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UNIT II

IDENTIFICATION OF BLOOD

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of the fact that the latter rely on at least some knowledge of the chemcial composition of blood. The fact that the knowl- edge of blood's chemical composition was very incomplete, and characterized by notions that have not stood the test of time, may make this distinction somewhat arbitrary. But it seems possible, nevertheless, to distinguish between the pre- 19th century commentaries which are of purely historical interest, and those methods which appeared after 1800, and were based on what most people would regard as chemical priniciples, rather than on purely empirical observations having no theoretical framework whatsoever. One of the oldest books in English having to do with bloodstains is W. D. Sutherland's Blood-Stains: Their Detection, and the Determination of Their Source (Sutherland, 1907). The book is, in fact, one of the few in any language up to its time which treats what we now call forensic serology (in its broadest sense) exclusively. Sutherland, a Major in the British Indian Medical Service at the time of the book's appearance, was obviously something of a scholar, in addi- tion to the fact that he had forensic casework respon- sibilities. The Preface of the book begins with the comment: "As there does not exist in any language of which I have knowledge a compendium of the modern tests by which the detection of bloodstains and the determination of their

source may be carried out... ." From this remark, and

from the nature and extent of his bibliographic reference list, it seems likely that Sutherland read a number of modem languages and probably Latin in addition of course to English. An interesting man, he later became a Lieutenant Colonel, and apparently stayed in India until he died in

1920. A brief obituary appeared in the British Medical Journal 2: 189 (1920). He was very interested in the precise origins of medico-legal examinations of bloodstains, noting at the beginning of the second chapter: I have endeavoured to ascertain when the medico-legal importance of blood-stains first came to be recognized, but without success. The older medico-legists, whose works are cited in the bibliography, treat of most things from miracles to slight wounds, but none make special mention of bloodstains. And, as we see, even so late as 1834 the fourth edition of a popular German textbook contained no special reference to them, although in 1817 Orfila had dealt with the chemistry of the blood in his textbook of medical chemistry, and in 1808 Jacopi is said to have managed by the aid of the microscope to distinguish bovine from human blood. It is from the discussion which took place at the Academie Royale de Medecine in 1828 that I infer that for a considerable time the French experts had busied themsleves with the question of the detection of bloodstains.

The discussion to which Sutherland referred may be one which appeared in the Journal generale de medecine de chirurgie et de pharmacie in 1828 (Raspail, 1828a; O d l a , 1828a; Raspail, 1828h Raspail, 1828~).It was based on a serious disagreement which had arisen between Raspail and Orfila over the value which was to be attached to the results

of a series of chemical tests proposed by Orfila (1827a) for the identification of blood in medico-legal investigations. Mathieu-Joseph-Bonaventure Orfila was one of the most prominent medical scientists of his time. Born in Spain April 24, 1787,Orfila began his studies in that country, but completed them in France, receiving his doctorate at Paris in

1811. He remained in France until his death in 1853. He enjoyed a brilliant career, had considerable prestige, and his opinions and pronouncements carried a good deal of weight in the scientific community. Orfila was primarily a tox- icologist; Prof. Dr. Muaoz, in a Spanish-language biograph- ical tribute to OrMa, published in 1956 in Quito, Ecuador, called him the "founder of modem toxicology". A biograph- ical sketch of Orfila's career appeared at the time of his death as well (Chevallier, 1853). There is no doubt that Orfila was one of the important figures in the development of legal medicine. At various times he focused his attention on blood and body fluids, and his contributions in this area are among the earliest systematic studies that can be found in the published literature. The earliest methods for medico-legal identification of blood were chemical ones. Lassaigne is said to have pub- lished a paper on the discrimination of bloodstains and rust spots in 1825. This paper could not be located, though there is no doubt of its existence. In 1827, Orfila published a systematic series of tests for the recognition of bloodstains on various substrata, and their discrimination from rust spots, iron citrate, and a number of red dyes (Orfila, 1827a). He mentioned Lassaigne's work, but without giving a specific reference. Orfila said that, in order not to be accused of scientific plagiarism, he should point out that he had been engaged in this work since 1823. Orfila's tests were based primarily on the solubility of bloodstain components in water, and the behavior of the aqueous extract toward a number of reagents, litmus paper, and so forth. Raspail disagreed that Orfila's criteria were sufficient to establish the identity of blood in a medico-legal investigation, and said that a stain could be constructed from ovalbumin and a red dye which would give a completely convincing set of false positive results using O d a ' s tests. He noted further that there might exist other materials which would give results similar to the ones given by blood. OrEla responded to these objections in a point-by-point refutation which appeared in the discussion cited above (1828a), as well as in a separate paper (Orfila, 1828b). Orfila was con- vinced that he was correct, but Raspail did not accept the refutations as is clear from the discussions in the Royal Academy. It appears that Orfila's opinion on the matter prevailed to a large extent, at least in terms of acceptance by judicial tribunals. In 1835, Orfila and his colleagues J.-P. Barruel and J. B. A. Chevallier oublished a detailed remrt on a case in which they had beer; consulted. Three brothers named Boileau and a fourth man named Victor Darez were accused of having murdered a rural constable named Ho- chet. A number of suspected bloodstains were involved in the case, and the Court was interested in knowing whether cer- tain of these stains were of human or animal origin, and

