacum tincture first, and the peroxide after a short time if no reaction had taken place. The guaiacum test was the first catalytic test devised for forensic blood identification, and, except for the aloin test which enjoyed very little popularity, was the only one in use for about 40 years. It is often referred to in the literature as Van Deen's test. Some English authors have called it Day's test. The old literature on this test was extensively and excel- lently reviewed by Kastle (1909). Kastle and Loevenhart (1901) stated that the product of the reaction, the so-called "guaiacum blue", had the formula C20H2006 and that guai- aconic acid, G0H240h was the component of guaiacum resin which underwent oxidation. Guaiaconic acid can be sepa- rated, as it turns out, into a and B compounds (Richter, 1906), having the formulas W O , , and q,H4,0,, re- spectively. Guaiacum blue was said to be the oxidation prod- uct of the a compound, and to have the formula CZ2H24o9. The guaiacum test has been applied to the detection of blood in urine (Schumm, 1909) and feces (Messerschmidt, 1909) for clinical purposes. Medico-legal investigators were divided as to the relative value of the test as proof of the presence of blood. Buckmaster (1907) believed that, if the test were camed out .on boiled samples (to eliminate vegetable peroxidases), a positive result was meaningful. Negative results indicated the absence of blood with certainty. Jenne (1896) thought that a positive result in a carefully performed test, using proper controls, warranted the conclusion that the stain was "surely blood". Siefert (1898) stated that a positive guai- acum test indicated a "high probability" of the stain being blood, while a negative result insured that it was not. Hemp hi11 (1875) thought the test was a good and useful one, but did not clearly state that a positive result constituted conclu- sive evidence of the presence of blood. Schumm (1907) thought the test was trustworthy with certain precautions. A larger number of authorities did not believe that a positive result was to be taken as proof of blood, but they did think the test had value as a preliminary or sorting technique (Chapman, 1892; Delearde and Benoit, 1908; arobert and Hausser, 1938; Ewell, 1887; Macnamara, 1873; Marx, 1905; Sutherland, 1914; Wood, 1901). Other workers be- lieved that the primary value of the test was in eliminating stains that were not blood. They stressed the importance of negative results, which warranted the conclusion that blood was absent (Liman, 1863; Mialhe et al., 1873; Mecke and Wimrner, 1895; Palleske, 1905a; Siefert, 1898; Whitney, 1909). A few investigators believed that the test was virtu- ally worthless (Alsberg, 1908; Dervieux, 1910). Breteau (1898) said that very great caution should be used in inter- preting results because of the large number of substances that would give a positive test. The objections to the value of the test were based on the relatively large number of substances other than blood which had been reported to give positive results (Kastle, 1909). A number of inorganic elements and compounds, vegetable extracts, milk, gelatin, bile, gastric secretions, nasal mucus, saliva, pus, leather, soap, and certain typesof papers have all been reported to give false positive reactions. It must be said that the way the test is camed out, the nature of the guai- acum, and of the oxidant used all make a difference in this regard. Carrying out the test on boiled samples (to eliminate vegetable peroxidases), addition of the guaiacum and per- oxide in two steps (to eliminate inorganic oxidants), and the use of guaiaconic acid and H202 (to eliminate variability in the resin, ether and/or turpentine preparations) excludes the possibility of most, but not all, of the interfering substances. Workers who believed in the value of positive results recom- mended all these precautions, in addition, of course, to sub- stratum controls. Various claims have been made for the sensitivity of the test, it being difficult to compare the values in some cases because of the diierent ways in which they were expressed. Van Deen (1862) reported a positive test with a 1:40,000 dilution of whole blood in water. Mitchell (1933) and Kastle (1909) both say that Liman (1863) confirmed this result, but I have quite the opposite impression from Liman's paper. I understand him to say that he got a positive result with a 1:6,000 dilution of fresh blood in water, but that the reaction failed at dilutions of 1:40,000. However that may be, Schumm (1909) reported 1:40,000 to 1: 100,000 dilutions of blood in water and 1:20,000 to 1:40,000 dilutions of blood in urine as the limits of sensitivity. Nicolesco (1934) gave 1:20,000 as the value. The most extravagant claim is that of Vitali (1903) who said that a 1: 10" dilution of dessicated blood in water still gave a positive result; this value is greater by six orders of magnitude than any other published figure. I Expressed as a dilution of whole blood in water; the range most often quoted for sensitivity is 1:20,000 to 1:100,000. The measurements are obviously affected by the reagents used. The guaiacum test is now no longer employed as a cata- lytic test for the presence of blood in the forensic practice, having been supplanted by benzidine, phenolphthalin, etc. Schwarz (1936) used guaiacum as a substrate for testing the peroxidase activity of bloodstains in a series of experiments designed to correlate the color intensity with the age of the stain. In more recent times, guaiacol, which is o- methoxyphenol, and component of guaicum resing, has been employed as a staining substrate for haptoglobin (as the hemoglobin complex) in gels following electrophoresis (Reich, 1956; Queen and Peacock, 1966). 6.2 The Aloln Test The aloin test for identification of blood in stains (and in urine and feces) is, like the guaiacum test, of primarily his- torical interest. In many ways, it is quite similar to van Deen's test. Aloin is a mixture of pentosides in the extracts of aloes, a genus of plants of the Family Lilaceae. Barbaloin, the major ingredient, is a hydroxyanthroquinone derivative of glucose. The structure is shown in Fig. 6.1 (Merck Index, 1968). Hemoglobin and some of its derivatives catalyze the oxidation of this material by H202to yield a bright red product. Klunge (1882 and 1883) first noted that this test could be employed as a test for blood. A number of studies concentrated on chemical reactions of aloin, and the analogy between these and comparable guaiacum reactions (Neuberger, 1899; Schaer, 1900). Rossel(l901) noted that the test could be used for the detection of blood in urine. Buckmaster (1907) found that the test was less sensitive than the guaiacum test, but that the oxidation product (i.e. the color) was quite a bit more stable. The test was never as widely used as van Deen's, and not as much was written about it. Sutherland (1907) regarded it as a good confirmatory negative test. 6.8 The Phenolphthalln Test In 1901, Kastle and Shedd showed that preparations of cellular "oxidases" would catalyze the oxidation of phenol- phthalin to phenolphthalein in slightly alkaline solutions. At the time, the cellular enzymes responsible for the catalysis had not been purified to any great extent, nor had there been any attempt to systematize the enzyme nomenclature. The crude enzymatic preparations were often referred to as "ox- idizing ferments". Phenolphthalein is, of course, pink to red in alkaline solution, while phenolphthalin is colorless. Thus, the latter was an excellent artificial substrate for assaying the "ferments" because the colored oxidation product was soluble and readily quantitatable colorimetrically. Meyer (1903) utilized phenolphthalin to detect the "oxidases" in leucocytes. In particular, he found differences in this activity between normal and leukemic samples. He noted further that this test could be used for the qualitative and quan- Blood Z d e n t ~ i o t i o n ~ a l y t i c titative determination of blood in urine. The first un- equivocal suggestion that the test be applied to medico-legal blood identification was made later in 1903 by Utz. He reported that the test served well on bloodstains up to 1% years old, gave a negative reaction with rust, but, not sur- prisingly, gave "false positive" reactions with pus and other leucocyte-containing secretions. The test became known as the "Kastle test", the "Meyer test" or the "Kastle-Meyer test." It was soon quite clear that the test relied on the per- oxidase activity of hemoglobin. Kastle and Amoss (1906) showed that the catalytic activity of blood toward the per- oxide oxidation of phenolphthalin in alkaline solution was directly proportional to the hemoglobin content. The re- agent, phenolphthalin, was prepared from phenolphthalein by reduction in the presence of Zn and strong NaOH or KOH. Kastle (1909) recommended the precipitation of the phenolphthalin by acidifying the reaction mixture, and col- lection of the precipitate. This material was recrystallized several times from minimal alcohol by cold water, and stored as a solid. Liquid solutions of the compound were stable for a matter of weeks if kept dark, and stability was greatly increased by the presence of a small quantity of zinc dust. One of the advantages of this test, in comparison to guaiac and aloin, was that the reagent was a pure compound. Kastle, who was very partial to this test for blood, discussed its many aspects in great detail in hi review in 1909. DelCarde and Benoit (1908a) studied the phenolphthalin test and showed that it was positive with hemoglobin, methe- moglobin, hematin chlorhydrate, reduced hemoglobin, and old, putrefied blood. They got a positive test on a control bloodstain 26 years old, and believed that the test, properly controlled, was both sensitive and specific (1908b), in addi- tion to its usefulness in detecting blood in urine, feces and gastric juice. Boas (1911) indicated that the test was useful for occult blood. Powi-Escot (1908), however, thought that no value should be attached to the test for blood because saliva, pus, malt extract, vegetable extracts and the salts of heavy metals such as Co, Mn, Pb and Fe could give false positive reactions. Dervieux (1910) agreed with this view, suggesting that the test had no value at all, positive or negative. The phenolphthalin to phenolphthalein oxidation reac- - tion, and the structures of the latter compound in both acidic and basic solution, are shown in Fig. 6.2. Because phenol- phthalein is colorless in acidic solution but pink to red in basic solution, it has been widely employed as a pH indi- cator. Many have noted that the phenolphthalin test is more sensitive than either the guaiac or aloin tests. Del6arde and Benoit (1908a) and Nicolesco (1934) have indicated positive reactions with blood diluted 1:106, as has Girdwood (1926). Gettler and Kaye (1943) reported a sensitivity of 1:107 dilu- tion of whole blood, but of 1: lo6 dilution for old, decomposed blood. Glaister (1926a) noted that saline extracts of 1 year old stains reacted at 1 :212,000 dilutions but that a 1:800,000 dilution of a water extract of the stain gave a positive result. Kastle (1909) did sensitivity experiments by dissolving Figure 6.1 Barbaloin (1.8 dihydroxy - 3 - hydroxymethyl - 10 - (6 - hydroxymethyl - 3Ad - trihydroxy - 2 - pyranyl) - anthrone) 3.8 mg of blood in 100 d water as a first dilution and making three serial Ho dilutions in addition. These, he de- noted solutions (1). (2). (3) and (4), and they contained 38pg, 3.8 pg, 0.38 pg and 0.038 pg of blood per mQ,re- spectively. One mL of each solution was tested by the addi- tion of 2 d reagent. Solution (3) which contained 0.38 pg/mP could be readily distinguished from the control colonmetrically. Solution (4). the weakest, could not be, but Kastle noted that two independent observers (he, pre- sumably, being one of them) were able to distinguish the difference between solution (4) and the control by eye. In terms of dilutions of whole blood, therefore, since the assays were canied out in a total volume of 3 mP ,solutions (3) and (4) correspond to about 1:s x 106and 1:80 x 106parts blood to parts water. Kirk (1953) noted that 1:lV dilutions of blood gave a positive reaction within 3 sec while 1:s x 106 dilutions required 20 sec to do so. Glaisler (1926a) tested a variety of substances and body fluids, including rust, urine, saliva, semen, perspiration and milk, and got negative results with the phenolphthalin test. Kerr (1926b) took exception to GlaisteICs confidence in the method, noting that feces from patients taking aspirin gave a false positive test. Glaister (1926b) replied that, while he did not question the need for corroboration of the presence of blood, his own experience with the test had convinced him of its value in medico-legal cases. Girdwood (1926) noted that he did not think the test should be relied upon by itself as an indication of blood in stains, nor of occult blood in stool samples. Gettler and Kaye (1943) thought the test was more specific than guaiacum, benzidine or o-tolidine, and Gradwohl (1956) said that he preferred this test to ben- zidine. More recently, Higaki and Philp (1976) reevaluated . 