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January 1990 U.S. Department of Justice Volume 17, No.1 Federal Bureau of Investigation Crime Laboratory Digest

Review Article In This Issue Use of HPLC with Diode Array Spectrophotometric Detection in Forensic Drug Analysis

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T. w •• C'rr.' 1"""o:J 9.98 mjn Swjvel 45 deg 482 nm Tjlt 35 deg EDITOR ASSOCIATE EDITOR Barry L. Brown David T. Stafford Quantico, Virginia Memphis, Tennessee ASSISTANT EDITOR Denise K. Bennett Quantico, Virginia

EDITORIAL BOARD

William Anderson Dean Fetterolf Robert Sibert Reno, Nevada Quantico, Virginia Washington, D.C.

F. Samuel Baechtel Barry Gaudette Robert Spalding Quantico, Virginia Ottawa, Canada Washington, D.C.

Robert Cravey Robert Koons Irving Stone Santa Ana, California Quantico, Virginia Dallas, Texas

Harold Deadman Dale Moreau Phillip Wineman Quantico, Virginia Quantico, Virginia Rockville, Maryland

Peter DeForest Thomas Munson New York, New York St. Cloud, Minnesota

PUBLICATIONS COMMITTEE AMERICAN SOCIETY OF CRIME LABORATORY DIRECTORS

David T. Stafford*, Chairman Gerald Smith Rondal Bridgemon Memphis, Tennessee Knoxville, Tennessee Tucson, Arizona

Arnold Melnikoff Kenneth Jonmaire John Murdock* Missoula, Montana Lockport, New York Martinez, California

*These individuals also serve on the editorial board.

PUBLICATION POLICY The Crime Laboratory Digest is published quarterly by the FBI Laboratof'j in cooperation with the American Society of Crime Laboratory Directors (ASCLD). It is intended to serve as a rapid means of communication between crime laboratories, permitting information of in­ terest and value to be disseminated among crime laboratory scientists.

Inclusion of an article in the Crime Laboratory Digest in no way represents an endorsement or recommendation of any part of that article by the Federal Government, the Department of Justice or the FBI. Contributing authors assume total responsibility for the contents and accuracy of their submission. Que3tions or requests concerning an article should be directed to the contributing agency.

All submissions are subject to editorial review in accordance with the editorial policy established by the FBI Laboratory and ASCLD. The editorial staff of the Crime Laboratory Digest reserves the right to edit all articles for style, grammer and punctuation. Comments and let­ ters to the editor are encouraged and will be published when appropriate and as space permits. These should be forwarded to:

Crime Laboratory Digest - Editor FSRTC, FBI Academy Quantico, Virginia 22135 Contents

Message from the Assistant Director in Charge 1 of the FBI Laboratory

Editor's Column 2

Review Article: The Use of HPLC with Diode Array 5 . Spectrophotometric Detection in Forensic Drug Analysis

Barry K. Logan, H. Steve Nichols, G. Scott Fernandez and David T ... Stafford discuss the benefits of combining diode array ultraviolet visible spectrophotometric detection with HPLC for increased efficiency in drug analysis/identification.

Feature Article: The Effect of Luminol on the Serological 13 Analysis of Dried Bloodstains 131354

Robert R. J. Grispino examines the effects of luminol applications on the complete serological analysis of dried bloodstains and addresses the advantages and disadvantages of using the luminol test at crime scenes.

DNA Technology Seminar Videotape Order Form 24

Meeting Announcements 25

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Crime Laboratory Digest Subscriber Form 29 131353- U.s. Department of Justice 131354 Natlonallnstitute of Justice

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CRIME LABORATORY DIGEST Vol 17, No.1, January 1990 ISSN NO. 0743-1872 Mont. 24 11 Minn. VT 33 (488) (3,131) I (362) NH 76 (3,294) ~ OI'~ell:l. 20 S. Oak. (334) MA 102 (5,047) Wyo. CT 54 (2,7 45) 12 H13(~80) Ri 105 (12,218) (212) Neb. NJ 170 (9,331 ) 15 DE 139 (15,664) (466) MD 507 (24,642) Col. OC 521 (28,835) 31 Kan. (2,442) , I. 35__ .,..,,.

