Human Blood Identification: A Forensic Science Approach Francis X. Sheehan and Lawrence Kobilinsky John Jay College of Criminal Justice, City University of New York, 445 West 59th St., New York, NY 10019 The high school level forensic science elective in criminalistics has become more common in recent years. The teaching of criminalistics requires a thorough understanding of the nroblems inherent in the examination of nhvsical evidence and how the scientific approach is used toidentify and individualize (determining, if possible, the source of) the material under study. I t is most important that appropriate experiments be chosen so that the student will appreciate not only the techniques used hut the principles behind them. These experiments must be: (1)easily completed in a relatively short time, (2) safe for both students and faculty, (3) economical, and (4) well desiened. ntilizine sensitive and accurate meth---.-a
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In this DaDer we present a series of exneriments in which a stained fabric is analyzed to determineif blood is present, and if so, whether it is of human origin. These exneriments can be perf&med without expensive a d sophisticated equipment and require only a minimal expenditure on consumable supplies. The cost per student working individually is estimated to be $2.00. If the dried blood standards and reagents are prepared in advance, these experiments can be performed in five 45-min sessions, but it is recommended that more time be scheduled if feasahle. Because these experiments involve serological and immunological principles and techniques, i t is strongly advised that the instructor have some familiarity with serology andlor practical experience with such experiments as these before emnlovine . . -them in the classroom. Resuits can easily be misinterpreted by an inexperienced individual. Background
Presum~tiveTests When a stain is received for laboratory analysis it is first subjected to a very sensitive, but relatively non-specific, presumptive test to determine if the stain in question might be hlood. Presumptive blood tests are zenerallv catalvtic tests that involve the use of an oxidizing reagent, such as hydrogen peroxide, and an indicator that changes color (or luminesces) when oxidized as a result of the peroxidase-like activity of hemoglobin. Some of the best known tests include: leucomalachite green (LMG), phenolphthalin, and luminol. The reduced forms of these compounds are colorless, whereas the oxidized products are not. Because the LMG and phenolphthalin tests are quick and sensitive, they are generally nerformed either at the crime scene or in the laboratow. The iuminol test is most often used in field investigation. ~ a g test h takes onlv about 30 s to ~erformand the result is easilv visible to the unaided eye. ~ i n E hemoglobin e acts as a catalyst, only a verv minute amount of samnle is necessarv for the reaction to prbceed. As a general rule, if the stain is [isible, then there is nrobahlv more than enouzh samole to obtain a reaction. In thk case where sample s i z e s extremely limited, observation can be performed usinz a stereomicrosco~e.Kirk renorted a positive phenolphtha1;n test within 3 s, with a blood sample which had heen diluted 1:lOO.OOO ( I ) . Phenolphthalin can be produced by refluxing phenolphthalein in a strong alkaline solution in the presence of excess zinc until the pink alkaline phenolphthalein solution fades and 542
Journal of Chemical Education
becomes colorless. This test has often been incorrectly called the phenolphthalein test. I t is recommended that both an alkaline and an acidic presumptive test be performed on separate portions of a suspect stain to rule out pH effects. Phenolphthalin reagent is, as described above, prepared in an alkaline medium, whereas LMG reagent is generally prepared in a solution of glacial acetic acid and water (21). Consequently, phenolphthalin and LMG reagents can be used as complementary presumptive tests. A positive presumptive test indicates only that hlood may be present, and not that it actually is nresent. Unlike the LMG reagent which can be prepared in ahout 5 min. the nhenulnhthalin reaeent can take as long as 4-5 h to reflux. since crime laboratories need only prepare thereagent onceevery few months to maintain an adequate supply, and since the reflux requires minimal attention, its preparation time is not a real drawback. Furthermore, the phenolphthalin reagent is now commercially available. Another alkaline presumptive test, the luminol test, may be employed as a substitute for, or in conjunction with, the phenolphthalin test. An alkaline solution of luminol will chemiluminesce when it becomes oxidized. The mechanism of the reaction, which is greatly enhanced in the presence of blood, is unknown and several investigators have questioned whether hemoglobin