7 J.alkali the test for blood in terms of sensitivity, reagent stability and s-city. The reagent was prepared essentially as recorn- mended by Camps (1968), which follows almost exactly the original method of preparation used by Kastle (1909) except that the product is not isolated and recrystallized. Phenol- phthalein (2 g) is dissolved in 100 mQ water containing 20 g KOH and boiled with a reflux condenser in the presence of 20 g zinc powder until colorless. The resulting solution is kept in a brown bottle with some Zn dust present. The test was carried out in a number of different ways, with and without ethanol or methanol, and with peroxide or per- borate. A so called one-stage test amounted to the addition of the combined reagents to the sample; a two-stage test consisted of the successive addition of reagent and either peroxide or perborate; a three-stage test involved the addi- tion of the alcohol, the reagent, and the peroxide or per- borate successively. Benzidine was employed as a control, because the experiments were designed to evaluate phenol- phthalii as a substitute for benzidine in routine practice. It may be noted here that Camps (1976) recommended that the phenolphthalin test be substituted for the benzidine test. Dilutions of whole blood as well as stains prepared from them were tested. Stains were tested by applying reagents to a stained thread on filter paper. All results were recorded after 5 sec of observation. With liquid dilutions, ethanol or methanol, reagent and perborate, used in a one-stage test, proved to bt most sensitive (in ex- of 1:106). A one-stage phenolphthalin-perborate test was sensitive to about 1:105-1:106 dilutions, there being no advantage to a two- stage test with these reagents, nor to a three-stage test in- volving either alcohol. Reagent-peroxide combination, with or without alcohol, regardless of the number of stages, gave A L O HO. A TTF- 1'0 I Figure 6.2 Phenolphthalin Oxidation and Phenolphthalein 1954; Hunt et al.. 1960; Kerr, 1954; Lucas, 1935 & 1945; Mikami et al., 1966; Prokop, 1966; Simpson, 1965; Thomas, 1937). In 1964, the benzidine test was critically examined by Culliiford and Nickolls. They noted that by 1931, the test had fallen into disfavor, at least in some quarters, as a cer- tain test for blood, Glaister having written in that year in the 5th edition of Medical Jurisprudence and Toxicology: While some employ this test, it has the disadvantage that, like the Guaiacum test, it can only be of value as a negative test, in that if no colour reaction occurs- blue or green-on applying it to a stain, it indicates the absence of blood. Should the colour reaction take place, it only suggests the presence of blood, since gluten, many plant juices as horseradish, and hypochlorites will give the blue colour reaction, although these may give the reaction either before or without the addition of ozonised ether. We do not put our trust in this test. We have abandoned completely the Guaiacum and Benzidine tests for the reason chiefly that the reaction obtained in the presence of minute amounts of known blood is uncertain and doubtful, and also because a reaction may be produced by it by substances other than blood. These objections do not apply to the Kastle- Meyer test. On the other hand, Gradwohl (1954) wrote in his book, Legal Medicine, that positive reactions with benzidine (or phenolphthalin), assuming properly negative controls, do in- dicate "the presence of blood." This statement suggests that many laboratories regarded the test as considerably more valuable than did Dr. Glaister. CuUiford and Nickoh point out, as did Grodsky et al., (1951), that the judgment made about the test must be evaluated in the context of a number of variable parameters, such as the precise way in which the test was done (i.e., one-stage, two-stage, what controls were used, testing stains directly, testing extracts, etc.), the con- centration and purity of the reagents used, whether peroxide or perborate was used, and the experience and judgment of the person carrying out the procedure. The major sources of "false positives" were categorized by these workers as: (1) blood contamination; (2) chemical oxidants or catalysts; and (3) vegetable or fruit peroxidases. Contamination should not be a problem in practice if the reagents are pure, the glassware clean, and the examination area kept scru- pulously uncontaminated. The test is exceedingly sensitive, and contamination does not have to be very great to be a serious problem. Chemical oxidant interference is readily dispensed with by adding the reagent and the peroxide in successive steps; if color develops upon the addition of re- agent alone, the presence of a chemical oxidant is indicated. Chemical catalysts, which work only in the presence of the peroxide, are not eliminated by the two-stage procedure, but Culliford and Nickolls argued that reactions caused by these materials appear quite different to the experienced eye than do those caused by blood. The plant peroxidases are heat- labile, and testing samples that have been heated to 100' for a few minutes serves to differentiate them from blood, which still reacts readily after the heating step. It was further Blood Ident~$lcotwn-Catalytic shown that the majority of plant peroxidases which would give the reaction were quite labile in the dried state. These gave weak to negative benzidine reactions after three days in the dried state, whether the test was done directly on cloth, on a rubbing, or on an extract. Finally, these investigators described a simple electrophoretic procedure, carried out on 1% agar gels, for the differentiation of a great number of substances which could give misleading results in the benzi- dine test. The procedure is also described by Culliiford (1971). It should be noted that these authors took strong exception to the statements of Hunt et al. (1960) above, to the effect that the reporting of a positive presumptive test in a case where there was no additional material available for testing would be scientifically and morally incorrect, because it could be misleading. Failure to report such a result, Culliford and Nickolls argued, would be to usurp the prerog- atives of the Court, and in such a case as the one discussed by Hunt et al., the result should be reported with a suitable explanation of its meaning. Reports of the sensitivity of the benzidine test vary in the literature. Adler and Adler (1904) originally reported a sen- sitivity limit of 1:105 dilution of whole blood in water. Nicolesco (1934), Dtrobert and Hausser (1938) and Thomas (1937) all cite 1:200,000 dilutions as the limit. Grodsky et al. (1951), Hunt et al. (1960) and others have noted that the sensitivity quotations can be misleading, because the results depend on so many different parameters. Unless the technique and the reagents used are fully described, it is not at all certain that the results will be able to be duplicated exactly. Indeed, while it is easy enough to compare the sensitivities of reagents in terms of the maximal dilutions of whole blood which still give positive tests, it is not always clear how such values translate in their applica- bility to bloodstains. Hunt et al. (1960) noted great vari- ability in different lots of commercially obtained benzidine. Expressed as the highest dilution of blood, dried onto filter paper, which gave the test, the sensitivity ranged from 1:20,000 to over 1:150,000, and was even more variable if the amount of time required for the color to develop were taken into account. Grodsky et al. (1951) noted that stains made from 1:300,000 dilutions of blood came up within 10 sec while those made from 1: 100,000 dilutions came up within 1 sec. Akaishi (1965) reported a sensivity of only 1:12,800 for benzidine in stains made at that dilution, and noted that 20 sec was allowed for color development. It may be noted here that Alavi and Tripathi (1969) rec- ommended that blood testing in the field (e.g. at scenes etc.) could be done with benzidine-impregnated filter papers. The suspected material was moistened with water, the benzidine paper then pressed against it, and peroxide added to the paper from a sealed vial. The papers were said to be stable for up to 1 year and could be regenerated by soaking again in benzidine solution and drying. The foregoing discussion of the benzidine test, without which the sourcebook would obviously be incomplete, may nevertheless be almost purely academic from a practical point of view. That benzidine was a chemical carcinogen has Sourcebook in Fo& Serology, Immunology, and Biochemistry apparently been known for some time. Hunt er al. (1960) mention that the manufacture of Analar Benzidine was dis- continued in 1951 for that reason. Camps (1976) notes that phenolphthalin has replaced benzidine in routine practice. Rentoul and Smith (1973), in the 13th edition of Glaister's Medical Jurisprudence and Toxicology, suggest a saturated solution of amidopyrine in 95% ethanol as a benzidine re- placement. (See Section 6.6.8). Apparently, therefore, the use of benzidine has been largely discontinued in England. Higaki and Philp (1976) carried out their study of the phe- nolphthalin test (see Section 6.3) primarily to check its a p plicability as a substitute for the benzidine test, suggesting that benzidine has been abandoned in Canada as well. In this country, the use and manufacture of benzidine has become subject to extremely stringent restrictions and con- trols, according to regulations issued by the Occupational Safety and Health Administration of the Department of Labor (Code of Federal Regulations, 1976). While manu- facture and use have not been ordered to cease, the regu- lations and restrictions are prohibitively involved for a laboratory doing routine work. Regardless of the merits of the test, or the qualifications that should or should not be place on interpretation of the results obtained with it in medico-legal cases, it is probable that the substance will shortly be unavailable. Supervisors will probably be in- creasingly unwilling to place their examiners at risk by con- tinued use of the reagent, and the OSHA Regulations may also be sufficiently constraining that manufacturers will con- sider them prohibitive as well. It is likely, therefore, that the benzidine literature will shortly become a part of the archives of this field. 6.5 Leucomalachlte Green and Leucocrystal Vlolet Tests The use of the leuco base of malachite green as a blood testing reagent was first reported by Adler and Adler (1904), as noted above. The term "leuco compound", or in this case, "leuco base", comes from the literature of biological stains and dyes (Lillie, 1969). Compounds to be employed as stains or dyes obviously have to be colored. Although they differ greatly from one another chemically, all contain a chromo- phore group, a structure which renders them colored. They all share in common additionally the property of being reducible and reduction alters the chromophore group ren- dering the compound colorless. These colorless reduction products are referred to as "leuco compounds". Clearly, the leuco compounds are oxidizable to the dye forms. The "leuco bases" are particular types of leuco compounds, usually car- binols, and characteristic of the triphenylmethyl derivatives. In the original work, the Adlers used leucomalachite green and leucocrystal violet, in addition to benzidine, for blood detection in aqueous solution. The structures of crystal violet (hexamethylpararosanilin) and malachite green are shown in Figs. 6.5 and 6.6, respectively. Only the leuco base of the latter has been widely used in forensic practice. The leucomalachite green test had a sensitivity limit, like Figure 6.5 Crystal Violet benzidine, of 1:105 dilution of blood. Michel(191 la) recom- mended this test and said that it was more sensitive then phenolphthalin. Von Fnrth (191 1) utilized the test on blood- stain extracts prepared by digesting the bloodstained mate- rial with 50% KOH in ethanol, and extracting that solution with pyridine. The test was then performed on a piece of filter paper, moistened with the pyridine extract. Medinger (1933) strongly recommended the reagent, and tested vari- ous physiological fluids, plant extracts and inorganic com- pounds, all with negative results, provided the peroxide was added in a second step after no color had developed in the presence of reagent alone. White (1977) showed that leucoc~stal violet was as sensi- tive in detecting iron (111) mesoporphyrin IX as a spot on filter paper as tetramethylbenzidine, guaiacum and aminodiphenylamine. Alvarez de Toledo y Valero (1 935) review the test rather extensively, and tested a large number of organic and inor- ganic compounds for false positive reactions. He found that there are many chemical oxidants that will give the reaction in the absence of peroxide, as well as some that catalyze the Figure 6.6 Malachite Green reaction in its presence in much the same manner as does blood. The test is, therefore, not more specific for blood than most of the other catalytic tests. The sensitivity of the test was originally reported to be 1:1@ dilution of blood by Adler and Adler (1904). Alvarez de Toledo y Valero (1935) reported the same value, but Nicolesco (1934) quoted the sensitivity as a 1:20,000 dilu- tion of blood. Alvarez de Toledo y Valero noted that more dilute solutions of blood required longer times for color de- velopment, 1 :1000 dilutions being instantaneous, 1 :2,000 di- lutions requiring 15-20 sec, and so on until 55 sec was necessary at 1:105 dilutions and 25 min was needed for 1:2 X 10S dilutions. Grodsky et al. (195 1) reported that stains made from 1: 10' dilutions of blood came up in 15 sec, using a reagent prepared by dissolving 0.1 g leucomalachite green and 0.32 g sodium perborate in 10 mP of 2:l (v/v) glacial acetic acid in water. The benzidine test on the same sample, by contrast, came up in 1 sec. Hunt et al. (1960) similarly reported leucomalachite green to be less sensitive than ben- zidine, and apparently more specific. But the apparent in- crease in specificity was attributed to the lower sensitivity, and was thus not considered an advantage. 