Ariz. I N. Mex. 31 16 (3,408) (1,008)

Texas 212 Foreign Governments (15,803) 140~·i\ (1.978) . ~ ~;R Australia 2 (297) Bahamas 6 (87) British West Indies 4 (903) .. ~.- ,Q Canada 4 (420) '(.\ \j '-} Cayman Islands 1 (95) Hawai~ Jamaica 1 (12) 101 (13,269) {>

FBIIDOJ IFeature Miele I

The Effect of Luminol on the Serological Analysis of Dried Human ~stains

Robert R. J. Grispino Serology Unit FBI Laboratory Washington, D. C. 20535

To a great degree today, crime laboratories In 1936, Gleu and Pfannstiel confirmed are being provided with crime scene evidence Albrecht's observations regarding luminol and which has been treated with luminol reagent blood. They noted that luminol would not mixtures by law enforcement personnel at the chemiluminesce in the presence of boiled veg­ scene. Laboratory examiners are then requested etable peroxidases and observed that luminol to perform a complete serological analysis of mixed with pure hematin produced the most the questioned samples. However, uninformed brilliant blue luminescence. crime scene personnel sometimes opt to use the The first proposed forensic use of luminol as luminol test as a preferred field blood test at a preliminary blood test was reported by Specht all crime scenes. Since the reagents are com­ in 1937. He sprayed blood on bushes, stone mercially available in packaged crime scene kits, walls, rusty fences, furniture, stone steps the luminol test is susceptible to abuse and mis­ and a garden. After allowing the blood to use by untrained officers. This article discusses remain exposed to the elements for 14 days, the effects of luminol sprays on the complete Specht sprayed a luminol reagent mixture onto serological analysis of neat dried bloodstains. In the blood and photographed the results. 'fhe addition, the advantages and disadvantages of mixture was 0.1% luminal in 5% aqueous sodium the luminol test are addressed, and suggestions carbonate (Na2C03) with a 15% solution of HP2 are offered concerning the proper application added immediately before spraying. All blood­ and protocol of the luminol test at crime scenes. stained areas glowed with blue light for 10 to In summary, the results of this study demon­ 15 minutes. Blood was also detected in water, strate that the use of luminol denatures most soapy water and sewage. The luminol test blood enzymes after a short exposure, thus worked well with both fresh and old blood­ causing further serological comparisons to be stains; in fact, the older the bloodstain, the questionable. more pronounced the positive reaction. Proescher and Moody confirmed Specht's findings in 1939 using Specht's spray mixtures. REVIEW They detected bloodstains on paper, fabrics and iron pipes exposed to the elements for 3 years, with 3-year-old putrefied blood exhibiting bril­ The compound 3 aminophthalhydrazide liant luminescence. In addition, they observed (5- amino 2, 3- dihydrophthalazine 1, 4- dione) that dried and decomposed blood elicited a was first synthesized by Schmitz in 1902. At the stronger and longer lasting luminol reaction than time of synthesis, he noticed that the compound fresh blood. When the luminescence disap­ exhibited a strong blue fluorescence in aciti so­ peared, it could be reproduced by application lutions. In 1927, Lommel first observed a blue of fresh luminol spray. Dried bloodstains were after oxidation of the com­ made luminescent many times. Fresh dried pound in alkaline solution. His work was never bloodstains were made more luminescent by published, but Albrecht, one of his coworkers, spraying the blood with 1 to 2% hydrochloric confirmed and published Lommel's original acid solution before luminol application. The findings in 1928. Albrecht also pointed out that luminol reaction was elicited with both animal blood and fresh potato juice caused the chemical and human blood. Hematin was detected in a 8 to exhibit strong luminescence in the presence of dilution of 1:10 • Most importantly, Proescher (HPJ. In 1934, Huntress et and Moody (1939) made the following emphatic al. termed this compound luminol as a producer supportive statement on luminol's application to of light. forensic serology: 13

