Blood Stain Typing by ElectrophoresisAn educational service of:PO Box 752Beaumont, TX USA 77704-0752C310/09(6)

Section 4Final Notes itan III and the Super Z Applicator System are fast, easy and practical forTforensic investigation of many different proteins and enzymes. The examples in this booklet will give the investigative laboratorian specific techniquesfor some assays and the equipment and reagents are easily modified forother uses. Please contact the authors at SEMO Regional Crime Laboratory or HelenaLaboratories if you need assistance or have information to share.Handbook for Rapid Blood Stain Typing UsingElectrophoresis for the Crime LaboratoryPrepared by:R.C. Briner, Ph.D.C. R. Longwell, M.S.For more information, call Helena Laboratories.Toll free 800-231-5663.28SEMO Regional Crime LaboratorySoutheast Missouri State UniversityCape Girardeau, MO 637011

AcknowledgementsThe following students have provided valuable assistance in the development of these methods: Retha (Matthews) Edens1, Alice Abbot2, Randall Webster3, Yvonne (Moll) Matthews4 and Ralph Willis5. We especially appreciate Ms. Donna Pruneau6 for reviewing and updating this manual (February, 1989). These procedures arein use in course FS552 (Blood and Body Fluids), an elective in the Science Division at Southeast Missouri StateUniversity, Cape Girardeau, Missouri. The authors would like to thank Helena Laboratories, Beaumont, Texas,for support of this project. Portions of this work were supported by the Research Council, Southeast MissouriState University, Cape Girardeau, Missouri. We would also like to thank Ms. Patty Butler7 for typing the originaldraft of this handbook.Peptidase A (Pep A)(For Negroids)1. Tank Buffer: Same as for GLO-I and EsD2. Gel Buffer: Same as for GLO-I and EsD3. Starch/Agar Gel: Same as for GLO-I and EsD4. S ample Preparation and Application: Soak dried blood stains for about 15 minutes indistilled water and apply to gel in the same manner as GLO-I and EsD. Run a knowntype 2-1 as a standard.5. E lectrophoresis: 250 volts for 45 minutes. Amperage at the start about 10 mA, at theend about 6 mA.6. Reaction Buffer (Same as PGM reaction buffer)3.64 gm Tris, pH 8.0Dissolve in 500 mL of distilled water. Adjust to pH 8.0 with concentrated HCl.7. Reaction Mixture40 mg L-valyl-L-leucine20 mg L-amino acid oxidase (crude snake venom)10 mg MTT (light sensitive)4.0 mg PMS (light sensitive)10 mL reaction buffer8. V isualization: Overlay 0.2 gm agarose (Sigma Type II) dissolved in 10 mL of reactionbuffer. Heat the agarose until dissolved. Allow the agarose to cool to 55 C before adding thereaction mixture. This is quickly poured over the gel. Incubate the gel for 1 hour at37 C.The phenotypes appear as blue bands on a light yellow background.Figure 13 – Peptidase A (PepA)Anode ( )Application point1. 11 West Adams, Cahokia, IL.St. John’s Mercy Hospital Clinical Laboratory, St. Louis, MO.Hannibal Police Department, Hannibal, MO.P.O. Box 945, College Station, TX.Crime Laboratory, Missouri Southern College, Joplin, MO.SEMO Regional Crime Laboratory, Southeast Missouri State University, Cape Girardeau, MO.Secretary, College of Science and Technology, Southeast Missouri State University, Cape Girardeau, MO.Cathode (–)Sample number12 3 4 56 7 8Electrophoretic patterns of three types of Pep A:Type 1-1: Samples 1, 2, 3, 7, 8 and 9Type 2-1: Samples 4 and 6Sample 5 is a suspected 1 2-1 mix from a vaginal/seminal sample27