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).

.%u#book in Fonmic Semlogy, Zmmunology, and BiochemirZry

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 6 5) discussion here, is that of the interconvertibility of the vari- 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-determinedhemoglobin 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-

L -^ -^ --^ -

Blood Iden@cation-Qwal T e a

Table 4.1. Side Chain Structures of Some Porphyrins

Porphyrin Substituents Type 1 Type^ Ill

Etioporphyrin 4 M. 4 E 1.3.5.7-M 2,4,6,8-E 1,3,5,8-M 2,4,6,7-E

Mesoporph yrin 4 M , 2 E , 2 P 13,5,7-M 2,4-E 6,8-P 1,3,5,8-M 2.4-E 6.7-P

Protoporphyrin 4 M , 2 V , 2 P 1,3,5,7-M 2,4-V 6&P 1,3,5,8-M 2,4-V 6.7-P

Coproporphyrin 4 M. 4 P 1,3,5,7-M 2,4,6,8-P^ 1,3,5,8-M 2,4,6,7-P

U roporphyrin 4 A , 4 P 1,3,5,7-A 2,4,6,8-P 1,3,5,8- A 2,4,6,7-P

Deuteroporph yrin 4 M , 2 H , 2 P 13,5,7-M 2,4-H 6.8-P 1.35.8-M 2.4-H 6.7-P

Hematoporphyrin 4 M , 2 H E , 2 P 1.3.5.7-M 2.4-HE 6,8-P 1,36,8-M 2.4-HE 6.7-P

J

Abbreviations used in the table: Numbering corresponds to Fig. 3.2. M- methyl E- ethyl P- propionic acid V- vinyl HE- hydroxyethyl A- acetic acid H- hydrogen

rihemoglobin). Methernoglobin does not bind oxygen, but will bind a number of other ligands, such as hydroxide, cyanide, azide and nitrite (Kiese, 1954). Figure 4.6 sum- marizes the interrelationships between the various deriva- tives (Lemberg and Legge, 1949; Pritham, 1968).

4.21 Introduction. Parkes (1852) reported that, in examining micro- scopically the residual matter in a bottle which had con- tained partially putrefied blood, had been rinsed with water, and allowed to stand for a time, needle-like crystals could be

observed in abundance. These crystals were insoluble in

water and in strong acetic acid, but soluble in what I pre- sume to be KOH. The crystals could be reprecipitated with strong acetic acid, but were less satisfactory and less abun- dant than the original crystals. Parkes noted that around this same time Funke had reported similar crystals from blood- water mixtures using horse spleen blood and fish blood. Drabkin (1946) has suggested that Funke may have been looking at hemoglobin crystals. Kalliker (1853- 1854) re- ported that he had observed crystals in dog, fish and python blood in 1849. These were soluble in alkali and in acetic and nitric acids, and he said they were identical to Funke's crys- tals. Parkes subsequently attempted to prepare crystals similar to those which he had discovered by accident, but did

not again obtain themin the same quantity. He did note that

a number of different types of crystals are obtainable from putrefyins blood, but could not identify them. He did not think they were identical to the hemoglobin crystals of Vir-

chow nor to the albumin crystals of Reichert.

The preparation, microscopical and spectros~opicexam- ination of crystallhe forms of hemoglobin derivatives have occupied a great deal of attention in the development of

methods for the medico-legal identification of blood. Many

methods have been devised, and most are b a d on the prep

aration of either hematin or hemochromogen crystals. Many

authorities have regarded crystal tests as methods of cer-

tainty in the identification of blood in stains (Beam and

Freak, 1915; Brunig, 1957; Bertrand, 1931; Casper, 1861; Chidi, 1940, Derobert and Hausser, 1938; Gonzales et al, 1954; Guarino, 1945; Lopez-Gomez, 1953; Lucas, 1945; Mueller, 1975; Olbrycht, 1950; Rentoul and Smith, 1973; Schleyer, 1949; Smith and Fiddes, 1955; Sutherland, 1907). Others who have considered the crystal tests in detail have been less explicit about the issue of whether the tests should

be considered certain or not (Ziemke, 1938; Kirk, 1953).