CRIME LABORATORY DIGEST Vol 17, No.1, January 1990 "Luminol does not interfere with the The c:hemical basis of the luminol test is an spectroscopic, chemical or precipitation oxidatjon reaction. An alkaline solution of lu­ tests for the definite identification of minol will oxidize in the presence of HP2 and blood. Hematin and hemochromogen a hematin-catalyzed peroxidase system. Hydro­ crystals of dried blood were obtained gen peroxide is the oxidizing agent. Blood is a after rep~atedly treating blood with hematin-catalyzed peroxidase system. The re­ luminol. The precipitin test can be sulting oxidation reaction is visualized by a blue applied if the blood is not decomposed." chemiluminescence. In 1985, Thornton and Maloney reviewed the Lytle and Hedgecock (1978) experimented chemistry of the luminol reaction'. Figure 1 is with the effects of alkaline luminol/sodium an amalgamation presented by Thornton of the perborate (NaB OJ mixtures on blood. They work of a number of researchers on a proposed concluded: oxidation pathway for luminol. Not all of the steps of the oxidation pathway have been ".... luminol is relatively nondestructive elucidated to date. The important points are: to the surroundings (it is noncorrosive and nonstaining) and to the blood (it 1) Oxygen is required. does not prevent subsequent identi- 2) The anion of luminol is the reactant. fication tests or ABO blood grouping 3) Free radicals are involved which provide the analysis although it does interfere with 40 to 70 kcal./mol of chemical energy needed electrophoretic analysis of.. .. erythrocyte for chemiluminescence (Brune:irett et al. acid phosphatase and phosphoglucomutase)." 1972). 4) Nitrogen is the product of the reaction. They recommended luminol as "a good field test" 5) The entire conversion pathway is unknown. which was "sensitive and reasonably specific for blood." The luminol test is known to be extremely MATERIALS AND METHODS sensitive to the presence of blood. In 1986, Thornton et al. stated that the unaided eye could The luminol spray regimen utilized two detect blue chemiluminescence of luminol in separate mixtures consisting of the following: 4 blood diluted 1:10 • With the use of an infrared starlight scope, blue chemiluminescence of Luminol Mixture 1 (Specht 1937) luminol could be detected in blood solutions 6 6 of 1:10 to 5:10 • Part 1 - 0.1 g luminol and 5 g Na2C03 In the luminolliterature (Gundennann 1965; carbonate in 50 ml distilled water. Schneider 1970; Gaennsslen 1983; Kraul and Meyer 1941; Wei and White 1971; Roswell and Part 2 - 0.7 g NaB02 in 50 ml 95% ethanol. White 1978), researchers have reported false positive results (that is, chemiluminescence of Luminol Mixture 2 (modification) luminal in the absence of blood) from alkaline luminol in the presence of iron, , hypo­ Part 1 - 0.1 g luminol and 5 g Na2C03 in chlorites, manganese peroxide and ferricyanide. 90 ml distilled water. False negative results were obtained with lu­ minol sprayed on cadmium-coated materials bearing blood. Cadmium has been employed as a corrosion resistant coating for materials. Parts 1 and 2 were always prepared separately in The chemistry of the luminol reaction should advance and were only mixed together immedi­ be reviewed so that the results of these experi­ ately prior to use. Application to the questioned ments can be better understood. Chemilumines­ stains was made directly via an aerosol sprayer cence is defined as the production of light by or indirectly via dropper bottle to a distilled chemical means. It occurs when an electron water dampened cotton swabbing of the stained moves from an excited state to a ground state. area in question. Luminol mixtures were The electron is raised to an excited state by applied as a spray in a completely dark, well chemical energy. It requires a chemical reaction ventilated room. which will supply at least 40 to 70 kcal./mol Positive and negative controls were employed energy :t$ in tht'i case in free radical reactions. It in all tests; these consisted of known human does not require excitation by radiant energy as bloodstains, a copper penny, sterile cotton swabs does fluorescence (Wildes and White 1973). and washed cotton sheeting. 14 0 OW OH- H 0 o~ 2 2 NH N· ~-N • I • I • ~H ~H

NH2 NH2 0 NH2 0

LUMINOl LUMINOl lUMINOl MONOANION RADICAL * O· 2 ~. N- .. • •

LUMINOl lUMINOl ELECTRONICAllY RADICAL ENDOPEROXIDE EXCITED DIAN ION ANION o

# O· r +hv42Snm /,~~ o NH2 H H GROUND STATE 3 AMINOPHTHALATE

pH optimum 10.4 - 10.8 for light emitting reaction

3+ 4+ • • Fe + H 0 • Fe + OH + OH (H 0 ) .....11{..... -...~ O· + H 0 2 2 t 2 2 2 2 fromt peroxidase Hematin like activity

Figure 1. Chemistry of luminal (Thornton and Maloney 1985).