Figure 11 – Glyoxylase-I (GLO-I)Table of ContentsAnode ( )Section 1General InformationI.II.III.IV.V.VI.VII.Application pointCathode (–)Sample number1 2 3 4 5 67 8Electrophoretic patterns of three types of GLO-1:Type 1-1: Samples 5Type 2-1: Samples 1, 3, 4, 6 and 7Type 2-2: Samples 2 and 8IntroductionFactors Influencing ResultsSample Extraction and StorageMaterialsElectrophoresis TechniquesResults and DiscussionReferences4555699Section 2Typing of Dried Blood Stains With CelluloseAcetatePart I: IsoenzymesPhosphoglucomutase (PGM)10Erythrocyte/Vaginal/Seminal Acid Phosphatase(EAP/VAP/SAP)12Adenylate Kinase (AK)14Adenosine Deaminase (ADA)16Carbonic Anhydrase II (CA-II) and Hemoglobin18Lactate Dehydrogenase (LDH)21Glucose-6-Phosphate Dehydrogenase (G-6-PD) 22Part II: ProteinsGroup Specific Component (Gc)23Haptoglobin23Figure 12 – Esterase D (EsD)Anode ( )Section 3 Typing Dried Blood Stains Using Agar StarchGelsApplication pointCathode (–)Section 4IsoenzymesEsterase D (EsD) and Glyoxylase 1 (GLO 1)Peptidase A (Pep A)2427Final NotesSample number12 3 4 5 67 8Electrophoretic patterns of two types of EsD:Type 1-1: Samples 1, 3, 4, 5 and 6Type 2-2: Samples 2, 7 and 8263

Section 1General InformationI.Introduction In the past decade, progress in developing methodologies for typing blood factors usingisoenzyme studies has made blood stain evidence one of the most important investigative tools available to forensic scientists. Originally, all procedures for isoenzyme typinginvolved electrophoresis on starch gels. At the present time, four major electrophoreticsupport media or utilized: starch gel, agarose, polyacrylamide gel and cellulose acetate.This presentation is a result of the research performed in our laboratories directed towardadaptation of isoenzyme typing of blood factors by electrophoresis on supported celluloseacetate membranes (CAM). raditionally, the only method for identifying human blood samples in the crime laboratoryTwas typing by the ABO system. Using the ABO system, the most rare blood type encountered is AB, which is found in only 3% of the population. While the percentage of personshaving blood type AB is very low, almost 50% of the human population has type O. It is apparent from these statistics that a system for more definite identification of blood systemsis mandatory if blood typing is to be used in a crime laboratory. In the early 1970s, research was initiated to improve the system for determining the typeof human blood found on dried blood stains. In addition to the ABO system, other systemswere utilized. The erythrocyte MN and Rh (primarily RhD) antigen systems were used. Protein studies involved hemoglobin and haptoglobin. Several enzyme systems were utilized;the most useful of these being phosphoglucomutase (PGM), adenylate kinase (AK), erythrocyte acid phosphatase (EAP), esterase D (EsD), and adenosine deaminase (ADA). Initialwork was performed by Bryan J. Culliford of the Metropolitan Police Forensic Laboratoryof London, England.1 Determining blood types using a combination of the systems cited,greatly diminishes the occurrence of a particular type in the population. Although electrophoresis was the method of choice in determining the proteins present in a blood stain, thetime involved became a prime concern since starch gel (the only media used at that time)electrophoresis procedures often required 15 to 30 hours for completion. he EsD bands appear as white fluorescent bands on a dark background. Use an EsDT2-1 as a control. After visualizing EsD, mark the position of the number 2 band of theEsD pattern. The gel is covered with GLO I reaction mixture beginning with this positionand incubated again.b. GLO I: 20 mg reduced Glutathione50 µL 40% Methyl glyoxal7.5 mL GLO I reaction buffer Soak mixture into Whatman paper cut to fit the size of the gel from the second bandof the EsD pattern to the edge of the plate (approximately 6.5 cm x 10 cm). The filterpaper is placed on the gel beginning at the cathode side, gently rolling down the restof the paper, being sure no air bubbles remain under the paper. Incubate 30 to 40minutes at 37 C. Heat 0.2 gms agarose (Sigma Type II) in 20 mL distilled water until dissolved. Allowthe agarose to cool to 60 C and then add 2 drops of iodine solution. (Iodine solutioncontains 1.65 gms of KI, 2.94 gms of I2 dissolved in 30 mLs of water.) Mix the agarose/iodine mixture. This mixture will be a light orange color. After the electrophoresisgel has been incubated as above, the