Dalla Voh (1932) took the position that the microscopical methods used to examine the crystals in routine forensic

practice were inadequate to insure proof. Rather more

sophisticated crystallographic analysis than would be routine-

ly practical would be needed, in his view, to establish with certainty the presence of blood by these methods.

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. t w o 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

Soumebook in Forensic Serologv, Immunology, and Biochemistry

According to Oustinoff (1929), who advocated the iodide crystals, Strzyzowski had prepared hematin iodide in 1902. Guarino (1945) employed iodoform in alcohol for the prepa- ration of the crystals, and Lopez-Gomez (1953) reviewed, in some detail, the use of bromide and iodide salts in hematin crystal test reagents. Lopez-Gomez and Cantero (1942) are said to have carried out systematic studies on bromide and iodide crystals. Gouillart (1939) studied the formation of hematin iodide crystals in great detail. There is some con- fusion in the literature regarding the preparation of hematin fluoride crystals, Welsch and Lecha-Marzo ( 1912a) seeming to advocate their use, while Leers ( 1910 and 1912) believed that other halogens were greatly preferable. In any case, there does not seem to have been much subsequent use of fluoride-containing reagents. A number of authorities have recommended solutions containing O.lg each of KC1, KBr and KI in 100 mL glacial acetic acid for the production of Teichmann crystals (Nippe, 1912; Kirk, 1953; Smith and Fiddes, 1955). Fiori (1962) said that there is no particular advantage to preparing hematin crystals froq halogens other than chloride. An extremely thorough study of the formation of both hematin and hemochromogen crystals was published by Mahler in 1923. He studied bloods from different species, including human, mostly as dried stains under a variety of different conditions and using a number of different meth- ods. In these expriments, Mahler compared among other things the method of Wachholz ( 1901 ), utilizing alcoholic solutions of strong acids, with that of Nippe (1912) which called for a solution that is 0.1% (w/v) in KI, KBr and KC in glacial acetic acid. Mahler got his best results from a combination of the two methods, wherein the blood-stained fragment was first warmed with alcoholic glacial acetic acid, and then warmed again following the addition of the Nippe reagent. A similar set of comparative experiments was done by Kerr and Mason (1926). Some of the methods they studied were the same ones that Mahler had considered, but there were some differences. They preferred the method of Suth- erland (1907), according to which a drop of saline is evapo- rated by heating on a clean slide, the stained fragment then being placed on the residue, and a drop of glacial acetic acid added. After applying a cover slip, the preparation is heated gently until bubbles just appear. It is then set aside and crytallization allowed to proceed. Blood or bloodstains heated to temperatures in excess of 140' to 145' will not yield Teichmann crystals (Katayama, 1888; Hamrnerl, 1892; Wood, 1901). Be11 (1892) discussed the hematin test in his review, and described the techniques then being used in this country by Formad (Formad, 1888) and by Prof. Tidy. Wood (1901) discussed his own experi- ences with the test in a paper read to the Massachusetts Medico-Legal Society. Any substance or condition which causes hemoglobin or hematin to form its decomposition product, hematoporphyrin (iron free hematin), he said, will interfere with crystal formation. Heat, long exposure to sun-