15 Sixty-two dried bloodstains were tested to (Lattes 1927; 1928). Conclusive identifications determine how sensitive these procedures would were made only when the forward and reverse be in our laboratory. Stains of neat group A tests agreed. Both techniques relied on the eval­ blood were tested ranging from 100 1'1 of blood uation of microscopic agglutination of indicator to serial dilutions of the same blood from 1:10 to erythrocytes and corresponding ABO antibodies. 8 1:10 • The stains were on washed cotton sheet­ The strength of agglutination was evaluated ing, #1 Whatman filter paper and gtass micro­ according to the following criterion: scope slides. All stains were air-dried in a ven­ tilating hood. +4 - one solid grape-like cluster aggregate of Forty-five dried human bloodstains consisting erythrocytes of 100 1'1 of neat group A, AB and 0 blood (15 +3 - several large agglutinates each type) were deposited on the same fabric, +2 - medium size agglutinates on a clear paper and glass materials. These stains were background ,-, exposed to luminol mixtures 1 and 2. An addi.­ +1 - small aggregates (triplet or doublet cell tional 80 swabbings were developed. The spec­ clusters) imens were then analyzed serologically through o - no agglutination the following stages: 5) P,,)lymorphic Blood Enzyme Analysh. ~ 1) Phenolphthalein Preliminary Blood Test - (Wraxall et al. 1978) Following the Kastle-Meyer protocol (Kastle Phospboglucomutase (PGM) - Isoelectric 1909; Kastle and Shedd 1901), reduced phenol­ FocusiIilg (IE F) sub typing (Budowle 1984a; phthalein is applied first to a damp swabbing of 1985; Budowle et al. 1986) the suspected stain; then a 3% solution of HP2 is applied to the swab, all via dropper bottle. A Erythrocyte Acid Phosphatase (EAP) - IEF positive result consists of a swab color change (Budowle 1984b) from colorless to pink/red. Esterase D/Glyoxalase I (EsD/GLO) - aga.r 2) Hemochromogen Confirmatory Blood Test - electrophoresis (Budowle 1984 b; 1985; Budowle and Gambel 1988) Hemochromogen crystal formation is micro­ scopically observed and evaluated ~fter appli­ Peptidase A - agar electrophoresis (Parkin 1978) cation of Takayama reagent and heat to a por­ Adenosine Deaminase/ Adenylate Kinase tion of the luminol treated and untreated (ADA/AK) - agar electrophoresis (Murch et al. bloodstains (Takayama 1912). 1986)

3) Origin Determination - 6) Serum - Antihuman serum was produced in rabbits Haptoglobin (HP) - Polyacrylamide Gel after the ID<;'tnod of Proom (1943). Buffered Electrophoresis (Budowle and Chow 1985) saline extrac~ of luminol-sprayed and unsprayed human bloodstains were reacted Group Specific Component (Gc) and Transferrin against the prepared rabbit antihuman sera (Tf) - Immunofixation (Alper and Johnson 1969) according to the protocol of Ouchterlony (1948; 1949a; 1949b; 1949c; 1968). Identity precipitin Duplicate sets, 15 for each group, of group A bands creating a fused chevron effect were bloodstains (neat to 10-8 dilutions), neat group indicative of a positive result (see following). AB and neat group 0 stains on washed cotton sheeting, paper and glass materials were stored 4) ABO Grouping - at room temperature for 2 months. These items were considered aged stains for the purposes of Forward testing of ABO antigens was accom­ this study. They were tested using the same plished using the absorption-elution technique protocol as the fresh stains. (Kind 1960a; 1960b, Outterridge 1962; 1965a; Blood used in this study was collected volun­ 1965b). Reverse testing of ABO antibodies was tarily via finger prick from employees of the accomplished via the Lattes crust technique FBI Laboratory.

16 RESULTS After the initial stains were sprayed with the luminol mixtures, they were also tested using the phenolphthalein protocol. In these cases, phe­ Sensitivity Evaluations: nolphthalein detected blood in neat to to-2 dilutions only. Accordingly, bloodstains which Three separate regimens were utilized in were luminol treated in the fieJd could diminish luminol sensitivity testing. Neat group A dried by one order of magnitude the ability of the bloodstains and serial dilution stains were: phenolphthalein test to detect the presence of blood. 1) aerosol sprayed directly with luminol mixture I or 2; 2) rubbed with a distilled water dampened Serological Analysis: cotton swab (the swab with the transferred stain was then sprayed with luminol mixture Neither luminol mixture had anv observable 1 or 2); or effect on hemochromogen crystal formation in 3) touched with a clean damp swab as in dried bloodstains, a well established confirm­ regimen 2, except luminol mixture I or 2 atory test for the presence of blood. was applied to the swab via dropper bottle. Regarding blood origin determination tests, neither luminol mixture had any observable The resuits of the luminol sensitivity experi­ effect on precipitin line formation between ments are summarized in Table 1. rabbit antihuman sera and extracts of treated Both luminol mixtures presented similar sen­ dried human bloodstains, when compared to sitivity results. They both detected the presence untreated control stains. Spurious Liesegang of blood in neat to 10-1 stain dilutions of' group bands were not noted in the Ouchterlony plates. A blood. The manner of application of luminol The results of the experiments questioning the (direct spray of stain or indirect application via effect of luminol in ABO testing in dried blood­ dropper bottle to swabbing of stain) had no real stains are summarized in Table 2. effect on its sensitivity in detecting blood in Using the absorption-elution ABO forward fresh dried stains. The results were the same testing technique, the following erythrocyte regardless of stain deposition on fabric, paper or antigens can be detected in dried bloodstains: glass. A duplicate set of fresh bloodstains was also tested with phenolphthalein test reagents Group A stain - A and H erythrocyte applied via dropper bottle. The phenolphthalein antigens test detected blood in neat to to-3 stain dilutions Group B stain - Band H erythrocyte antigens 3 but not in dilutions greater than to- • These Group AB stain - A, Band H erythrocyte results were also the same on all three stained antigens materials. Group 0 stain - H erythrocyte antigen