agarose/iodine mixture is quickly poured ontothe gel starting at one corner of the gel allowing it to flow over the entire gel surface.The phenotype appears as blue bands on a light yellow background. The heterozygote (2-1) has three blue bands present and should be used as a control. A secondagarose/iodine mixture may be applied to the gel if the blue bands appear weak in thefirst gel. This is done after removing the first agarose/iodine gel. In the mid 1970s, workers at the University of Pittsburgh proposed a method for multipleenzyme separation on a single electrophoresis plate. This work was completed under thedirection of B. Wraxall.2 By covering a particular portion of the starch gel and developingeach portion specifically for the desired enzyme, it was possible to identify several enzymesystems at the same time. Additional work by Wraxall developed a multiple systems usinga starch/agarose gel. ince that time, the research of Dr. B. Grunbaum and P.L. Zajac using the BeckmanSMicro-zone System and adapting the starch gel procedures to Sartorious cellulose acetate membranes has drastically reduced the time required. The Titan III Cellulose AcetateElectrophoresis System from Helena Laboratories is now being used successfully by theSoutheast Missouri Regional Crime Laboratory. Methods for identifying genetic markersare performed on supported cellulose acetate electrophoresis plates. The results are discernable and readily reproducible. Studies on all the enzyme systems can be completed inless than eight hours.425

Section 3Typing Dried Blood Stains Using Agar Starch GelsII.IsoenzymesEsterase D (EsD) and Glyoxylase I (GLO I) This procedure should be performed within two days of receiving sample. Maximum sample age is 10 days. After 10 days the sample may be too deteriorated to produce a readableresult.1. Tank Buffer: HEPES, pH 7.55.95 gm HEPES 0.05M (N-2 hydroxyethyl piperazine-N-2 ethanesulfonic acid) Dissolve in 400 mL distilled water. Adjust to pH 7.5 with 40% NaOH. Q.S. to final volume of 500 mL with distilled water. The pH of the tank, plate and reaction buffers is extremely important. All enzymes havean optimal pH for activity. Denaturation of the enzymes may occur if reagents with the incorrect pH are used. Improper reaction temperatures have the same effect. Correct ionicstrength of the buffers is also very important.3. Starch/Agar Gel1% Agarose, Sigma Type V (low EEO)2% Hydrolyzed starch (Connaught Labs, Willowdale, Ontario, Canada).Available from Fisher, Cat. No. S-676. Dissolve 0.3 gm agarose and 0.6 gm starch in 30 mL gel buffer. After dissolving, de-gasthe solution prior to pouring into 8 cm x 10 cm x 2mm deep plastic plates. Gel must sitovernight before use. roper voltage and amperage are extremely important to good electrophoretic resolution.PVoltage should remain constant, allowing the amperage to start low (about 2 milliamperesper plate) in order to avoid overheating the system. The temperature of the electrophoresis plate rises as the amperage rises. If the temperature exceeds the optimal range, theenzyme will denature.4. Sample Preparation and Applicationa. Soak dried blood stains for 15 minutes in 0.1N Clelend’s reagent, pH 8.0. Run aknown type 2-1 as a standard (GLO and EsD).b. Eight wells (1 mm x 4 mm, 3 mm apart) are cut in the gel 2 cm from the cathodic endwith a metal template. Apply 5 µL of blood in each well using a micropipet.6. Reaction Buffera. EsD: 0.41 gm sodium acetate, anhydrous (0.05M). Dissolve in 100 mL distilledwater. Adjust to pH 6.5 with 1% acetic acid.b. GLO I: 2.42 gm NaH2PO4, anhydrous (0.2M)1.31 gm Na2HPO2, anhydrous (0.072M)Dissolve in 100 mL distilled water, adjust to pH 6.2 with phosphoric acid. Several variables influence the results obtained with an electrophoretic procedure. Theseinclude sample preparation and application, cellulose acetate plate preparation, pH andionic strength of the buffers, voltage, amperage, and color development techniques. Thecellulose acetate