6.6 Other Cablytlo Tests Over the years a number of catalytic tests have been pro- posed for the detection of blood in stains, or of blood in feces or urine, which have enjoyed only limited use, or about which there is not a great deal of literature. All these tests are briefly discussed together in this section. 6.6.1 Peroxide It should be mentioned that peroxide was sometimes used as a reagent for blood detection, especially in the older lite- rature. That blood possessed a peroxidase activity, and act- ing as a catalase was thus capable of evolving oxygen from peroxide, has been known since the experiments of Schanbein (1863). All the catalytic tests are based on this principle, as discussed in the preceding sections; but all have relied upon the coupling of the peroxidase activity to the oxidation of a compound which formed a colored product. Zahn (1871) specifically noted that peroxide could be used to detect blood in stains, though it is not clear that he was aware of Schttnbein's work. If peroxide is brought into contact with a bloodstain the peroxidase reaction takes place after a minute or so, and is evidenced by the formation of large numbers of tiny bubbles. Gantter (1895) suggested that the test had substantial value if negative, i.e., was a good indication of the absence of blood. Sutherland (1907) re- ferred to the test as the Zahn-Gantter Test, and noted that many of the substances now known to give false positive catalytic tests, such as vegetable extracts, gave this test as well. Cotton (1904) studied the evolution of oxygen in the presence of H202 from the blood of a number of different species. Palleske (1905b) also conducted studies on the test with different bloods. A positive test could be obtained with a drop of blood in 1500 mQ of water, which, if we assumed that 20 of his drops would equal 1 d,would represent a Blood Identi$iition-CbtaIflic sensitivity of a 1:30,000 dilution. Sutherland (1907) thought the test was useful when negative, except where the stain had been heated above 120". Leers (1910) presented the test as a presumptive one, noting that other substances than blood gave positive results. He called the test "Die Vorprobe mit Wasserstoffsuperoxyd~' or, simply, "the preliminary test with hydrogen peroxide." 6.6.2 Eosin In 1910, Ganassini proposed the use of an eosin reagent, prepared from crystalline eosin by heating in strong base, and collection and washing of the acid precipitate. This re- agent in alcoholic solution in the presence of strong alkali and H202 gave a momentary yellow to red colored product. He believed the reagent to be specific for blood. Belussi (191 1) disagreed, noting that other substances gave positive tests, and that the sensitivity of Ganassini's test was far lower than that of benzidine. 6.6.3 2,7-Diaminotluorene There is a brief report in the literature by Schmidt and Eitel (1932) on blood identification using 2,7-diamino- fluorene (also called 2,7-fluorenediarnine). This report has mainly to do with a problem concerning the stability of the reagent, but the implication that the reagent was in use for the detection of blood is quite clear. The structure of 2,7-diamonofluorene is indicated in Fig. 6.7. The 7th edition of the Merck Index indicates that the reagent is used to determine halides, nitrate, persulfate and several metals. It seems likely, therefore, that these might be expected to give false positive reactions in the test fir blood ;sing this-re- agent. Figure 6.7 2,7 - Diaminofluorene 6.6.4 Rhodamine B In 1917, Fuld recommended the use of a Rhodamine B reagent for blood detection. The reagent was prepared from Rhodamine B (Fig. 6.8) by reduction in base in the presence of zinc. This reagent detected blood at a dilution of 1 :lo7, according to Fuld. Alke (1922) studied the reaction in some detail, and reported that it is not given by semen, saliva, urine or a number of other biological substances. The usual inorganic oxidants will oxidize the reagents in the absence of H202. The reaction was negative with rust, and Alke re- ported a sensitivity limit of 1:105 to 1:106 dilution of blood. Ziemke (1938) notes that an investigator named Diels showed that chlorophyll gives the test. The reaction is appar- ently still positive with blood which is putrefied, or which has been heated above 200'. Sourcebook in Fore- Serology, Immunology, and Biochemistry Figure 6.12 Amidopyrine H202 detect Fe(II1) mesoporphyrin IX as a microspot on filter paper. 6.6.12 Diphenylamine In 1970, Woodman proposed the use of diphenylamine as a catalytic test reagent for blood in feces. The test was sensitive to about 1:4000 dilutions of blood in water and the oxidized chromophore is a green color. The reagent is also suitable for detecting hematin compounds on paper or cellu- lose acetate membranes following electrophoresis, and was recommended because it is not carcinogenic. 6.6.13 Fluorescin Fluorescin is the reduced form of fluorescein, the latter being 9-(o-carboxyphenyl)-6-hydroxy-3H-xanthen-3-one. Fluorescein is soluble in alkali hydroxides and carbonates at room temperature, and exhibits an intense green fluorescence. In 191 0, Fleig proposed using fluorescin to detect blood in the urine of patients. He prepared the reagent by reducing fluorescein in the presence of KOH, zinc dust and heat. He said that it was a more sensitive reagent than phenolphthalin, and suggested that it could be used to detect blood in dried stains as well. We have conducted studies on the use of fluorescin as a presumptive test reagent, and have found that it is entirely satisfactory (Lee et al. 1979). We were unaware of Fleig's paper until quite recently. Fluorescin can be prepared from fluorescein in the same way that phenolphthalin is prepared from phenolphthalein. We find that the reagent is stable for months if kept over some zinc dust at 4'. We dilute it to about 1:60 with water for use, and the dilute solution is not as stable as the stock one. Water is preferable to ethanol as a diluent. A positive test can be obtained on blood dilutions up to l:107. It works on bloodstains on many different substrata. 6.7 Lumlnol Test The luminol test is based on the fact that a number of hemoglobin derivatives greatly enhance the chemilumi- Figure 6.13 3, 3', 5.5' - Tetramethylbenzidine nescence exhibited by luminol upon its oxidation in alkaline solution. According to Proescher and Moody (1939), the compound was first synthesized by A. Schmitz in Heidelberg in 1902, under the direction of Prof. T. Curtius. Curtius and Semper (1913) then synthesized it in a different way, and referred to Schmitz's earlier work. But none of these workers noticed the chemiluminescence properties of the molecule. The intense blue chemiluminescence of the compound dur- ing its oxidation in alkaline solution was first observed by W. Lommel in Leverkusen (Germany) who brought it to the attention of H. Kautsky. Kautsky apparently interested H. 0.Albrecht in looking into the properties of the phenom- enon, the results of the investigation having appeared in 1928. Albrecht found that a number of oxidizing agents, which could be used in alkaline solution, brought about the luminescence, that H202 alone brought about only a feeble luminescence, and that luminescence was visible in a dark- ened room even at luminol concentrations of lo-' M. Fer- ricyanide or hypochlorite greatly enhanced the luminescence obtained with H202, as did plant peroxidases and blood. Albrecht suggested a mechanism for the reaction as well, and this matter is discussed in more detail below. Luminol is 3-aminophthalhydrazide (Fig. 6.14). In 1934, Huntress el al. reported a method for the synthesis of the compound from 3-nitrophthalic acid and hydrazine sulfate, and named it "luminol". A year later, Harris and Parker, in the same laboratory, published studies on the quantum yield of the chemiluminescence. Gleu and Pfannstiel (1936) ob- served that crystalline hemin produced an especially intense luminescence, a fact soon confirmed by Tamamashi (1937). Specht (1937a, 1937b) did an extensive series of studies intended to design a useful medico-legal blood identification test based on luminol chemiluminescence. Old as well as recent bloodstains were examined, and able to be detected reliably using luminol reagent. Specht made two solutions, and noted that either one worked well: ( I ) 0.1 g luminol, 5 g Cam,, and 15 mQ 30% H202 in 100 mL H20, (2) 0.1 g luminol in 100 mS! 0.5% aqueous sodium peroxide. He tested a variety of substances for their ability to enhance peroxide- luminol luminescence in the same manner as did bloodstains. These included milk, coffee stains, semen, saliva, urine, feces, dyes, moldy bread, leather, fabrics, oils, varnish, wax, shoe polish, wood, grass, leaves and a number of metals, and all gave negative results. The reagent could be used in solu- tion or sprayed onto suspected surfaces of all types using an atomizer. The spraying of luminol reagent onto bloodstain Figure 6.14 Luminol (3-aminophthalhydrazide) material was said not to interfere with subsequent crystal or spectral tests, nor with serological tests for species or blood groups. The luminescence lasts for quite a while, at least a matter of minutes, and under proper conditions can be photographed to provide a record of the location of stains. Specht recommended the test strongly for medico-legal ex- aminations, and believed that it was quite specific for blood. Proescher and Moody (1939) looked into the test fairly extensively, using commercial luminol at a concentration of 0.1 g in 100 mi? 5% Na2C03. This reagent solution was indefinitely stable. The test was performed by adding 15-20 mi? 3% H202 or 1 g sodium peroxide to 100 mQ luminol solution just prior to making the test. For the detection of fresh blood in solution, the hemoglobin was converted to hematin by the addition of concentrated HC1 to the sample, which was then boiled briefly to destroy vegetable per- oxidasts. The test solution was then made alkaline again with sodium carbonate, and luminol reagent added. For bloodstains, the surfaces were 6rst sprayed with 1-2% HC1, sprayed again after 10-15 min with sodium carbonate solu- tion, and finally with luminol-peroxide reagent. Proescher and Moody regarded the test as extremely useful, but pre- sumptive. The test could be given by hypochlorites, fer- ricyanide, and several other inorganic substances. Many of the substances with which Specht (1937a, 1937b) had ob- tained negative results were tested, and the negative results c o n h e d . Bloodstains were found to give more intense and longer lasting luminescence than fresh blood, and could be made luminescent many times by allowing the sprayed re-. agents to dry, and then re-spraying. McGrath ( 1942) recommended the luminol test for use in forensic blood detection. He noted, as had others, that older stains gave more intense and longer-lived luminescence than fresher ones, because more met-Hb and hematin has formed in the older stains. He believed the test to be quite specific for blood, having obtained negative reactions with serum, bile, pus, semen, pleural fluid, earth, feces, fresh and spoiled veg- etable material, various paints, metals, wood and shoe pol- ish. He nevertheless cautioned that the test should not be used as a final, specific test for blood by itself. The main disadvantage of the test, in McGrath's mind, was that it had to be camed out in the dark. Kraul et al. (1941) noted that they regarded the test as a good presumptive one, but that it was not specific for blood. They also determined the wave- length of maximum chemiluminescence to be at 441 nm, with a shift to longer wavelength, 452 nm, in the presence of Blood Zdent~$iation-Gzta&tic old blood. Schneider (1941) reported that a number of iron chlorophyll derivatives give luminescence with luminol- peroxide in sodium carbonate solution. Grodsky et al. (1951) described a number of studies on the luminol test. They recommended a reagent consisting of 0.07 g sodium perborate in 10 mQ water, to which is then added 0.01 g luminol and 0.5 g Na2C03. The order of addi- tion was important because the perborate is more soluble in water than in sodium carbonate, while the opposite is true of luminol. Reagents for the preparation of this reagent were incorporated into a field test kit recommended by the au- thors. The test was considered to be quite specific for blood, no false positives having been observed with a variety of mat* that affected other cataIytic tests. A noteworthy exception was copper salts. Grodsky et af. found that most brass, bronze and similar alloys which contain copper gave the reaction, a very important consideration if one is dealing with locks, door handles or other fixtures constructed of these materials. Indeed, Steigmann ( 1941) recommended the use of luminol for the detection of copper, as well as iron and cobalt, ions. He noted in 1942 that the reagent could be used for peroxide determinations as well. Zweidinger et al. (1973) evaluated a number of types of film for the photography of bloodstains sprayed with luminol reagent. Various reagents, made with peroxide or perborate, were also tested. In aqueous solutions, the peroxide yielded a more intense, but shorter-lived chemiluminescence than the perborate. Similar