Table 1. Sensitivity Evaluation

Blood Lum 1 Lum 2 Lum 1 Lurn 2 Lum 1 Lum 2 Dilution Sl!!n Spray Dropper Dropper ill Spray + pH Spray + pH neat + + + + + + + to-I + + + + + + + 10-1 + + + + + + + 10-3 wk + wk + wk 10~ wk wk + to-5 10-6 to-7 10-1 wk "" weak positive reaction (less intense glow than +) pH - phenolphthalein preliminary test 17 Table 2

ABQ Forward Tl:~inK Anti2~ns ABQ Reverse Tl:ning Antibodies A It H A l! Q A +4 0 +4 0 +4 0 ALl +2 0 +4 0 +4 {) AL2 +1 0 +4 0 +4 0 AB +4 +4 +4 0 0 0 ABLI +4 +2 +2 0 0 0 ABL2 +4 +2 +2 0 0 '0

0 0 0 +4 +4 +3 0 OLI 0 0 +4 +4 +3 0 OL2 0 0 +4 +4 +3 0

L 1 == Luminal Mixture 1 L2::o: Luminol Mixture 2

The treatment of neat group A, AB and 0 Band H erythrocyte antigens was detected in all dried bloodstains with either luminol mix.ture treated samples. Both luminal mixtures permit­ failed to impair the correct conclusion regarding ted the detection of A, Band H erythrocyte the presence of appropriate autologous erythro­ antigens; however, the agglutination of Band H cyte antigens. It would, however, be incorrect antigens was much less intense in treated sam­ to state that the detection of erythrocyte anti­ ples than untreated samples (+4 agglutination in gens was unaffected by the luminol treatments. untreated samples and +2 agglutination in lumi­ Luminol mixtures applied to neat group A dried nal treated samples). The A erythrocyte antigen bloodstains resulted in a diminished capacity to agglutination was unaffected by luminol treat­ detect A erythrocyte antigens as compared to ment and was identical to that observed in un­ untreated samples. Luminol/perborate (mixture treated samples (+4 agglutination). 1) allowed the detection of the A and H eryth­ The ability to completely detect the presence rocyte antigens in A stains; however, the A of H erythrocyte antigens in group 0 dried erythrocyte antigen agglutination reactions were bloodstains was unaffected by either luminol much less intense than those observed in un­ mixture when compared with untreated samples untreated samples (+4 agglutination in untreated (+4 agglutination in all H antigen rows). samples and +2 agglutination in treated samples). Using the Lattes crust technique, the fol­ Luminol/peroxide (mixture 2) also allowed the lowing antibodies can be detected in dried detection of A and H erythrocyte antigens in A bloodstains: stains; however, the A erythrocyte antigen agglutination reaction was of lesser intensity Group A stain - anti-B antibody than that observed after the luminol/perborate Group B st!!.in - anti-A antibody tl'eatment (+2 agglutination with mixture 1 and Group AB stain - no ABO antibodies +1 agglutination with mixture 2). The agglu­ Group 0 stain - anti-A and anti-B antibodies tination reactions for H erythrocyte antigens remained constant for both mixtures and identi­ The treatment of neat group A, AB and 0 cal to the untreated samples (+4 agglutination). dried bloodstains with either luminol mixture A similar loss of the ability to completely had no influence on the detection of appropriate detect autologous erythrocyte antigens was also serum antibodies. The agglutination reactions observed in the analysis of luminol treated neat observed were identical in treated and untreated group AB dried bloodstains. The presence of A, samples. 18 Polymorphic Blood Enzyme Analysis Gc and Tf:

