Human Blood Typing: A Forensic Science Approach Part II: Experiments Lawrence Kobilinsky and Francis X. Sheehan John Jay College of Criminal Justice, City University of New York, 445 West 59th Street, New York, NY 10019
In the previous article, we described the methodology available to the forensic seroiogist for typing a human bloodstain in the ABH rrounine svstem. In this artirle wedescribe several e x p e r i m e k t i a t k k t r a t e some of these techniques. These exneriments are easilv comnleted in a reasonablv short time providing that tb;studeLts are adequately pared by lecture material and readings. They are also economical, and equipment requirements are minimal. Old, "outdated", hlood can often he obtained from a hospital. A transmitted-light microscope, although not require& would be helpful for instructional purposes. Overview Students should first become familiar with the method of ABO blood grouping and be able to recognize the hemagglutination reaction. This will reauire small ouantities (0.2 mL) of wet blood and antisera (anti-A,anti-H,and anti-H Iectin). The simnlest method. using a elass microsco~eslide ( 3 in. X 1 in.) al;d toothpick to mix the reagents, will he described helow. The student should also be a i \ w a wet blood samule for h i m h e r to blood group. T h i s l o o d sample should'be supplied in a small test tube that is appropriatelv coded. .. If the student successfully drterm&s ;he hlood group of the unknownsample, the instructor s h d d thensupply him/ her with a pre-coded blood-stained cotton swatch. ~ h e s e stains should be approximately 3-5 mm in diameter. Stains for the Lattes test should be prepared not longer than two weeks prior to analysis. Stains prepared for the absorptionelution test have no such limitation. The swatches of cotton fabric can he obtained by cutting out squares (1 in. X 1in.) from an old lahoratorv coat. The stained swatch should be placed into an envelope, sealed, dated, and initialed by the instructor. After the student is supplied with the envelope, helshe should make out an "evidence receipt" indicating the time and date that the envelope was received. This will maintain the integrity of the "chain of custody". The student should be informed that additional "evidence" will not he supplied and that if the sample is lost, or consumed, the analysis is over. The reason for this requirement is that nhvsical evidence must be nreserved whenever nossible so that additional analyses caihe performed by anoiher scientist (Brady versus Maryland (373 U S . 83) and U S . versus Bryant (439 F.2d 643)).' When the experimental analysis is completed, the student should return the remaininp portion of tGe swatch to the same envelope, seal itwith tape;sign and date the envelope, and return i t to the instructor. The instructor should ihen return the receipt form to the student. Thestudent should include the form with his written report. If the student feels that the entire sample must heconsumed, this should be reported to the instructor before it is used. Throughout the analysis, unknowns should be treated identically to controls. The experiments presented below are based on forensic principles of analysis and on the scientific method. The instructor may wish to omit some parts and to stress others
' Sheehan, F. X.: Kobilinsky, L. J. Chem. Educ. 1984, 61,542. 624
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to suit the needs of h i s h e r students. Cautionary note: It is strongly recommended that all students be supplied with disposable, high-quality (8-mil gauge), latex surgical gloves and that all unused blood samples, fresh or dry, he autoclaved and disnosed of nrooerlv. This will minimize the potential biohazard that ~ I W &companies ~ ~ S blood analysis. We further recommend that all students he instructed in safe techniques prior to handling blood, typing sera and other serological reagents. Contact with blood and blood products should always he minimized since infectious agents may he present. This principle should he adhered to even if the~hloodhas been presrreened for pathogenic agents by the hlood hank or hospitul serology lahorators. The experiments should he performed on a laboratory bench covered with a disposable absorbent diaper, and after completion of the experiment the table top should be cleaned with a strong disinfectant.
Reagents To Be Prepared Physiological saline is prepared by bringing 0.875 g of NaCl to a total volume of 100 mL with distilled water. This concentration of NaCl is isotonic with erythrocyte contents. Phosphate-bufferedsaline (PBS),pH 7.2, is prepared by addition of 5.68 g NalHPOa (dibasic)and 2.72 g KHPPO,(monobasic)to 2.5 L of saline. The pH is brought to 7.2, and saline is used to bring the final volume to 3 L. Materials Obtained Commercially A n t i - A and sn1r.B hlood typing serum. O~ganonTeknika Curp fHCA1. One Technuhg) Court, hlolwrn, P A 1'1355, catalog q T ' 1 118 and 7-1-128, resot~tivel\,.Alternate source: Onho lliaenostic Systems, Inc., R&&, NJ 08869. Ulex europaeus lectin to the H antigen, Organon Teknika Corp. (addressabove),catalog #771-152. Alternate source: Sigma Chemical Co., St. Louis, MO 63178, catalog #L5505. Reference cells (Al, As, B, AB, and O), Organon Teknika Corp. (address above);request Confirmcells, catalog #303-429 and Spectrogen pooled 0-type cells, catalog #303-739. Alternate source: Ortho Diagnostic Systems, Raritan, NJ 08869. Request Affirmagen A and B cells and Selectogen type-0 Cells. Serological test slides, Hema Slides #I, Cat#hts-001, Bionea Medical Industries, 1716 Ocean Park Blvd., Santa Monica, CA 90405. -~~~~~
Microscope slides, coverslips, lens paper, wax china marking pencil, Pasteur pipets, Fisher Scientific, 52 Fadem Rd. Springfield, NJ 07081. Cotton thread, No. 8, mercerized. Nail polish or non-water-soluble glue. Equipment Required Incubator set at 56 'C. Transmitted-lieht microscoue of magnification of 400X. . caoable . Table-top clinical centrifuge. Orbit shaker (optional).Labline Instruments Ine., Melrose Park, 11.~
Refrigerator.