derivatives act as catalysts or as "accelerators" to produce the intense blue luminescence. This reagent can easily be prepared in about 10 min before the class begins. The test must be conducted in the dark in order to observe the resulting luminescence. An adjoining closet or, if available, a "viewing box" which can be built from a cardboard carton, would be appropriate for testing. Prooer Controls I t is important that the students be made aware of the need for . Droner . controls. as well as how tolimit the number of false reactions. For example, false negative results may occur if a strong reducing agent, such as ascorbic acid, is present on the substrate being tested. Similarly, false positives may occur in the presence of strong oxidizing agents or vegetable peroxidases. When the luminol test is used it should be remembered that if a stain is on material which contains whitening agents (which are common in detergents) the stain may appear to fluoresce, resulting in a false positive interpretation. The effect of false positive and false negative observations on the interpretation of the results can be minimized if proper controls areused. The presence of a strong reducing agent may be discovered by adding a known blood sample to a portion of the unstained material, allowing i t to dry, and-noting whether a false negative results after testing. The presence of strong oxidizing agents may be determined by adding the peroxide-free presumptive reagent first and noting whether or not a nositive reaction occurs at this noint. If a (false) nositive rea'ction occurs, the test is invalid:~f no visible readion occurs until the hydrogen peroxide is added in a subsequent step, can i t then be concluded that the stain contains blood? Not necessarilv. Veeetable neroxidases can nroduce false positives which cannot be overcome by using this two-step
nrocedure since neroxidases are enzvmes which can catalvze bxidations with 'hydrogen peroxide: Fortunately, vegetible peroxidases are easily inactivated by heating the sample prior t o testing. Of course, positive and negative controls are essential in forensic lahoratorv work. because the historv and treatment of submitted evidentiary samples are usually completely unknown. Confirmatory Tests
Because many substances, organic and inorganic, will react with the test reagents to give false positive results, the criminalist should confirm the presence of blood whenever possible usine a much less sensitive, but more specific, test. Two such test;are generally utilized, namely thk Teichmann and the Takayama crystal tests. One of the oldest confirmatory tests for the presence of hlood in a dried stain was proposed in 1853 by L. Teichmann (2). He found that hlood that had been desiccated and then heated with concentrated acetic acid developed characteristic crystals, which he called "hemin." Hemin is the crystalline chloride of heme, the prosthetic group of hemoglobin. Nippe modified the original method by adding potassium halides to the acetic acid reagent to facilitate crystal formation (3).It is the Nippe modification that is now recognized as the Teichmann (hemin) test. Teichmann suggested that hemin crystal solubility be tested to compliment the morphological ohservation of crystal formation. The analvst comnares the nroduct crvstals from a suspect stain with the characteristic crystalline heme derivative obtained with a known blood samnle Fir. . (see . - 1). . The optical properties of these crystals are examined with a polarizing microscope. Teichmann crystals are anisotropic, pleochroic, and exhibit symmetrical extinction. If a polarizing microsco~eis not available, these properties can he verified with an &dinary light mic~oscope~eq"ipped with a rotating staeeand adanted with a ~olarizinafilter set. Pleochroism is theueasiest characteristicto determine with a standard microscope since one needs onlv rotate a piece of polarizing material below the substage cindenser add note whether the color of the crystals change as the plane of polarized light is rotated 90'. The crystals formed in the hemin test are small and sometimes difficult to locate, and thus the analyst may at times report a ialse negative ohservation. Another method was developed in which a crystalline heme derivative, pyridine hemochromogen, is formed. These crystals were first produced in 1893 bv Zacharias Donoeanv ( 4 , . He mixed eaual amounts of defihXnated blood a n i p&dine, and thenieduced the hlood with ammonium sulfide. Even thoueh it was Donoeanv who suggested that pyridine hemochromogen he used f i r medico-leeal " nurnoses. . . i t is Takavama's name which is now used synonymously with hemochromogen crystal testing. Takavama's reaeent. . unlike all other hemochromogen reagents, contained glucose. The suspect stain is broken into small fraements on a microscope slide, treated with 1-2 drops of