plates must be soaked in the individual system plate buffer prior to sample application. The soaking buffer is usually a dilution of the tank buffer. The correct dilution of plate buffer is essential for good resolution and for prevention of high amperage andexcess heat. The plate must be soaked gradually from the bottom up to ensure saturationwithout air bubbles. Air bubbles in the cellulose acetate impede the movement of the electric current throughthe plate. Once trapped in the plate, air bubbles cannot be removed and the plate must bediscarded.2. Gel Buffer: 1:14 dilution of tank buffer, pH 7.4.Dilute 10 mL of tank buffer with 140 mL distilled water.5. Electrophoresis: 250 volts for 30 minutes. It is very important to keep the electrophoresis chamber very cool during the run. Thetank buffer must be kept cold until ready for use. To keep the chamber at a low temperature, another lid or metal pan is filled with ice and placed on top of the closed electrophoresis tank. Plate is run face down on sponge wicks. Amperage at start 8 to 9 mA;at the end, about 10 mA.Factors Influencing Results he amount of time required for electrophoresis varies with the enzyme system underTstudy. None of our procedures require more than one hour. The technique used for developing the cellulose acetate plate after completion of electrophoresis is critical. The two basic types of development are colorimetric and fluorometric.After development, results must be recorded and filed. All plates are photographed withblack and white Polaroid film.III.Sample Extraction and Storage o preserve enzymatic activity, any materials containing dried blood stains should be froTzen. At the time of testing, cut a 0.5 cm x 0.5 cm piece of stained material from the originalsample and place in a well with 2 to 3 drops of solvent. Distilled water is routinely usedas the solvent; however, Cleland’s reagent must be used for phosphoglucomutase anderythrocyte acid phosphatase assays. Samples extracted with purified water may besoaked overnight in a humidity chamber at 4 C. Samples extracted in Cleland’s reagent(dithioerythritol; available from Sigma) are stable for 3 to 5 hours and should not be storedovernight. The procedure from making Cleland’s reagent is included in the PGM procedure.7. Reaction Mixture and Visualizationa. EsD: 4 mg MU-Acetate (4-methylumbelliferyl acetate)1.0 mL acetone (spectral grade)5 mL reaction buffer Dissolve MU-acetate in about 1.0 mL of acetone and immediately add 10.0 mL of thereaction buffer. Soak into Whatman 3 MM paper 4 cm x 10 cm. Lay this paper over aportion of the gel from the origin toward anode. Leave at room temperature for about5 minutes. Remove the paper after incubation at room temperature and read EsD byusing UV light.245

IV.V.MaterialsTitan III Cellulose Acetate System available from Helena Laboratories.HardwareCat. No.TITAN plus Power Supply (110V)1504Zip Zone Chamber1283Super Z Applicator Kit4088Development Weight5014Titan Photo VuBox Camera5048Bufferizer5093IOD Incubator, Oven Dryer (110V)5116Micro-Hood8009ConsumablesTitan III-Lipo Cellulose Acetate (60 x 76 mm)3900Electra HR Buffer5085Supre-Heme Buffer5802 Electra B1 Buffer5016LD Vis Isoenzyme Reagent5909Hemolysate Reagent5125Blotters50345081Zip Zone Chamber WicksDevelopment Slides5008Titan Plastic Envelope5052Titan Identification Labels5006Helena Marker50002. Plate Buffer: Same as tank buffer.3. S ample Preparation and Application: Extract blood stains with 3 drops of 6Murea (3.6 gm: 10 mL H2O). Place all of extract in a small plastic centrifuge tube.Fill the tube halfway with chloroform. Vortex to mix. Centrifuge the mixture in amicrocentrifuge at high speed. The chloroform washes down the debris in thestain. Extraction of stains with Hemolysate Reagent (Helena Cat. No. 5125) will eliminate aging bands which appear above the Gc bands. This will allow BC typingto be performed on old stains.