ABO Blood Grouplng of Wet Blood Grouping of wet blood in the ABO system is generally performed in one of two ways, namely the slide method or the test tube method. In both cases hemagglutination results when antieens and antibodies form associations known as lattice formations. The observation of bemagglutination appears slightly different, however, in the two situations. In the slide method, three standard microscope slides are cleaned and labeled "A". "B", and "0". (Depression well slides may also he used.) A circle about 1.0 cm in diameter is drawn in the middle of each slide with a wax china marking pencil. The blood sample to be typed is slightly diluted with physiological saline and washed by centrifuging a t low speed tonellet thenarticulate comnonents of the blood. The suoernatant is discarded, and saline is added to the cells such that the final concentration is ao~roximatelv3-5% of the initial concentration. The color ofthe solution\ill appear pinkish. Alternately, a single drop of blood is obtained by finger puncture with a sterile lancet and is added to 1.0 mL of saline. A single volume of this "unknown blood" is placed in the center ofthe circle on each of the slides. An equal volume of antiserum specific for the A antigen is added to the hlood on slide "A",and using a clean pipe.an equal volume of antiserum specific for the B antigen is added to the blood on slide "B". To the hlood on slide " 0 is added an eaual volume of Ulex europaeus extract containing the lectin {hat specifically recognizes the H antigen. Care must be taken so as not to cross-contaminate the slides. Reference blood s a m ~ l e (4-6% s v/v) that can be commercially obtained may be used for positive controls. If the reference samples show signs of bemolysis (erythrocyte damage due to transport or aging), prior to use the blood sample should be washed by low-speed centrifugation followed by addition of physiologic saline and gentle mixing. This washing should be performed twice. The purpose is to remove hemoglobin which may have been released by erythrocyte lysis. Excess hemoglobin in solution could coat the cell surface and interfere with antieen-antibodv comolex formation. A volume of each reference blood (50-100 p i ) is treated identically to the unknown blood sample. Hemagglutination with only the appropriate antiserum (or lectin) will verifv that the antisera and lectin reagents - are functioning properly. After the two reaeents have been oioetted into the circle on each slide, the contents are mixed-with a toothpick (use a fresh toothpick for each sample so as to avoid cross-contamination), and the slide is gently rocked and tilted to insure eood mixing. Hemaezlutination should be observed within i-2 min that the antigens present combined with the homologous antibodies. The appearance is similar to clumping of red particulates. I t should be noted that occasionally the agglutination of erythrocytes on the A slide is slower than exoected. This can hannen when the ervthrocytes are from ibe Ap subgroup. ~elis'ofthis type react more slowly than cells of other types. The same additions can be performed in test tubes instead of on slides. After the two volumes are combined and mixed, the reaction is allowed to take place for several minutes and the test tubes are gently centrifuged (1000-2000 rDm for 2 m i d to pellet the particulates. ~ 6 tubes e are removed from the centrifuge, and the tube is carefully tapped to disrupt the pellet. The appearance of the red pellet and the ease of disruption indicates whether hemagglutination has taken place. Table 1lists the expected results of these tests. Lanes Crust Test Procedure The student is given three small crusts that have formed from the same blood sample. Each crust is placed on a clean A coverslio is microscooe slide labeled "A". "B". and "0". placed gently over each crust: ~ e f i r e n c eblood cells (as'described above) are washed in physiological saline and centri-
Table 1. ABO Grouplng 01 Wet Blood by the Sllde Method Anti-A
Blwd Group
Anti-B Antiserum
Antiserum
Anti4
Lenin*
*me+ indicates mat the hemagaluiinatlonreastlon can oocur. bui it is not always Ob8WBd.