results were obtained if the reagents were made up in 95% ethanol, it beiig necessary in this latter case to basicify the solution with 0.02 M KOH since sodium carbonate is quite insoluble in ethanol. A number of dif- ferent photographic films were investigated, along with the effects of varying f/stop, exposure time and development conditions. It was possible to obtain good photographic records of bloodstains on items of evidence, and the pro- cedure was recommended for adoption in routine practice. One of the claims that has been made for the luminol test, particularly by those recommending sprayed reagents, is that its presence does not interfere with subsequent confirm- atory blood tests or serological tests (Specht, 1937a, 1937b; Proescher and Moody, 1939; McGrath, 1942; Grodsky et al., 1951 ). Srch (197 1 ) reported, however, that the presence of luminol reagent on a sample may interfere with the Taka- yama test, the determination of ABH agglutinins by the method of Lattes, and the absorption-inhibition test for ABH agglutinogens. It did not interfere with the benzidine test, nor with species determination by the precipitin test. Schwerd and Birkenberger (1977) c o n h e d Srch's finding that luminol-peroxide spray can interfere with ABO group ing by inhibition technique, especially in small stains. Mixed agglutination could still be used, but did not work as well as on unsprayed controls. The precipitin test was not affected by the spray reagent. An advantage of the luminol test is its great sensitivity. Albrecht (1928) stated that chemiluminescence was obtain- able at luminol concentrations of 10.' M, and Wegler (1937) reported luminescence at 10" g/mQ luminol, or about Sowwbook in FO- Sr~bgv,Zmrnunologv, and Biochem&y 5.6 X 10" M (the MW of luminol is 177.16). The more usual way of expressing sensitivity, of course, is in terms of maximal dilutions of blood which still give positive reactions at some constant reagent concentration. Most authors have used 0.1% (w/v) luminol solutions, corresponding to about 0.056 M. Proescher and Moody (1939) said that the test was sensitive to 1:lV dilutions of hematin. Grodsky et ai. ((1951) obtained luminescence lasting at least 15 minutes within 5 sec of spraying the reagent on stains made from 1:5 X lo6 dilutions of blood. Weber (1966) quoted a sensitivity of 1: 10' dilution of blood using a photomultiplier tube detection system. Bujan (1948) attempted to take advantage of the fact that luminol gives its intense luminescence with hematin, which is formed upon bloodstain aging. The luminescence in- tensity, measured photoelectrically, could be correlated with the age of the bloodstain, or with the amount of blood that was present in the stain. The luminol reaction is somewhat more complex than those involved in the phenolphthalin, benzidine, leuco- malachite green, and other catalytic tests. M i l e it is proba- bly not wrong to refer to the luminol test as a "catalytic test", it is not mechanistically a catalytic test in quite the same way as are the others. In dilute acid solution, luminol is relatively insoluble, and has the structure shown in Fig. 6.1 5(a). This compound gives a strong blue fluorescence with UV light. The tautomeric forms shown in Fig. 6.15(b) exist in alkaline solution, and it is these which produce chem- iluminescence upon oxidation. Albrecht (1928) proposed a mechanism for chemiluminescence (Fig. 6.16), in which the phthalazine (I) was oxidized to form a diimide compound (11). This material would be hydrolyzed in the basic solution to yield the phthalic acid compound (111) and N2H2, which reacts with an additional mole of I1 to form nitrogen, IV and light. Compound IV, it willbe noticed, is the form of luminol which exists in acid solutions (Fig. 6.15(a)), and thus pre- sumably a partial regeneration of starting material. Tama- mushi and Akiyama (1938) studied the reaction and their results were consistent with this mechanism, but Stross and Branch (1938) obtained results using fast-flow methods which could not be explained by it. Other studies were done by Sveshnikov (1938) whose results suggested a prior hydro- lysis, with luminescence being due to the oxidation of a hydrolysis product. Kubal(1938) and Plotnikov and Kubal (1938) investigated the spectral changes associated with the reactions. In the presence of rhodamine or fluorescein, some of the chemiluminescent energy is apparently absorbed by the dyes, which then fluoresce at wavelengths longer than that of the luminescence. This phenomenon, they called chemauorescence. Baur (1940) said that the decay of lumi- nescence of luminol in the presence of hemin and peroxide followed a bimolecular rate law. Weber and co-workers car- ried out extensive studies on the luminescence reaction, and state, among other things, that substances which greatly increased the peroxide oxidation-dependent luminescence, such as chlorhernin, met-Hb and ferritin, do not, strictly Figure 6.15 Luminol Structures in Solution speaking, act as catalysts under all conditions (Weber, 1942; Weber et at., 1942; Weber and KrajGaovic, 1942). Appar- ently, these compounds are best thought of as "acceler- ators", which may act catalytically. Weber et 01. (1942) suggested that the products of the initial oxidation reaction included 0 2 and reduced accelerator. If the reduced acceler- ator could be reoxidized by 0 2 , the compound would be acting catalytically, while if the reoxidation were not possi- ble, the accelerator would have acted as a reactant. The subject is complex, and it may be that the mechanism is not the same with every "accelerator" or catalyst. Shevlin and Neufeld (1970) studied the mechanism of the femcyanidc- catalyzed luminescence of luminol, and proposed the scheme shown in Fig. 6.17 to explain their data. It should be noted that the femcyanide acts catalytically in this scheme. The exact role of the catalysts or acoeIerators is not clear from Albrecht's scheme (Fig. 6.16). White and Roswell (1970) reviewed the chemiluminescence phenomena characteristic of organic hydrazides generally, including luminol. Isaccson and Wettermark (1974) noted that the mechanism of lumi- no1 oxidation in aqueous solution has still not been satis- factorily elucidated in spite of many studies. It may be mentioned, finally, that Weber (1966) proposed an improved reagent for blood testing. Stock solutions were: (A) 8 g NaOH in 500 mQH20, or 0.4N;(B)10 d 30% H202 in 490 mQ H20, or 0.176M; (C)0.354 g luminol in 62.5 mQ 0.4N NaOH to a fmal volume of 500 d with water, or 0.004M luminol. To make up testing reagent, 10 d of each of these solutions is mixed with 70 mQH20. It will be noted that the Na2C03 used by others is here replaced by NaOH, and that the luminol and peroxide concentrations are very much lower. Thisreagent works well with both fresh and dried blood, whereas the older reagents did not readily react with fresh blood because there was too little met-Hb or hematin present. The fact that this reagent serves with fresh stains is explained by the rapid conversion of Hb to met-Hb and/or hematin in the strong base. The reagent is more sensitive than the older ones because of the lowered H202 and luminol concentrations. Higber concentrations of these compounds tend to be inhibitory. 6.8 Catalytic Tests-4eneral Conddemtlonr There can be no doubt that most authorities have consid- ered the catalytic tests as presumptive when used alone. Their value is ascribed to the ease and rapidity with which Blood I&naYu:ation-Immnologiccrl und Other SECTION 7. OTHER TESTS 7.1 lmmunologlcslTests Wlth Antl-Human Hamoglobln The first antiserum to a hemoglobin was reported by Leblanc (1901). He prepared anti-cow Hb sera in rabbits, with which a precipitin reaction could be obtained. Ide (1902) confirmed these results, although his antisera were hemolytic as well. His student, Demees (1907) was able to prepare a non-hemolytic anti-Hb precipitin antibody by us- ing a more thoroughly purified Hb as immunizing antigen. Not all investigators obtained identical results in the early investigations, however. Gay and Robertson (1 9 13) concluded that globin alone was not antigenic, but when combined with casein into what they referred to as "globii-caseinate", an antibody could be raised which fixed complement (see sec- tion 1.3.5.3) with the antigen as well as with casein alone. Browning and Wilson prepared antibodies to guinea pig glo- bin in rabbits in 1909. In 1920, they enlarged these studies somewhat, noting that ox globin was antigenic as well. The anti-guinea pig serum was quite species specific, but the anti-ox globin was not. Ford and Halsey (1904) were unable to raise precipitin antisera to purified, crystalline dog or hen hemoglobin in either rabbits or guinea pigs. In 191 9, Schmidt and Bennett got similar results. Unable to obtain any precipitin antisera against pure, crystalline dog hemo- globin, they concluded that hemoglobin was not antigenic. Klein (1904,1905a) conducted extensive experiments on the immunization of rabbits with serum, whole red blood cells, and red cell extracts. Antibodies were obtained to the red cell extracts, which he called "erythropr'bipitine"; these antibodies he believed to be different from the anti-serum antibodies (obtained by immunizing with serum). Klein was the first to suggest (1905b) that the anti-red cell extract serum should be employed in medico-legal investigations. %Hebelieved the'antisera to be species-specific, and that their use would combine into one procedure the determination of the presence of blood and its species of origin. Leers (19 10) agreed with this viewpoint. He prepared specific "erythro- precipitin" antisera of his own, and he discussed the use of these reagents in carrying out medico-legal tests for blood. The issue of hemoglobin's antigenicity began to be settled in 1922, when Hektoen and Schulhof prepared precipitating antibodies against extracts of cow, dog, goat, guinea pig, horse, human, rat, sheep and pig red cells. Some of these were species-specific, while others showed cross reactions. The following year, they obtained many of the same antisera in species-speci6c form, and concluded that the red cell pre- cipitinogen giving rise to these antibodies was in fact hemo- globin. They suggested that the antisera would be useful in solving medico-legal cases. Higashi (1923) independently arrived at the same conclusions, suggesting that the antisera be called "hamoglobinopr~zipitin", and recommending that they be used in forensic practice. Heidelberger and Landsteiner confirmed these findings in 1923. They knew of Higashi's work, but had apparently not yet seen Hektoen and Schulhofs 1923 paper. Sera produced with crystalline hemoglobin, they said, reacted species-specifically with ho- mologous antigen. The antisera worked equally well with homologous met-Hb, HbCN and HbCO. Fujiwara (1928) carried out studies on anti-hemoglobin precipitin sera in parallel with anti-serum precipitin sera. He obtained species-specific anti-Hb sera, which had titers as high as 1:40,000 against whole blood or hemoglobin. Some years later, there was recurrent interest in antisera to hemoglobin, especially with the objective of developing immunological methods for differentiating the various human hemoglobin variants, which were coming to be known. These studies were also done as an immunological approach to discovering the differences in structure among the human hemoglobin variants. The subject of hemoglobin variants itself is involved, and will not be discussed here (see section 38). Darrow et al. (1940) prepared antibodies to cord blood and to adult blood. The former, if absorbed with adult cells, could be rendered specific for cord blood hemoglobin (Hb-F; fetal hemoglobin). Antibodies to adult hemoglobin (Hb-A) reacted with both adult and fetal blood, and absorption of the antisera with either adult or fetal cells brought down all the antibodies. Aksoy (1955) prepared rabbit antibodies against cord blood, but found that in most of the prepara- tions, absorption with adult cells precipitated all the anti- body. In only one case was the serum rendered specific for Hb-F by absorption with adult cells. In 1953, Chernoff published a pair of papers, the first of which (1953a) reported the preparation of anti-Hb-A, anti- Hb-F, anti-sickle cell hemoglobin (anti-Hb-s), and anti- guinea pig Hb, using Freund's adjuvant for the first time. The anti-Hb-F was specific for cord blood, but the antisera to Hb-A and Hb-S cross reacted. The second paper (1953b) reported the use of the specific anti-Hb-F to measure the amount of fetal hemoglobin in adult blood. Kohn and Payne (1972) described a radial irnmunodiffusion procedure for the determination of Hb-F, using a commercial antiserum. Goodman and Campbell (1953) showed that rabbit antisera to Hb-A and Hb-S did not differentiate them well, but that antisera prepared in chickens could be used to do so. Major Sourcebook inForen.uk Serology, Zmmunologv, andBiochemistry differences in reactivity between anti-Hb-A and anti-Hb-F were also confirmed. Ikin et al. (1953) prepared agglutinins to cord blood cells, which showed good titers only if used in the presence of 10% albumin and at 37". Diacono and Castay (1955a) obtained antibodies from guinea pigs which could be used to distinguish between adult and fetal red cells, and they showed further (1955b) that the anti-Hb activity was not identical to the hemolysin activity. Boivin and his collaborators studied the reactions of anti-Hb-A and anti- Hb-F with a number of variant hemoglobins using