reagent, covered wlth a coverslip, and heated until hubbies herin to amear. If hlood is present, the stain will change color from hro& to red and, within minutes after cooling, begin to crystallize (see Fig. 21. T o prevent evaporation of the reagent during heating, Sottolano and De Forest recommend heating the slides in a pressure cooker ( 5 ) .Other investigators have ~uggestedmo&fying the Takayama reagent to make it less volatile. However, the proper technique of heating a slide can easily be mastered without these modifications to produce excellent crystals each time. Students exnerimentine with ammouium sulfide (instead of glucose) may find that its use can result in more false negatives due to the vellow color of ammonium sulfide which can mask the cherry-red color of the hemochromogen crystals. The Takayama test is perhaps the best crystal method availahle today for routine hlood identification for it yields ~
~
positive results where other methods, such as the Teichmann &st, fail. Unfortunately, even this test is not without fault. Confirmatory tests, in general, are time-consuming- (approximately20 min per sample) and require a fairly large amount of sample. In addition, a microscope is required to observe the result; therefore, this test is usually conducted in the laboratory rather than at the crime scene. An exnerienced criminalist will take about 30 min to oerform h i h the presumptive and confirmatory crystal testibut students will reouireahout 45-60 miu. If hoth tests are nositive, then the species of origin of the hlood becomes of primary imnortance. Manv defendants. when confronted with the pr&minary testing results, will claim that the stain examined was animal hlood and not human. A species test must always he performed even if the blood will subsequently he typed because, although a stain is typed in the ABO blood-grouping system, this does not necessarily mean that the hlood is human. ~
Species Test
As early as 1901, Uhlenhuth in a series of articles (6-10) reported that the hlood of different species could he differentiated with certainty by testing hlood samples with species-specific antisera. The antisera were prepared by introducing foreign hlood (antigens) into an animal, allowing sufficient time for the animal to produce antibodies against the foreign antigens, and then bleeding the animal to collect the antisera-containing hlood. If a portion of this antiserum were placed in a test tube with homoluguus serum (even if the serum were diluted 1:1000), a cross-lmked cmnplex consisting of specific antihodies and antigens would form, resulting in a precipitate. A moditicatiun of this procedure is periurmed hy layering diluted hlood over the dvnser antisrra in a test tube and after a short period of time determining whethrr a ring of precipitation occurs at the interface. The formation
Figure 1. The anisotropic, pleochroic hemin crystals of the Teichmann test.
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Figure 2. The pyridine hemchromogen test.
crystals formed
in the Takayams
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543
of a nrecipitate indicates that antibodies within the antiserum recoinized and reacted with the antigenic components within the hlood, thus the species identity is established. This technique is known as the precipitin ring test. It takes about 20 min and is commonly employed using capillary tubes rather than test tubes to conserve the expensive antisera. Unless time is extremely critical, the antigen-antihody rectiun should he sunductnl on a gel medium as Ourhwrlony pruposed ( 1 1 ) . Wells are cut in the gel at approximarely 1.5 nlni intevsls. The antieen (diluted hlwd samnle) is nlacrd in~onewell and the antihoiy in an adjacent well. he adtigens and antibodies can diffuse toward one another and. if honrologuus, wlll form a precipitate. Controls can be tested slmultnneuusly on the snme gel as illustrated in Figure :$.The hands o f precipitatiun that are iormed indicate the presence the or absenn! ot'o sveclf~cantigen. As Virure 4 illustrat~~ri, can indicate whether two antigens are precipitin identical or not. The presence of antigens with partial identity can also he observed using this technique. The Ouchterlony test is completed in about 24 h, hut the actual "hands-on" time required to perl~mnthis procedure is unly slightly longer than the ring test. i'unsrquentlg, a e recommend that t h Oucht~ erluny procedure be employed after blwd has I w n idenrified. 'I'his test is rchtively iinlple to periorm and highly i n t i ~ r ~ n a tivr. However, the student whohas tot hren exposed to in,. nlul~ulogi~al concepts such as antigens, antibodies, and ilnm m e c~mnlexeswill not understand or benefit from oer~~~~~~tor~ningthis test. A lessm on the principles l~ehindthis test should he pnwided to the students hefore the laburatury session begins ~~
~
~~
~
~~~
.