*4. Electrophoresis: 500 volts for 10 minutes; Initial amperage 2 to 4 mA/plate.5. Visualization (Immunofixation)a. After electrophoresis, the plates are covered with human Gc antiserum diluted 1:4 with physiological saline (2 mL serum 6 mL saline). Allow plateto soak for 20 minutes. The diluted antiserum can be reused and is stable about 1 month after dilution, depending on the number of times used. Store in the refrigerator.b. Remove excess serum from plate and soak overnight in physiological saline (0.85%).c. Stain for 5 minutes in 0.2% Ponceau S (v/v in distilled water).d. To destain, place plates in a solution of 5% acetic acid, agitate for 15 minutes. Pour out this solution and add fresh acetic acid, agitate for another15 minutes until the background becomes white. Plates may be washedovernight. The plates are then dried; the bands are dark pink on a whitebackground.A. Preparation of Titan III-Lipo Plates1. P roperly code the required number of Titan III-Lipo platesby marking on the glossy, hard side with a magic marker.It is suggested that the mark be placed in one corner ofthe plate so that it is always aligned with sample #1. he plates may be soaked in the Bufferizer (availableTfrom Helena Laboratories, Cat. No. 5093) or they maybe soaked in a laboratory beaker using the principle ofthe Bufferizer. Place the plates in the beaker in a verticalposition and allow the plate buffer to slowly flow down theside of the beaker from a separatory funnel or directlyfrom the buffer container. Alternately, fill a beaker withbuffer and slowly and steadily immerse the plates (vertical position) into the buffer.Group Specific Component (Gc)1. Tank Buffer: Tris/Glycine, pH 8.421.8 gm Glycine4.5 gm Tris Dissolve in 900 mL water. Adjust to pH 8.4 with NaOH. Q.S. to a final volumeof 1000 mL with distilled water.Electrophoresis Techniques2. S oak the plates for 20 minutes in the individual systemplate buffer. The soaking buffer is usually a dilution of thetank buffer. The plates must be soaked gradually fromthe bottom up to ensure saturation without trapping airbubbles in the cellulose acetate.Part II. ProteinsTwo suggested sources for Gc antisera are:Atlantic AntibodiesIn Vitro Research Sources, Inc.10 Nonesuch RoadP.O. Box 110P.O. Box 1032Benson, MD 21018Scarborough, ME 04074(301) 877-7110(207) 883-4154Figures 9 and 10 on page 22 illustrate the electrophoretic patterns of Gc.Haptoglobin (Hp) he stain extract prepared for the Gc procedure may be used for haptoglobinTdetermination on polyacrylamide gradient gel. However, before using the extract,prepare a 1:1 mixture with 40% sucrose. *Personal Communication: Willard (Bud) Stuver, Supervisor, Serology/Biology Section, Metro-DadePolice Crime Laboratory, Miami, FL 33125.623

Figure 9 – Group Specific Component (Gc)Anode ( )B. Preparation of the Electrophoresis Tank (Zip Zone Chamber)1. P rior to using the tank, fill the inner chamber halfway withwater and keep frozen until the tank is used.Ice2. W hen preparing for electrophoresis, pour 50 mL of tank bufferinto the cathode chamber and a 1:1 dilution (25 mL tank buffer/25 mL H2O) in the anode chamber. (Except when statedotherwise; check specific procedures.)BufferBuffer3. W et two Zip Zone Chamber Wicks in the tank buffer anddrape one over each support bridge. Be sure they makecontact with the buffer and that there are no air bubbles under the wicks. Do not allow the wicks to touch the ice in theinner chambers. Cover the tank until used.Application pointCathode (–)Sample number12345678Electrophoretic patterns of two types of Gc are demonstrated as follows:Type 2-1: Samples 1, 3, 5, 6, 7 and 8Type 2-2: Samples 2 and 4C. Sample Application1. F ill the wells of the sample well plate with blood stain extractusing a small capillary pipette.Figure 10 – Group Specific Component (Gc)Anode ( )2. P rime the applicator by depressing the tips into the samplewells 3 or 4 times. Apply this loading to a piece of blotterpaper. Priming the applicator makes the second loading muchmore uniform. Do not loa