Ouchter- lony gel diffusion and immunoelectrophoretic techniques. They showed that hemoglobins A, F, S, C and E showed a common antigenic determinant (Boivin and Hartmam, 1958b; Boivin et al.. 1959).They also showed that anti-Hb- A reacted with hemoglobin-A in the hemoglobin-haptoglobin complex in gels (Boivin and Hartmann, 1958a). In 1961, Muller et al. obtained a rabbit immune anti-human Hb serum, which did not discriminate between Hb-A and Hb-F. But it did not react with human serum, nor with animal hemoglobins, and these workers suggested that it could be very useful in medico-legal work for confirmation of species of origin of bloodstains. Likewise, Fiori and Marigo (1962) prepared anti-Hb-A and anti-Hb-F which were species- specific for human blood and recommended their use in forensic bloodstain analysis. The method was also described by F'iori (1%2) in his review. Adult bloodstains react only with anti-Hb-A, while fetal, or newborn bloodstains react with both antisera. Stains of human body fluids other than blood may react with anti- human serum serum, but will not react with the anti-Hb sera. Hematin does not react with the anti-Hb sera, so that with old bloodstains other methods of identification must be used. Mori (1967) prepared an anti-human Hb in goats and studied its reactivity and cross-reactivity with human and animal hemoglobins by immunodiffusion and immunoelec- trophoresis. At least four precipitin lines could be observed in the reaction between crude human Hb and its antiserum. The antiserum cross-reacted with monkey, horse and dog hemoglobin as well. Absorption with monkey hemoglobin rendered the antiserum human-specific, but it was clear that human Hb seemed to share some antigenic determinants in common with monkey and several other animal hemo- globins. Ohya prepared antisera to human Hb (1970a) as well as to non-hemoglobin red cell proteins (1970b) in rab- bits. The anti-Hb was found to cross react with monkey, dog and horse hemoglobin preparations. It could be shown that the cross reactivity'was due not to hemoglobin, but to car- bonic anhydrase in the non-hemoglobin red cell protein. The anti-human non-hemoglobin red cell protein sera readily cross reacted with many animal lysates, and it was shown that the cross-reacting protein was carbonic anhydrase. The carbonic anhydrases of humans and animals, therefore, do have antigenic determinants in common. If these are not rigorously excluded from the "crude hemoglobin" prepara- tions used for immunization, the "anti-Hb" thus raised will contain non-species-specific anti-carbonic anhydrase anti- bodies as well. Ohya cautioned that great care should be exercised in using anti-Hb sera for confirmation of species of origin. Fukae et uf. (1976) prepared antisera in rabbits against carefully purified human Hb-A and Hb-F. The anti- sera were then absorbed with human non-hemoglobin red cell proteins to render them species-specific. The anti-Hb-A was shown to contain approximately equal amounts of anti- a-chain and anti-@-chain antibodies, while the anti-Hb-F contained a preponderance of anti-y-chain antibodies. This observation accounted for the superior antigenicity of Hb-F in comparison with Hb-A. Baxter and Rees (1974a) tested a commercial anti-human Hb. They found that if the antiserum was used undiluted, it showed a titer of 1:8000 against human blood, 1:2000 against baboon blood, 154 against human serum and 1: 16 against human semen and saliva. The antiserum could, therefore, be rendered specific for blood by moderate dilu- tion, and further dilution would abolish the cross reaction with baboon blood (see section 16.8). These same in- vestigators (l974b) evaluated commercial anti-human Hb-F serum in combination with anti-Hb-A for the discrimination of HBA and Hb-F in medico-legal cases. The anti-Hb-A reacted with cord blood, even at blood dilutions of 1:6400, but the anti-Hb-F did not cross-react with adult blood at blood dilutions as low as 1: 100. Baxter and Rees recommen- ded this method in appropriated cases (e.g. infanticide), but cautioned that there are adults whose cells may possess Hb- F in small, residual amounts, or in larger amounts because of pathological conditions. This fact must be taken into ac- count in interpreting the results of precipitin tests using these antisera. The use of anti-Hb sera in bloodstain detection and assess- ment is, of course, very closely related to the use of anti- serum sera for species of origin determination. he latter is discussed in great detaii in a later section (section 16.1). The advantage of anti-Hb sera is obviously that it may serve to combine the identification of blood with the determination of species of origin into a single test. This point was made emphatically by De Forest and Lee (1977). They prepared a high titer anti-human Hb and tested its applicability in mediwlegal blood identification. The use of the reagent was highly recommended. The cross-reactivity of anti-Hb antisera among different species is of theoretical interest, as well as being of consid- erable practical importance. Immunological relationships between the same protein(s) of different species can be used to draw conclusions about phylogeny, and various evo- lutionary relationships between species. Thissubject will not be discussed here with respect to hemoglobin. It will be discussed in somewhat more detail in the sections dealing with determination of species of origin (sections 16.8 and 16.9). The relationship between the structure and evolution of proteins and their immunogenicity forms the basis for the application of immunological techniques to identification and species determination. 7.2 Chromatographic Methods In 1957, Fiori first suggested the use of paper chro- matography for the identification of blood by separating and locating hemoglobin and/or its derivatives. Whatman No. 1 filter paper was employed, using a solvent system of 2,4-1utidine:water: :2:1, in the presence of 20-30 mP concen- trated ammonia. Spots were detected with alcoholic ben- zidine (pH 4.4-4.6 with acetic acid) spray reagent. &values were of the order of 0.56-0.57 but were highly temperature dependent, and hematoporphyrin could be detected by its fluorescence under 366 mn light prior to spraying. Using this method, 5 nQ of blood could be detected (corresponding to about 1 pg Hb), though best results were obtained with 30 nQ of blood (Fiori, 1957). The disadvantages of this sys- tem were the relatively long development time required (12-14 hrs), and the fact that pre-saturation was required. In 1961, Fiori reported a modified procedure which was consid- erably faster. The solvent system employed was meth- anokacetic acid:water ::90:3:7, Whatman No. 1 filter paper was again used, and chromatography was carried out in the ascending direction. In this system the solvent front mi- grated about 12 cm in 1-2 hrs and pre-saturation was not required. After a run, the paper was dried in a 100" oven to inactivate vegetable peroxidases. The chromatogram was then examined under UV light to detect fluorescent materials which differed from hematin compounds. Examination at two wavelengths, 253.7 nm and 366 nm, was recommended. Hematoporphyrin could be identified at this stage. A two- step visualization reaction was used, the first consisting of spraying with 1% (w/v) benzidine in 96% alcohol, acidified with acetic acid. Spots which developed at this stage repre- sented chemical oxidants. The second step was the spraying of the paper with 3% H202 solution to develop the hematin compound spots. This procedure was also described by Fiori in his review in 1962. In running unknown samples, it was recommended that several dilutions of extract be run in the system in a preliminary chromatogram to determine the optimal concentration of extract to be used. A second run was then made using the optimal concentration of extract, and the proper controls. The Rf values for hematin com- pounds are about 0.70-0.71. Hb-A and Hb-F are not differentiated, nor are a number of animal hemoglobins. Most chemical oxidants have different Rf values. A few, such as CuN03, NiCl and CuS04, have similar Rl's but are differentiated by the two-step spraying procedure. These also give different colored spots than does blood. Rust does not interfere. Hematoporphyrin formed long "comets" in this system, and Fiori recommended use of the earlier luti- dine:water system if identification of this substance was re- quired. A control spot of 10-20 pQ of a 1:1000 dilution of whole human blood was run with each chromatogram. If oxidants are encountered, a second chromatogram sprayed with phenolphthalin H202 reagent, which might help to differentiate them, was recommended. This procedure, Fiori said, amounted to a method for rendering the benzidine test considerably more specific than it would be if it is carried out Blood Zdentifrcatn-Immunological and Other directly; but he suggested, nevertheless, that another, equal- ly sensitive and specific test for blood be run in parallel on case materials. An anti-Hb precipitin test in agar gels was thought to be the best choice. Farago (1966) reported a thin-layer chromatographic method for the identification of hematin compounds from bloodstains. Kieselguhr 250 p TLC plates were employed with a solvent system of methano1:acetic acid:water:90:3:7. 5 to 10 pl of water or saline bloodstain extract was spotted on the plate, with 10 pt of 1'70 whole blood as control. At W%",the plates were developed for 25 rnin, dried at 100" and sprayed with benzidine reagent and $0, in two suc- cessive steps. The R, of the hematin compounds was 0.79, and the method could detect 3-4 pg blood. There were many other studies on the paper chro- matography of hemoglobins prior to Fiori's (1957a) paper, but they were designed to gather information about various human hemoglobin variants and not as forensic methods of identification (Andersch, 1953; Berlingozzi et al.. 1953a and 1953b; Kruh et al., 1952; Penati et al., 1954; Sansone and Usmano, 1950a and 1950b; Schapira et al., 1953; and others). There have also been studies on the separation of human and animal hemoglobins, and on the applicability of the chroma- tographic patterns thus obtained to determination of species of origin of bloodstains. These are described in a subsequent section (section 17.3). Chromatography has additionally been employed as a de- vice for concentrating diluted bloodstains and samples, and for separating blood material from various debris. Frache (1939a, 1939b, 1941) used small alumina columns to con- centrate the blood substances in diluted samples in prepara- tion for subsequent identification and species of origin tests. In 1951, Kirk et al. proposed the use of paper chro- matography for the separation of blood from debris. It was noted that blood may be encountered mixed with a variety of materials, such as soil, leaves, wood products, and so on. It may also be extremely diluted or diffuse on surfaces from washing, or present in small quantities which occupy large surface areas, as for instance on a car in a hit-and-run case. The technique which Kirk et al. described is applicable to many of these situations, and involves placing a wet piece of filter paper in contact with the moistened blood-containing material. Water was used as the solvent. The capillary action of the migrating solvent carries and concentrates the blood material in a particular region of the filter paper strip, where it may then be subjected to identification, species and blood grouping tests. Obviously, diierent situations call for dii- ferent strategies in terms of the actual experimental set-up, and Kirk et al. discussed a number of these in the paper. Positive identification, species, and grouping tests could be gotten from very small amounts of material from irregular surfaces and/or which had been mixed with debris. Schaidt (1958) reported a very similar technique, which he had ap- parently devised independently. Fiori (1962) noted a clever modification of this approach. The filter paper strip was cut so that one end was pointed (V-shaped). The pointed end Sourcebook in ForensicSerology,Zmmunologv, andBiochemistry Furuya and Inoue (1966a) said that their rates of detection were variable. Neumann (1949) found that 23 examples of menstrual bloodstains showed negative results out of 248 examined. False positive results are apparently also possible. Oral cavity epithelial cells, which are morphologically very similar to vaginal epithelium, may contain small amounts of glycogen, although because the amounts of glycogen in the two types of cells are so different, it is not expected that this would present a practical problem (Mueller, 1953; Sakamoto, 1957). Popielski (1949), after some fairly careful investigations came to the conclusion that the finding of glycogen-positive epithelial cells was not necessarily diag- nostic for menstrual blood. He showed that morphologically similar cells could be obtained from the male urethra, and that substantial numbers of them (25 to 50 percent) con- tained glycogen. This fraction decreased if smears were taken at times of sexual excitation, but was even higher in the cells from post-gonorrhealurethral discharge. Cells from the female urethral orifice also showed the characteristic epithelial morphology, and many contained glycogen. Thus, if any of these types of cells were to be mixed with blood, the presence of glycogen-containingcells could easily be misin- terpreted. Other types of blood than menstrual also contain glycogen-positive cells. Furuya and Inoue (1966a) obtained positive results in about 54%of 133cases of bloodstains from menstrual blood, and blood shed during labor and abortion. They also showed (1966b) that blood shed during the puer- perium contained glycogen-positivecells. The puerperium is the period of approximately 40 days from birth to the com- plete involution of the maternal uterus. Blood shed during this time is sometimes called lochial blood. Glycogen- containingepithelial cells were found in bloodstains obtained from recently delivered women up to puerperal day 17. Thus, if nothing at all is known of the history of a bloodstain submitted for examination, some care must be exercised in interpreting the finding of glycogen-containing epithelial cells. 