light, and some organic solvents often cause dSculty. It is to bi noted, nonetheless, that Muller et al. (1966) reported a positive Teichmann test from bloodstains on clothes that had been dry cleaned. Schech (1930), in his studies of the effects of ironing bloodstains on cloth on the subsequent ability to detect and analyze the bloodstain, noted that he- matin crystals might be obtained if the iron had not been applied directly, but through a wet cloth for example. Rust and exposure, and especially the combination of these, inter- fere with the test (Sutherland, 1907). Among the many modifications of the test that have been proposed, a few others will be mentioned. Oustinoff (1929) thought that the incorporation of gum arabic into the re- agent improved the results. The heating step in the pro- cedure, if done, is very critical. Bertrand (1931) discussed this point, noting that it is possible to err either in the direc- tion of overheating or of underheating. He recommended a solution containing glycerol, apparently to lower the vol- atility of the reagent. Wachholz (1901) recommended a solution of a concentrated acid (lactic, sulfuric or acetic) in 95% alcohol because this boils easily, and reduces the chances of overheating. Sottolano and DeForest ( 1977) have described a technique utilizing Kirk's (1953) solution for hematin crystals which involves placing the test slides on a rack within a pressure cooker. Crystal formation is facili- tated by the increased pressure, and the danger of total evaporation is overcome. The technique is applicable to the formation of hemochromogen crystals using Takayama's solutions (see Section 4.2.4) as well. Beam and Freak (1915) proposed a technique which, they stated, rendered crystal formation much more certain. The essential ingredient of this modification is very slow evaporation. The material to be examined is placed in the bottom of a flat, arsenic sub- limation tube. A few drops of glacial acetic acid, containing 0.01 to 0.1% NaCI, are added, and a fine cotton thread is placed in the tube such that its lower end contacts the solu- tion and its upper end is near the top of the tube. The thread is moistened if necessary to insure that it is everywhere in contact with the side of the tube, and evaporation is allowed to proceed at its own rate, without heating. The process which may require from 12 hours to more than a day, is accompanied by capillary movement of the solution within the thread, the crystals forming along the thread's length. This technique, recommended by Lucas (1945), is capable of giving large crystals suitable for crystallographic analysis (Fiori, 1962), but may not be very practical for routine work because of the investment of time required. Although a number of materials and conditions interfere with the formation of hematin halide crystals, the age of the stain alone does not seem to be deleterious. Haseeb (1972) got a positive Teichmann test on a 12-year old stain kept on the laboratory bench in the Sudan. Beam and Freak (1915) obtained crystals from 10-year old human and 12-year old ovine bloodstains, as did Mahler (1923) from human stains over 20 years old. Dervieux (191 1) mentioned that he had obtained hematin-iodide crystals from a 4000 year old bloodstained cloth from a mummy.

Blood Identifin-Oys?al Tests

4.2.3 Acetone cblor-hemin crystal test

In 1935 Wagenaar recommended the preparation of ace- tone chlor-hemin crystals. A few drops of acetone are added to a fragment of bloodstain, followed by a drop of dilute mineral acid. Crystals forrn quickly at room temperature, even when the stains are old or the blood partially putrefied (Wagenaar, 1937). DQobert and Hausser (1938) discussed this technique in their reference book. Chiodi (1940) carried out extensive studies on it with human, and different animal bloods, and stains exposed to adverse conditions. He recom- mended that it replace the Teichmann test. In 1949, Schleyer showed that the test could detect as little as 2 to 8 pg hemoglobi, and that methemoglobin and putrefied blood would give a positive test. Hematoporphyrin and blood heated above 200' do not give the test. Apparently, crystals are obtained from bile as well (due perhaps to the bilirubin content), but not from the urinary or fecal pigments (proba- bly stercobilin, urobilin and urochrome) (Schleyer, 1949). Stassi (1945) reported that the test did sometimes fail in the presence of blood. While the Teichmann and Wagenaar crystal tests are at least valuable, if not conclusive, tests for the presence of blood in stains, the crystals are not always easy to obtain. Even in experienced hands, these tests sometimes fail in the undoubted presence of blood (Sutherland, 1907; Corin, 1901; Dalla Volta, 1932; Olbrycht, 1950; Stassi, 1945; Mahler, 1923).

4.2.4 Hemochromogen crystal test Hemochromogens are those compounds of ferroporphyrin (i.e., Fez+) in which the iifth and/or sixth positions of the hexacoordinate complex are occupied by the N atom of an organic base, such as pyridine, histidie, pyrrolidine, or var- ious amines. Hemochromogen was first prepared by Stokes in 1864. He obtained this material, which had a very characteristic s p e ~ trum by treating hematin with a reducing agent in alkaline solution. Stokes called the substance "reduced hematin." Hoppe-Seyler took up a number of further studies on the pigment finding, among many other things, that it bore as close a resemblance to hemoglobin as it did to hematin. He proposed that it be called "hemochromogen" (Hoppe- Seyler, 1877,1879), this name having essentially supplanted that given the compound by the original discoverer. In 1889, HoppSeyler prepared crystals by exposing hemoglobin to 100° temperatures in basic solution. These crystals have often been referred to in the literature as the first example of