Table 2. Expected Results for Lattes Crud Ten Crusts Reference Cells
A
B
AB
0
A, 0
+-
+-
-
-
n
-
+ +
fuged a t low speed to pellet the cells. The washing is repeated two times. Sus~ensionsof A,... B;and . 0 cells are nreoared a t a concentration of 0.1-0.2% in physiological saline. A small volume of AT cells is added to the crust on slide A. Similarly a small vb~umeof B cells is added to the crust on slide B, and a small volume of 0 cells is added to the crust on slide 0: In all cases the cell suspension is added such that the fluid slowly moves under the coverslip by capillary action. The slides should he placed in a humidified chamber kept a t ambient temperature (a Petri dish with a moistened towel placed on the bottom is adequate). The slides should not be touched for approximately 1-2 h. They can be left overnight if necessarv. The slides can now he examined microsconically to ohserve bemagglutination. This test will work onl; with "young" stains, e.g., up to 2 weeks old. The instability of agglutinins (antibodies) within the stain is responsible for this limitation. The expected results are seen in Table 2.
. .
Preparation of Stain Extracts For the absorption-elution test, extracts of unknown stains will have to be made. The simplest and gentlest way to prepare extracts of stains is to cut them into small pieces and place them into a test tube with a minimal amount of icecold saline. Maintain the test tube a t 4 OC overnight. If the stain is very old or resists extraction, then in place of the saline a 5%aqueous solution of ammonium hydroxide may be used. If this is found to be inadequate, then a 10% (vlv) solution of ammonia in ethanol may be necessary for efficient extraction. The ABH antigens are stable to these reagents and conditions. Absorption-Elutlon Procedure This procedure can be conducted in several ways including the test tube and thread methods. The thread method was developed by Howard and Martin in 1969.2 For this procedure, the student will require antisera to the A and B antigens and the Ulex europaeus lectin, which recognizes the H antigen. I t is important to use fresh and potent (hightitered) reaeents for this procedure. The titer refers to the dilution of the antiserum br lectin that can be added to the test ervthrucvtes and still oroduce hernaepllutination. The higherthe titkr of the antiserum, the moreuconcentrated are the specific antibodies in the solution. If outdated or lowtitered reagents are used, then false negative observations may result. The student (or instructor) should prepare 0.5% suspenHoward. H. D.: Martin, P. D. J. For. Sci. Soc. 1969, 9, 28. Volume 85 Number 7 July 1988
625
sions of A,, B, and 0 cells in physiological saline. I t is helpful to use serological slides for this procedure (see Materials section); however, a multi-welled glass plate can also he used. Twenty-one cotton (#a) threads, each 2 cm in length, are prepared. Care should he taken so as not to touch the threads with fingertips since body secretions may contain antigens that could contaminate the threads and confuse the results. The first three threads are allowed to soak in the suspension of A, cells, the second set of three threads is allowed to soak in the suspension of B cells, the third set of three threads is soaked in the suspension of AB cells, and the fourth set of three threads is soiked in the sus~ensionof 0 cells. A fifth and a sixth set of three threads a;e allowed to soak in the unknown stain extract. Unknowns should alwavs he done in duplicate when this method is used. Lastly the seventh set of three threads is soaked in saline. Positive and negative controls are most important and should always be included. The seven sets of threads are removed from the hathing fluid, are allowed to air dry, and are carefully glued to the slides so that each thread is placed in a separate well, and each set is maintained separate from each-other. The ends of the threads can he glued down to the slide with nail polish, hut care should he taken that the main portion of the thread is left untouched. Each set of three threads, once glued to the slide, should he labeled: anti-A, anti-B, and anti-H. (See figure.) Once the glue has hardened and the threads are known to he secure in position, a drop of the appropriate antiserum or lectin is added to the appropriate thread until all threads are covered. The slides are then carefully placed into a humidified chamber a t 4 O C overnight for proper absorption to take place. The next day, the threads are all rinsed with cold saline. This washing procedure is critical and should he done identically for all samples. I t is best to use a wash bottle so that a stream of saline can he directed a t each thread. Some scientists will olace the slide into a beaker of cold saline for 5 min and then repeat the washing proceduredescrihed above. After five or six washes. the rhreads are bloued with microscope lens paper, and ;drop of the appropriate reference ~
626
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Absorption-elution setup illustrating one of Me seven sets of threads.
Table 3.
Expected Results of the Absorption-Elutlon Test
Blwd Group
Hemagglutination wilh known cells A cells B cells 0 cells2
+ symbol indicates mat hernsgglutimtionm y a may not be seen. cells is added to each thread. Thus the thread that was soaked in known A blood and then treated with anti-A antiserum will now have a volume of A1 cells placed on it. The threads are now laced into a humidified chamber placed inside an incubator that is set at 56 O C for 15 min. hes slides are removed from the chamber and either hand-agitated or placed on a low-speed orbital shaker for up to 15min. The slides are examined for hemagglutination at 5-min intervals. The expected results are shown in Table 3.