8.1.2 Methods based on fibrinolytic properties That menstrual blood does not clot has been known for many years. Luginbuhl and Picoff (1966) quoted Hunter as having written in 1794 that: "In healthy menstruation, the blood which is discharged does not coagulate, in the irregu- lar or unhealthy it does". Formad (1888) also noted the property of incoagulability as being characteristic of men- strual blood. The property is of no direct value, of course, in bloodstains. Methods have been proposed, however, to take advantage of it indirectly. To help clarify the current think- ing about the reasons for the incoagulability of menstrual blood, and the forensicmethods of identificationbased upon this property, some background on the fibrinolytic enzyme system will be presented. The fibrinolytic system is actually quite complicated, still not completely understood, and its literature is immense. In 1959, now already some 20 years ago, Sherry et al., in their review, said that studies of fibrinolytic activity had resulted in a literature ". . . so vast that it would be impossible to attempt a complete survey within the confines of this report". Even at that, the review ran 40 pages and dealt only with the then current aspects. 425 references were cited. The interestedreader must, there- fore, be referred to the specializedliterature if more detailed information on this interesting subject is wanted (Hahn, 1974; Marder, 1968; McNicol and Douglas, 1976;Sherry et al., 1959; Verstraete et al., 1971). Blood clotting is an extremely complex process. Many factors are now known to be involved, and the nomenclature and terminology were most complicated and confusing until the early 1960's. At that time, a universal roman numeral designation was adopted for use by the international com- munity, largely through the results of an international com- mittee chaired by Dr. I. S. Wright (MacFarlane, 1976).The last step in the clotting reaction involves the conversion of fibrinogento fibrin. The fibrinolyticsystem,which is of inter- est to the present discussion, is quite obviously related to the clotting system, and in a way, represents its physiological converse. The fibrinolytic system acts to dissociate pre- viously formed blood clots, or to prevent their formation in the first place. The relationshipbetween these physiological systems is still not very well understood, but their functions clearly have enormous consequencesfor the health and well- being of the organism. There is a fairly widely held, but unproven, hypothesis that the fibrinolytic system is in a dy- namic equilibrium with the clotting system. In its barest essentials,the fibrinolyticsystem is represented in Fig. 8.1. Plasminogen is a heat-stable plasma protein, the molecular weight of which has been reported to be as low as 81,000 and as high as 143,000. Its activation to plasmin, the active fibrinolysin, is a proteolytic reaction, analogous to the activation of trypsinogen to trypsin, pepsinogen to pepsin, etc. A limited number of peptide bonds are split in the acti- vation reaction, and the reaction is irreversible. Activation is effected by so-called plasminogen activators, which may function in one of three ways: Acting directly on plasminogen e.g. tissue activator, and urokinase from urine Proteolytic enzymes acting normally e.g. trypsin and plasmin itself a By converting normally inert proactivator to activator e.g. streptokinase (SK) and tissue lysokinase Natural activators occur in blood plasma and in tissues. The tissues contain a soluble activator as well as a much more insoluble one. The latter may be extracted with thiocynarme and has been called fibrinokinase. Activatormay be found in milk, tears, saliva, bile and semen, but not in sweat. In addition, there are known to be naturally-occurring anti- plasmins and anti-activators. The active plasmin (fibrin- olysin) is responsible for the proteolytic degradation of fibrin in vivo. The incoagulability of menstrual blood is apparently due to the presence of activator. Menstrual discharge has no fibrin; it contains activator, plasmin, large amounts of pro- activator, and no plasminogen (Albrechtsen, 1956). Using Blood Ident.@xtion-Menstrual, Pregnancy and Fetal Proactivator 1tissue lysokinase bacterial kinases (e.g. streptokinase) Plasminogen I F Activator -b inhibitors ) inactive enzyme (Prefibrinolysin) (Fibrinolysin) (antiplasmins) I f fibrin ) fibrin degradation products I Figure 8.1 The Fibrinolytic System histological techniques, Luginbuhl and Picoff (1966) found that proactivator was present throughout the endometrial stroma at all stages of the menstrual cycle, but that the presence of activator was confined to the superficial layers of tissue at around the time of menstruation. There was little activator present during other phases of the cycle, and virtu- ally none at midcycle. Beller (197 1) noted that the fibrin- olytic breakdown products present in menstrual discharge showed the characteristics of fibrinogen degradation prod- ucts, rather than of fibrin degradation products. In 1949, Popielski had noted that the absence of fibrin threads in a bloodstain would indicate that it was not of menstrual origin, but said that this simple device could not be used if the stains had soaked into any type of absorbent substrate. Berg (1954) was apparently the first medico-legal investigator to take advantage of the fibrinolytic activity of menstrual blood for identification purposes. Hismethod con- sisted of incubating suspected stain extract with fresh human fibrin at 37" for 24 hrs or so. At the end of the incubation period, a micro-Kjeldahl assay for nitrogen was carried out to assay for the fibrinolytic products. If the value was sufficiently different from a nonfibrinolytic control, menstrual blood could be diagnosed. This method was also discussed by Berg (1957b), and in this latter paper, he said that he had been using this method since 1952. In 1959, Culliford reported an electrophoretic method based on the same principle as Berg's assay, but easier to carry out in terms of product detection. A strong extract of suspected stain was made, along with a similarly strong extract of capillary bloodstain as control. These samples (30 pQminimum) were incubated with human fibrin at 37" for 24 hrs. 20 pQof each sample was then applied to What- man # 1 filter paper and electrophoresed at 120- 150 V for 16 hrs. The bridge buffer consisted of 33 g sodium barbital, 19.5 g sodium acetate, 205 mQ of 0.1N HC1, all in a 32 final final volume with water. The strips were dried at 105" and stained with azocarmine B. An extra band, due to the fibrinolytic products, could usually be seen in the menstrual blood samples. The presence of the band was noted to be variable, however, even in known menstrual bloodstains, and samples were encountered in which the band was absent altogether. Karnimura (1 96 1 ) confirmed Culliiord's obser- vations using almost the same technique, except that he employed bromphenol blue as a protein stain. Positive re- sults were obtained in stains up to 6 months old, and in putrefied bloodstains, but bloodstains that had been heated to 60" for 30 min, or that had been in water, gave negative results. Kamimura also noted that bloodstains from blood shed at parturition in induced abortions was indistinguish- able from menstrual blood by this method. In 1947, Permin first reported the use of what came to be called the fibrin plate method for the assay of fibrinolytic activity. The method was refined by subsequent workers (Astrup and MDllertz, 1952; and others), and can be used for the assay of any of the components of the fibrinolytic system. The method consists simply of preparing purified fibrinogen, and suspending a quantity of it in buffer in the presence of a small amount of thrombin. This mixture is placed in a Petri dish, and allowed to form a gel. The material whose f ib~oly i tcactivity is to be tested is placed on the surface of the plate, directly on the gel. After incubation, usually about 24 hrs at 37", the digested fibrinogen can readily be seen. If semiquantitative results are wanted, the sample can be placed on the gel in such a way that the digested area is amenable to measurement. It has been common practice to use beef or ox plasma to prepare the purified fibrinogen. In 1962, Shiraishi utilized this method to demonstrate the fibrinolytic activity of menstrual blood, and to identify men- strual bloodstains. The fibrin plate was prepared by dii- solving 1 mQ of fibrinogen solution in 2 mP barbiturate buffer (O.lM, pH 7.8) and 20 pQthrombin in a small (4.5 crn diameter) Petri dish, shaking gently for 3-5 sec to mix,and allowing formation of the white gel. The plate was then incubated 30 min at 37" before use. The fibrinogen was prepared from beef plasma, washed, and dissolved in the barbiturate buffer. In the first paper (1962a), Shiraishi Sourcebook inF o e Serology, Z~mmunologv, and Biochem&tty showed that menstrual blood serum contained large amounts of plasmin, it being able to dissolve the fibrin plate even at dilutions as high as 1: 1000. The maximum dilution of men- strual blood serum which would dissolve the plate varied with the day of the menstrual period on which the sample was collected. The 1: 1000 value occurred on the second day and was the highest, while the lowest dilution, 1:100, oc- curred in samples from Day 5. It could similarly be shown that circulating blood was always negative in the fibrin plate test unless streptokinase was added (see Fig. 8.1). In the presence of streptokinase, circulating blood serum would dissolve the fibrin plate at dilutions as high as 1:640,000. Similarly, streptokinase greatly increased the dilutions at which menstrual blood serum would dissolve the plate. The second paper (1962b) dealt with the identification of men- strual bloodstains. One thread from a 1 cm2 stain was ade- quate to demonstrate fibrinolysis on the plate. The technique was extremely simple: the thread was placed onto the fibrin plate surface, the plate incubated, and examined. Stains that were kept two years at room temperature gave positive re- sults, as did stains left a month under water and stains heated to 70-100' for an hour. Shiraishi said that neither blood shed at abortion or delivery, nor lochial blood gave a positive test. Considerably less encouraging results were obtained by Schleyer (1963) in his study of the fibrin plate techique for menstrual blood identification. Only about half the 39 men- strual bloodstains examined gave positive results on the fibrin plate described by Astrup and Mullertz (1952). The presence of natural plasmin inhibitors was discussed as a possible reason for the false negative results. Schleyer was, therefore, less enthusiastic about the techique than Shiraishi had been, and stated that the results only had any meaning when they were positive. In 1973, Whitehead and Diva11 conducted experiments to determine the amounts of "soluble fibrinogen", that is, high MW fibrinogen breakdown prod- ucts, in menstrual blood. They used a tanned red cell hemag- glutination inhibition immunoassay (TRCHII) (Fox et al., 1965; Merskey et d,1%6; Hahn, 1974). It was found that menstrual blood contained significantly more soluble fibrin- ogen, expressed as a percentage of total protein, than did capillary blood. Mixtwes of capillary blood and semen or capillary blood and vaginal secretions in stains were also higher in soluble fibrinogen than capillary blood alone, but not has high as menstrual blood stains. Whitehead and Diva continued this work (1974), conducting an irnmunoelectro- phoretic study of the controlled degradation of fibrinogen and fibrin by plasmin, and its applicability to the identi- fication of menstrual bloodstains. Their experiments were based on earlier work by Nussenzweig and Seligmann (1960), who carried out the first systematic irnmuno- electrophoretic study of fibrinogen and fibrin degradation by plasmin, and by Berglund (1962) and Dudek et al. (1970). The last-mentioned investigators used CM-Cellulose chro- matography to separate the products. Marder (1968 and 1971) has also studied the problem extensively. The scheme for the degradation of fibrinogen which emerges from these studies is indicated in Fig. 8.2 (Marder, 1971). Whitehead and Divall's experiments gave results in ac- cord with the scheme in Fig. 8.2. The first stage of de- gradation (fibrinogen conversion to Fragment X, plus A, B and C) was reached after 4 min digestion. Fragments Y and D could be seen after 10min of digestion, and after 45 min, Fragment Y had disappeared and only Fragments D and E were visible. The digestion of fibrin was studied as well, and followed a pattern quite similar to that of fibrinogen, except that the rate was slower. Fragments A, B and C do not react with the antiserum to fibrinogen, and thus are not detected in this immunoelectrophoretic system (Nussenzweig and Seligmann, 1960). 