~~
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known human blood
n
sohe (blank)
0n 0 w
a -human
sera
known rabb~tblood
V
substrote extract Figure 3. Ouchterlony gel illustratingthe precipitin panern that distinguishes a sample that is known to be blood from an unknown, but suspect,stain labeled "A,"
Unknowns If an unknown is to he sundied .. to the students it should he a stain -3 mm in diameter on a substrate such as cotton. An old lahoratorvcoat can be cut into swatches for this numose. The student; should learn and develop their tech&ues on known samnles until thev -reauire . as small a sample as ~ o s s i ble. It is always easy to perform a micro-technic& on ;large s a l n ~ l ehut it is far more difficult to adapt a macro-technique to ahma11 sample. Additional time wili he required for siudents to achieve nroficiencv with minute samples. The unknown should he ceated as if it were evidence which, if lost, destroyed, or consumed in the analysis, cannot he replaced. A student asking his instructor for another sample is equivalent to a forensic scientist asking the criminal to recommit a crime in order to produce more evidence, and therefore this practice should not he permitted. The sample should he placed in an envelope, initialed, dated, and sealed with clear tape, and then given to each student. The students in return for the "evidence" should submit an "evidence receipt form" which the teacher should collect to maintain the inteeritv - .of the "chain of custodv." The student can remove the rv~denceby carefully cutting a new opening in the e w e Iopr with scissors. He or she wuuld then initial and date the opminp. After the analysis is completal, the rwnnining purtlon 01 the samule is returned to the mvelone which is then sealed with clear tape. U ~ o nthe return of the re-sealed envelove, the instructor sho"ld return the signed evidence receipt tlirrn tothe student indicatiw that the evidencc hns been returned. T k form should bitaped into the student's notebook on the last page of the report. If the student intends to use the entire sample during the analvsis a "reauest to consume sample" form should he suhmittid hrfnre ihe analjsii is conduc&d to comply with rulings such as Brndy versus Maryland (373 1'583) and U.J.versus Bryant (439 @.2d643) where the court suppressed the results of an analysis after the prosecution knowingly failed to preserve a questioned sample (or failed to disclose their inability to do so). Such a request by the student should not be easily granted for the courts have clearly decided that every attempt must he made to preserve discoverable evidence. The student should exnlain clearlv un the reauest form whv the samnle. . . albeit small, must be completely consumed. Cautionary Note: Because of the potential biological hazards which accompany hlood analysis, it is strongly recommended that all students he provided with disposable latex gloves and that unused bloodstained materials be autoclaved and properly disposed. In addition all students should he instructed regarding safe technique in handling hlood and/or serological reagents such as typing sera, especially when they are derived from human sources. Safety goggles should he used while conducting these experiments.
Materials IAI
ICI
I Dl
(El
Figure 4. The precipitin patterns that resun from antigens in the upper wells: A) identical, B) partially identical. C) partially identical. D) nan-identical. Panern E is mare complex and results from a combination of the reactions illustrated in A and 0.
544
Journal of Chemical Education
Reagents to be Prepared
Phenolphthalin reagent. Add 2.0 g phenolphthalein (CzoHlsOn), 20 g potassium hydroxide, 20 g powdered zinc, and 100 ml distilled water tu a 250-ml (or larger) round-bottomedflask which has a ground glass top. Place two water-cooled condensers on top, and reflux using a heating mantle for 3 4 hours or until the solution becomes colorless. This stock solution should be stored in the refrimrator in a dark
dent. Luminol Reagent. Add 0.1 g luminol (5-arnino-2,3-dihydr0-1.4phthalazinedione) and 5 g anhydrous sodium carbonate to 100 ml distilled water, stir to dissolve, and dispense into dropper bottles, alluwing 3 ml per student.
Leuco-Malachite Green Reagent Prepare 66% aceticacid hy diluting66 ml of glacial acetic acid with 33 ml of distilled water. (For convenience large batches can he prepared and ran be stored indefinitely.) Refore class, prepare 0.1% (wlv) leuco-malachite green (p.p'-henzylidenrbis-(N.N-dimethylaniline)) in 66% acetic acid. Dispense into dropper bottles, allowing 3 ml per student.
Teichmann Reagent Add 0 1 g p t a + i u m < hloride. 0 I c ~r,taithr naked qr.olnainn pierr