hemochromogen crystals. Leers (1910) noted that Hoppe-

Seyler's student, Trasaburo-Araki reported obtaining simi- lar crystals in 1890. Gamgee (1898) said that HoppeSeyler had not in fact obtained hemochromogen crystals, as had becn reported in the textbooks for a number of years: It is quite erroneous to state, as is asserted in all text- books, that Hoppe-Seyler succeeded in separating haemochromogen in a crystalline condition. He only succeeded (at most) in obtaining crystals of the CO-

compound, and concluded that haemochromogen itself must be a crystalline body, but he never asserted that he had actually obtained the crystals, and a promise made in 1889 to describe the assumed crystalline haemochro- mogen, though implying that he had already obtained the body in this condition, was never fulfilled. More- over, in the last systematic account of haemochromogen which he published in 1893 [Hoppe-Seyler and Thier- felder, 18931 Hoppe-Seyler does not refer to its being crystallime, but, on the contrary, speaks of it (as he had done in 1889) as separating in the form of a violet-grey powdery precipitate. Copeman (1890) observed that hemochromogen crystals forrn from hemoglobin crystals upon long standing. Menzies (1895a) noted that allowing blood to stand in a water bath for some days in the presence of the chloride, bromide or iodide salts of potassium gave rise to a substance which could be converted to hemochromogen upon addition of a reduc- tant, (NHJ2S. He further showed that ammonium sulfide would convert acid hematin to hemochromogen (Menzies, 1895b). Hllfner (1899) used hydrazine hydrate to convert alkaline hematin to hemochromogen. Donogany (1893a) working in Budapest was the first in- vestigator to note that pyridiie reacted with hemoglobin to form hemochromogen crystals, and that these formed within a few hours if the material was placed on a microscope slide and sealed with a cover slip. He showed that this reaction occurred with dried blood, and suggested its application to the problem of medico-legal blood identification. In a sub- sequent paper (1 897), Donogany suggested that the pyridine hemochromogen test would be useful for the determination of blood in urine, noting at the same time that he had first published hi observations on pyridiie hemochromogen in the Hungarian literature four years earlier (Donogany, 1893b). Hemochromogen crystals may be obtained from either acid or alkaline solutions. Fiori (1962) stated that crys- t a b t i o n from acid solution was first carried out using acetic acid, pyridine and pyrogallol (1,2,3-benzenetriol), quite possibly a reference to the papers of Welsch and Lecha-Marzo (1912) or of Lecha-Marzo (1907), the latter of which was cited by Gisbert Calabuig (1948) who pro- posed the use of an improved solution: 0.5 m4 glacial acetic

acid, 1.5 d ,pyridine and I mP 2Vo ascorbic acid.

It is far more common in the literature, however, to find descriptions of crystallization from alkaline solution. The older methods consisted of treating the stained material with

pyridine and ammonium sulfide (Bbrker, 1909; Dc Domini-

cis, 1902 and 1911), following upon the work of Menzies (1895a). Lochte (1910) suggested a modified version of the reagent containing NaOH and alcohol. Klirbitz (1909) and apparently Lecha-Marzo (1908) recommended extraction of the stain with alcoholic iodine solution prior to adding the

pyridine-ammonium sulfide reagent. Leers (19 10) used this

method, but apparently employed an aqueous iodine solu- tion. Alkaline solutions of pyridine containing hydrazine hy- drate (Mita, 1910) or hydradne sulfate (De Dominicis,

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 SO 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 i n g virtu- ally indistinguishablefrom 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 /mP gave 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 concentrationsof 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. It 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 is alwaysnecessary 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 (1870) 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

Blood Zdent@ation-Spectml and Mkmxopical

WAVELENGTH, A. 10000 9000 8000 7000 6000 5000 4000 3000

WAVELENGTH, A.

• Oxyhemoglobin -,-.-.-^ Carboxyhemoglobin -----. Hemiglobin - Hemiglobin cyanide

............ Hemoglobin

Figure 5.1 Absorption Spectra of Some Hemoglobin

Derivatives (after Lemberg and Legge, 1949)

(Reprinted by permission of lnterscience

Publishers, Inc.)

Soumbook in Forensic Ser010gy.Immunology, and Biochem&v

1

2

3

4

5

I

OXYHEMOGLOBIN (Varying Concentrations)

HEMOGLOBIN

6 CO-HEMOGLOBIN

7

8

I

ALKALINE HEMATlN (Dilute and concentrated)

9 HEMOCHROMOGEN

10 METHEMOGLOBIN

11 ACID HEMATIN

12

13

14

! O 1! l 0 I ..II ' I "0 I

I

1 o .I 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.