102 menstrual bloodstains were exam- ined in the system. First stage degradation products were found in 56, and second stage products in 18. No third stage degradation patterns were observed, and 28 stains gave no precipitin arcs at all. The absence of precipitin arcs in 28 of the 102 stains, it was said, might be due to the low levels of degradation products in some stains. The predominance of Stage I and Stage I1 patterns, and the absence of Stage I11 ones, in the positive sample might be explained by the inter- vention of plasmin inhibitors present in endometrial tissue. 24 samples of circulating bloodstains were tested, and none gave precipitin arcs. 8.1.3 Immunological methods A few attempts have been made to prepare specific anti- sera for the differentiation of menstrual blood. Sudo (1957) reported that he could prepare antisera to menstrual blood in rabbits or sheep, and by suitable absorptions, obtain an anti- serum which detected menstrual blood in stains up to 2 years old. Domestic fowl were not suitable for making this anti- serum, he said, because the anti-hemoglobin titer was too high. Harada (1960) made antisera to human and horse fibrinogen. The anti-human serum reacted with menstrual blood, and not with capillary blood, but it also reacted with the blood of victims of sudden death. 8.1.4 Methods based on menstrual blood toxicity There have been a few reports that menstrual discharge contains various toxins, and a few investigators have indi- cated that these might be used as the basis for a test for the presence of menstrual blood in stains. In 1927, B'6hmer noted that menstrual blood contained a substance which greatly retarded the growth of seedlings of Lupinus mu- tabilis plants (1927a). Neither cord blood nor cadaveric blood had this effect. He further indicated (192%) that menstrual blood inhibited glucose fermentation by yeast cells, but cord serum showed this effect as well. Yamaguchi (1958) reported that menstrual blood serum contained a substance that was toxic to mice, and that the concentration of the toxin increased during successive days of the menstru- al period. It does not appear that Yamaguchi was describing the same substance that had been reported earlier by Smith and Smith (1940 and 1944). The latter toxin resided not in detecting HCG in bloodstains representing 100-200 pP blood. The hormone could be detected in bloodstains from pregnant women from 45 days following the last menstrual period to parturition. 8.2.2 Methods based on pregnancy-associated proteins The subject of pregnancy-associated proteins (as distinct from pregnancy-associated enzymes, which are considered in sections 8.2.3 and 8.2.4) was opened by Smithies in 1959. In some 10% of serum samples from recently delivered women, or women in late pregnancy, a protein band was observed in the haptoglobin region following starch gel elec- trophoresis. This observation was confirmed by Afonso and DeAlvarez (1963) who noted that the protein was not equiv- alent to cystine aminopeptidase. They also pointed out that Giblett had independently observed the protein at about the same time as Smithies. Smithies called the region of the gel to which the protein migrated, the "pregnancy zone", and the term "pregnancy zone protein" has persisted. The protein is especially characteristic of the 2nd and 3rd trimesters of pregnancy. Afonso and De Alvarez (1964) found the protein in 10% of sera from the first trimester, 6990 of those from the 2nd. and more than 80% of those from the third trimester. The protein was found to be an a2-globulin, and was not observed prior to the ninth week of gestation. It was not identical to ceruloplasmin, thyroxin-binding protein, nor transcortin. Following the initial observations, the subject became somewhat complicated because, not only were additional pregnancy-associated proteins discovered, but many of the observations were independent, each group applying its own separate nomenclature. Some of the confusion has recently been cleared up, but the field is very active, and rapidly developing, In 1966, Bundschuh reported a new antigen in serum, detected by variously absorbed horse immune antisera, which could be found in almost 78% of women, but in only about 22% of men. The antigen appeared to be inherited, but its expression was under the influence of sex hormones in some way. This antigen was named "Xh". MacLaren et a l . (1966) found a similar protein, and called it Pa- 1. Kueppers (1969) studied Xh further, noting that he had found it in 97% of women and 88% of men, using anti-female-serum serum absorbed with pooled male serum. He also found that the purified protein had a sedimentation coefficient of 12.2s and an isoelectric point of about 4.75. Dunston and Gershowitz (1973) studied the antigen and found that, in addition to the sex dependence of its expression, there was some age dependence of expression in females as well. They argued for the Xh designation, saying that the "Pa-1" desig- nation implied more about the protein in terms of standard nomenclatures than was actually known. In 1969, Margolis and Kenrick reported that "pregnancy zone protein" occurred not only in pregnant women, but also in those taking estrogenic oral contraceptives. They found that the protein had a MW of 450,000. Beckrnan et a l . (1970) found that the protein was not present in cord blood, and they confirmed (1971) that women taking estrogenic oral contraceptives had the protein in their sera. Bohn (1971) reported that four proteins could be detected in preg- nant serum by immunodiffusion with antisera made from placental extracts. One of these was identical to human placental lactogen (HPL). Two others were PI-glycoproteins and one was an a2-glycoprotein. One of the 8,-glycoproteins was pregnancy-specific, occurring in urine and in serum. The other 8,-glycoprotein and the a*-glycoprotein could be found in non-pregnant serum but were elevated in pregnancy or in the presence of estrogenic oral contraceptives. The arglycoprotein, Bohn said, was identical to pregnancy zone protein, and to Xh. In 1972, having found that the nonpregnancy-specific bl-glycoprotein and arglycoprotein were elevated in some disease states as well, he proposed to name them 81-AP-glycoprotein and at-AP-glywprotein, where "AP" stood for "acute phase". By 1973, Bohn was referring to the pregnancy-specific 8-glycoprotein as "SPl", while the non-pregnancy-specific 8-glycoprotein was "SP;' and the or2-glycoprotein was ''SP?. "SP" in these designa- tions stood for "Schwangerschaftsprotein", i.e., "pregnancy protein". By 1975, the number of different names being applied to the a2-glycoprotein by the above-mentioned and still other authors had reached such absurd proportions that a large group of workers in the field jointly agreed that this protein would henceforth be called "pregnancy associated a2- glycoprotein" or "a2-PAG (Berne et al.. 1975). This pro- tein is the same one that has been called "pregnancy zone protein", pregnancy-associated a,-globulin (Hasukawa et al., 1973), pregnancy-associated globulin (Horne et at., 1973), alpha-2-pregnoglobulin (Berne, 1973), pregnancy- associated a-macroglobulin (Stimson and Eubank-Scott, 1972), and has been described additionally by Than et a l . (1974) and by von Schoultz and Stigbrand (1974). The MW has been reported to be as low as 360,000 (von Schoultz and Stigbrand, 1974; Bohn and Winckler, 1976), and as high as 760,000 (Than et aL, 1974). In 1972, Gall and Halbert first reported finding four anti- gens in pregnancy serum by immunodifTusion using anti- pregnancy-plasma serum, exhaustively absorbed with non-pregnant plasma. These were soon named "pregnancy- associated plasma proteins A, B, C and D , or PAPP-A, -B, -C and -D (Lin et al., 1973).B and C were 8-globulins while A and D were a,-globulins. The MW of A, C and D were determined to be 750,000, 110,000 and 20,000, respectively. It became clear almost immediately that PAPP-D was human placental lactogen (HPL),and that PAPP-C was identical to Bohn's SPI protein ( L i et al., 1974a, 1974b). None of the PAPP's were equivalent to the pregnancy asso- ciated a2-glycoprotein (Lin and Halbert, 1975), and the amounts present in the placenta were shown to be, from greatest to least, D (or HPL) > B> C> A (Lin et al., 1976). PAPP-A and PAPP-C, as well as HPL, are probably synthesized by the placental trophoblast cells (Lin and Hal- bert, 1976). Lin et al . (1976) showed, finally, that PAPP-B and PAPP-D (i.e. HPL) disappear within a day of delivery. Sotmebook in Forensic Semlogv, Znununologv, and Biochemktry The level of PAPP-A drops rapidly within a few days of delivery and becomes undetectable at 4-6 weeks post- partum. PAPP-C levels fall rapidly too, being undetectable at 3-4 weeks postpartum. In summary, the proteins which have had more than one name, but which are identical, are: (1) pregnancy-associated a2-glycoprotein, with many former names; (2) PAPP-D = HPL; (3) PAPP-C = SP,. PAPP-A is apparently preg- nancy specific, and is not immunologically identical to other known pregnancy-associated proteins. SP2, a 8-glycoprotein, remains unique, though liot pregnancy-specific. Curiously, Than et al . (1974) reported a M W of 760,000 for their protein, which they said was identical to SP,, i.e., a2-PAG. All other reports of the MW of a2-PAG have been in the neighborhood of 500,000 except that Bohn and Winckler (1976) reported 360,000 for the purified protein. The 760,000 value of Than et al. (1974) is very close to the MW of 750,000 reported for PAPP-A although this could cer- tainly be a coincidence. Lin et al. (1974b) insisted that PAPP-A is not equivalent to any of the SP proteins. Towler et al. (1976) showed that the levels of HPL and of the specific B1-glycoprotein (probably equivalent to SPl) cor- relate well with gestation stage. These, as well as perhaps PAPP-A, seem to offer the best prospects for application to the forensic diagnosis of pregnancy in bloodstains. 8.23. Methods based on leucine aminopeptidase and cystiw aminopeptidase Leucine aminopeptidases (EC 3.4.1.1 ) are a-aminopep tide amino acid hydrolases, which hydrolyze L-peptides, splitting off N-terminal leucine residues which have a free a-amino group. The enzymes hydrolyze a fairly broad range of substrates, and may also show esterase activity as well. Cystine aminopeptidases are very similar enzymes, except that, as the name implies, they prefer substrates having N- terminal cystine residues. These enzymes have usually been assayed using artificial substrates, L-leu-8-naphthylamide' for leucine aminopeptidase, and L-cys-S-S-cys-Bnaphthyla- mide for cystine aminopeptidase. The cystine amino- peptidase (hereinafter, CAP)enzymes do show leucine ami- nopeptidase (hereinafter, LAP) activity, but LAP does not hydrolyze the cystine substrate. In 1961, Page et al., using vertical starch gel electro- phoresis, noted that LAP could be detected in all sera, but that two CAP enzymes in serum, CAP, and CAP2, were characteristic of pregnancy. CAP was not found in non- pregnant, nor in fetal serum. The CAP is believed to func- tion physiologically as an oxytocinase. Oxytocin is a hormone, elaborated by the mammalian neurohypophysis. A cystine-containing nonapeptide, its function is to elicit smooth muscle contraction, as of the uterus during birth, and the ejection of milk in lactating females. It is very closely related structurally to another neurohypophyseal hormone called vasopressin. The various reports of "LAP iso- enzymes" in pregnancy, in addition to the enzyme found in all normal sera, may actually represent reports of the appar- ently pregnancy-specific CAP enzymes (Rowlessar et al., 1961; Smith and Rutenberg, 1963). This is partucularly a possibility in the studies in which CAP was not separately assayed, because, as noted above, CAP possesses LAP activ- ity. The serum LAP pattern is also altered in various pathological states, especially in hepatic and biliary diseases (Kowlessar et al., 196 1 ). Robinson et al. (1966) noted that the CAP enzymes were found only in pregnant serum, and not in the sera of non-pregnant women, even those on es- trogenic oral contraceptive therapy. In 1970, Gladkikh reported that bloodstains from preg- nant or puerperal women could be discriminated on the basis of an additional, slow LAP band following electrophoresis. It seems probable that the slow band represented one of the CAP enzymes. The band could be seen in bloodstain extracts from women, from the 8th to 10th week of gestation until about 30 days postpartum. The enzyme could be detected in stains up to 50 days old. The slow band was not seen in bloodstains from men or from non-pregnant women. Some fetal sera, but not bloodstain extracts from fetal blood, showed a similar but qualitatively different slow band. Oya and Asano (1971 ) reported very similar results after electro- phoresis on Oxoid cellulose acetate membranes, noting that the enzyme appeared in the last half of pregnancy, and also characterized retropiacental blood. In 1974, Oya enlarged his studies, noting that the resolution of the usual and pregnancy-specific LAP enzymes was not terribly good on cellulose acetate membranes, but that L-methionine in- hibited the usual serum enzyme, but not the pregnancy- specific one, with L-leu-Bnaphthylamide as substrate. By running paired samples, and staining with and without L-met, the bands could be resolved. Oya et al . (1975a) em- ployed polyacrylamide disc gel electrophoresis to examine these enzymes in serum and in placental extracts. In this study, a CAP assay was incorporated, and it was clear that the two slower bands had CAP activity as well as LAP activity, while the fastest band had only LAP activity. The fast LAP band was the enzyme found in all normal sera, was heat-stable and L-met inhibitable. The CAP bands were heat-labile, not inhibited by L-met, and seemed to originate in the placental lysosomes. The bands were detectable in bloodstains after about the 4th month of pregnancy, and in stains up to about 2 weeks old (Oya et al., 1975b). It was recommended that a phosphocellulose column be employed to remove the excess hemoglobin when examining blood- stains for CAP enzymes. 8.24 Method based on allr4line phosphatase Alkaline phosphatases (EC 3.1.3.1) are widely occumng enzymes with broad substrate specificities. They catalyze the hydrolysis of orthophosphoric monoesters at alkaline pH optima, and are systematically named orthophosphoric monoester phosphohydrolases (alkaline optimum). Serum contains a number of alkaline phosphatase enzymes derived from different tissues, including liver, intestine, bone, and in pregnancy, placenta. Some aspects of the alkaline phos- phatase enzymes are not pertinent to the present discussion, and are not taken up here. These include the relationship between intestinal enzyme expression and the ABO blood groups and secretor loci (SchrefRer, 1965; Beckrnan and Zoschke, 1969), and the fact that placental alkaline phos- phatase exhibits polymorphism in human beings (Beckman and Beckman, 1968; Beckman, 1970). That plasma contains alkaline phosphatase activity has been known since the work of Kay (1930a, 1930b). Fishman and Ghosh (1967) said that the French investigator, Coryn, first noted in 1934 that the activity of the enzyrne is elevated in pregnancy. The enzyme in maternal circulation during pregnancy was shown to differ from the other alkaline phos- phatases in its immunological properties, K,,, for various substrates, electrophoretic mobility, inhibition by L-phenyla- lanine and heat stability (Posen et al., 1969). By 1967, it had become clear that the heat-stable alkaline phosphatase of pregnancy plasma was of placental origin (Fishman and Ghosh, 1967; Posen, 1967). The placental alkaline phos- phatase is stable to at least 55' for 2 hours, is almost 90% inhibited by 10 mM L-phe, has a pH optimum of 10.6 in 18 mM phenylphosphate and a K, of 0.51 for phenyl- phosphate (Ghosh, 1969). In 1973, Oya et al. showed that the heat stable alkaline phosphatase could be detected in bloodstains from the blood of women in the latter half of pregnancy, the blood shed at delivery, or puerperal blood. The assay of total alkaline phosphatase and heat-stable alkaline phosphatase (that which survived 56' for 30 min), using p-nitrophenylphos- phate as substrate, was recommended for the medico-legal diagnosis of pregnancy from bloodstains. Stafunsky and Oepen (1 977) recommended a slightly modified, but similar procedure. Results could be obtained in bloodstains stored up to 19 months. A disadvantage of the method is the rela- tively large amounts of sample required. Stafunsky and Oepen extracted stains from 1 to 3 cm2 in size. Fishman et al. (1972) said that the level of the placental enzyme in- creased exponentially in maternal serum as a function of gestation time. Thus the amount of enzyme to be expected will depend on the progress of the pregnancy. In bloodstains, the age of the stain is probably inversely related to the amount of active enzyme as well. Older stains, or stains from persons whose pregnancies have not progressed very far, or a combination of these circumstances, might, therefore, re- quire prohibitively large amounts of sample in order to ob- tain unequivocal results with the usual spectrophotometric assay technique. There is another consideration, which is of enormous irn- portance in the interpretation of results of heat-stable alka- line phosphatase assays in bloodstains of unknown origin. Fishman et al. (1968a, 1968b) first described an alkaline phosphatase in the serum of a patient named Regan with a bronchiogenic carcinoma. This enzyme in every way resem- bled the placental enzyme, and was found in serum, in pri- mary tumor tissue and in its metastases. The enzyme in the plasma of patients with neoplastic disease came to be called the Regan isoenzyme, and is immunologically and biochem- ically indistinguishable from the heat-stable alkaline phos- Blood Idenn@ca&n-Memtmai, Pregnmrcymd Fetal phatase of the placenta (Fishman, 1969). Fishman has recently (1974) reviewed this entire subject excellently. 8.3 Identlficatlon of Febl and Blood trom Chlldren 8.3.1 Fetal hemoglobin Fetal hemoglobin (Hb F) is best distinguished from the hemoglobin of adults (Hb A) by electrophoresis. The test for Hb F is probably the simplest way of diagnosing fetal, or early childhood, blood. Before an electrophoretic method had been worked out, it was common practice to discrimi- nate Hb F from Hb A on the basis of their differential sensitivity to alkali denaturation. Hb A is quite alkali-labile, whereas Hb F is relatively resistant, the denaturation usu- ally being detected spectrophotometrically. This method was applicable to bloodstains (Huntsman and Lehmann, 1962; Culliford, 1964; Watanabe, 1969). Pollack et at. (1958) successfully separated Hb A from Hb S (sickle-cell hemo- globin) in a medico-legal case in Massachusetts using paper electrophoresis, and suggested that it would be very de- sirable to have such a method for the separation of Hb A and Hb F as well. Wraxall provided such a method in 1972, which was simple, reliable and was performed on Sartorius cellulose acetate membranes. Wilkens and Oepen (1977a) fully confirmed the usefulness of Wraxall's method with bloodstains from 160 cord blood specimens on glass, wood, paper and textiles. The technique is also fully described by Culliford (1971). Immunological methods, using anti-Hb F, have been employed as well (Baxter and Rees, 1974b), and were fully discussed in Section 7.1. It must be noted that there are a few adults whose red cells contain abnormal amounts of fetal hemoglobin, and this fact must be kept in mind in interpreting the results of tests for Hb F. 8.3.2 Methods based on a,-fetoprotein In 1956, Bergstrand and Czar reported that they had found a high concentration of a protein in fetal serum that did not occur in maternal serum. This protein eventually became known as a,-fetoprotein, or a-fetoprotein. Nishi and Hiraki (197 1) reported that the protein had a MW of 64,600 and gave its amino acid composition. Masopust et al. (1971) said that the MW was 76,000, the isoelectric point 5.08, and that the protein had no associated sialic acid or lipid. The protein occurs normally in fetal serum, but is found in the serum of adult patients suffering from malignant tumors, especially hepatomas. It is clinically useful in the latter re- gard as a diagnostic tool (Abelev, 1971). Patzelt et al. (1974) detected a-fetoprotein in bloodstains by what they referred to as "immunoelectroosmophor~sis" (crossed-over electrophoresis), using a specific rabbit im- mune serum. The test was positive in bloodstains stored up to 3 months, and was carried out on 1% agar gels in veronal- sodium acetate buffer at pH 8.1. Thomsen et al. (1975) noted that there was a definite, inverse relationship between gestational age and the serum concentration of a-feto- protein, and that this fact could be used as an aid to the Sourcebook in Forensic Serology, Immunology, and Biochemistry quotients were higher in fetal bloodstains than in adult bloodstains. In the other method, the ratio of the OD of the 540 nm peak to the 500 nm trough in metHbCN, obtained by treatment with "transformation solution," was correlated to stain age up to 15 weeks. Kind et al. (1972) proposed a method whereby the spectrum of the stain was determined in the visible region from 490 to 610 nm. Vertical perpendic- ular lines were then dropped at wavelength values of 490, 560, 578 and 610 nm, and the points of intersection of the perpendiculars with the spectral trace at 490 and 610 con- nected by a straight line (as shown in Fig. 9.1). Points of Wavelength Inrn.1 Figure 9.1 Scheme for Determination of a-ratio (after Kind et al., 1972) intersection were identified by letters as shown, and a quan- tity called a could be calculated, according to: OD., - ODb, a = ODdf - 0D.f where OD,,, ODk, etc., represented the changes in optical density represented by line segments ab, bc, etc. The a ratio was found to decrease in some 200 bloodstains, which had been stored at room temperature in the dark for up to 8 years. The ratio underwent a change of from about 1.50 to about 0.80 over the course of some lo5hours of aging. There was considerable scatter in data points from samples of the same age group, however. An improved ratio, called q,was proposed by Kind and Watson (1973). a, is calculated in precisely the same way as is a,except that two of the wave- lengths at which the perpendiculars are d ropp l are changed as shown in Fig. 9.2. The spectra were determined in ammo- niacal extracts of bloodstains, rather than directly in stains mounted in paraffin, as had been done in the 1972 experi- ments. The a,ratio was seen to decrease in a consistent way for bloodstains aged up to 15 years, and there was less scat- ter in samples of the same age group. Kiihler and Oepen (1977) reported a lengthy series of experiments on 85 blood- O.D. Wavelength (nm.) Figure 9.2 Scheme for Determination of as- ratio (after Kind and Watson, 1973) stains on various substrates aged up to 5 years in which the methods of Kind er al. (1972) and of Kleinhauer et al. (1967) were tested. It was found that the variations of the parameters defined by the previous workers within an age group exceeded those between samples of different age groups. Kbhler and Oepen concluded, therefore, that neither of these methods was suitable for reliable estimation of bloodstain age in practice. Nuorteva (1974) pointed out that in decaying samples, which are found to be maggot-infested, an estimate of the age of the material could be made on the basis of knowledge of the length of the life cycle stages of the insects whose larvae were present. Rajamannar (1977) looked at the serum protein pro6le by immunoelectrophoresisin stains as a function of their age, from 15 days to one year. The presence of precipitin arcs representing y-, B2M-, b2C-, B2B-, 8,-, a,-and a2-globulins and albumin was followed with time. A characteristic pat- tern of disappearance of various proteins at test points along the time line could be constructed.. Albumin, a,- and arglobulins were absent at 15 days of age. At 30 days, &M-globulin disappeared, with &C-globulin disappearing at 60 days, &B-globulin at 150 days, and 6,-globulin at 300 days. All the proteins were undetectable at 365 days of age. It should be pointed out that Sensabaugh et al. (1971) found albumin to be detectable by its immunological reaction in a dried blood sample eight years old. The apparent disappearance of albumin in a compara- tively fresh bloodstain in Rajamannar's (1977) experiments can be accounted for on the basis of a change in the electro- phoretic mobility of albumin in aging bloodstains. Beginning almost immediately when blood dries, and progressing steadily with time, the amount of albumin which appears at the "albumin position" on electrophoretic support media de- creases, while there is an apparent increase in the amount of "gamma globulin." Using monospecific precipitating sera, it can be shown that albumin does not denature; rather it migrates differently, and appears at the y-globulin position. Quantitation of the amount of protein detectable with pre- cipitating antisera in the "albumin" position and the gamma globulin "position" indicates that the ratio is approximately proportional to the age of the stain from which the extract was obtained. A preliminary account of this work has ap- peared (Lee and De Forest, 1978). Further studies are in progress in our laboratory, using two-dimensional immu- noelectrophoresis (Laurell electrophoresis technique), to gather additional data about this interesting change in the albumin molecule (Lee,H.C.,.R. E. Gaensslen, B. Novitch BIood Idmt~n-Bbo&ahAge and R. Fossett, in preparation). The alteration in electro- phoretic mobility of albumin, and perhaps of other serum proteins, does not appear to be restricted to aging blood- stains. We have noticed that it can occur in aging serum samples as well, and others (e.g. Heftman et al., 1971) have reported the same effect. Antisera specific for a particular serum protein must be used in examining bloodstain extracts because one cannot use the electrophoretic mobilities of these proteins in fresh serum as a guide to identity. Shinomiya et al. (1978) used imrnunoelectrophoresis to estimate the age of bloodstains. They found that stain age could be correlated to the number of precipitin arcs de- tectable in stain extract following immunoelectrophoretic analysis. SourcebookinFowndf SeroIogy, Zmmunologv, andBiochemisrty REFERENCES FOR UNlT I1 Abelev, G. I. 1971. a-Fetoprotein production by normal liver and liver tumors. irx H. Peeters ( 4 . ) Protides of Biological Fluids, Proc. of the 18th Colloq., 203-209, Pergamon Press. Oxford and New York. Abelli, G., B. Viterbo and M. Falagario. 1964. The immunological diagnosis of pregnancy with specimen of blood stains. Med. Sci. Low 4: 115-118 Adler, 0. and R. 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