PGM Subtyping: All samples were identified as Gc type I. The untreated group A and 0 stains were The untreated group A bloodstain was identi­ identified as Tf type C while the untreated AB fied as PGM subtype 2-2+, while the untreated stain was identified as Tf type CD. In the Gc AB and 0 stains were PGM subtype 1+2+. After immunofixation system, all luminol treated treatment with either of the luminol mixtures, samples were readable with light discrete bands the 2-2+ bands were still readable, but intensity noted. In the Tf immunofixation system, all was reduced compared to the untreated control luminol treated samples were readable, but sample. New spurious bands or unexplained streaking was prevalent in the gel. The band enzyme activity were not noted on the electro­ staining was much lighter than in untreated phoretic plates. samples. New spurious bands or unexplained The PGM subtype bands 1+2+ in the other activity were not noted. after luminol treatment stains were barely readable after treatment with in either the Gc or Tf systems. the luminol/perborate mixture. New spurious bands or unexplained enzyme activity were not noted. The luminol/peroxide treatments ren­ Aged Stains dered unreadable PGM activity in the AB and 0 samples. Duplicate sets of the stains were stored for 2 months at room temperature to simulate the EAP: aging of forensic samples. These stains were subjected to the same serological protocols as the The untreated group A stain was identified fresh samples described previously. The results as EAP type B, the AB stain was EAP type A of these experiments are provided. in Tables 3 and the 0 stain was EAP type BA. All luminol and 4. treated samples showed an intense fluorescence Aging did not reduce the sensitivity of either under ultraviolet light at the anode with no luminol preparation. Hemochromogen crystal cathodic migration. Thus, these typing results formation and antihuman sera precipitation were were considered inconclusive for reporting unaffected by either luminol mixture. purposes. Using the absorption-elution ABO forward testing technique, many similarities were noted Other Blood Enzymes: in the effects of luminol on aged stains as com­ pared to fresh stains. A diminished capacity to Enzyme activity in the following enzyme detect A erythrocyte antigens was observed after systems was not detected after treatment with luminol treatments of aged A stains (+4 agglu­ either luminol mixture: tination in untreated aged stains and +2 agglu­ tination in luminol treated aged stains). As EsD/GLO Peptidase A ADA/AK Table 3. Sensitivity Evaluation of Aged Stains

Serum Analysis Lum 1 Lum 2 HP: ~ Spray ill The untreated group A stain was identified as neat + + + 10'\ + + + HP type 2-1, the AB stain was HP type 2 and 10'2 the type 0 stain was HP type 1. After treatment + + + 10'3 + + wk with either bminol mixture, heavy brown 10-4 wk wk wk streaking was noted in the polyacrylamide gels. lO's to 10-3 New spurious bands were not observed in lu­ minol treated samples. Th,e brown streaking failed to interfere with the ~orrect reading of wk = weak positive reaction (less intense glow than +) Haptoglobin types. pH - phenolphthalein preliminary test

19 Table 4. A.ged Stains

ABQ FQrward TYl!in.:; Anti.:;en~ ABQ R~'ferse TYl!inK Antibodies A J! H A J! 0 A +4 0 +4 0 +4 0 ALl +2 0 +4 0 +4 0 AL2 +2 0 0 0 +4 0 AB +4 +4 +4 0 0 0 ABLl +4 +4 0 0 0 0 ABL2 +4 +4 0 0 0 0 0 0 0 +4 +4 +4 0 OLI 0 0 +4 +4 +4 0 OL2 0 0 +4 +4 +4 0

Ll ... Luminol Mixture 1 L2 = Luminol Mixture 2

described earlier, similar results were also noted Regarding serum proteins, HP type 2-1 was with fresh bloodstains after luminol treatment. readable in aged treated stains; however. HP Luminol treatment of group A and AB aged type 2 and HP type 1 were unreadable due to stains resulted in a greatly diminished ability intense brown streaking in the gels. Protein to detect the H erythrocyte antigen. In aged A activity was not observed in luminol treated stains, the luminoljperborate mixture 1 had no aged samples in the Gc/Tf immunofixation effect on the detection of the H erythrocyte systems. These systems were typeable in fresh antigen (+4 agglutination). In the same aged A luminol treated stains. stains. the luminol/peroxide mixture 2 rendered the H erythrocyte antigen completely undect­ able (no agglutination was noted). In aged AB DISCUSSION stains, both luminol mixtures again destroyed the ability to detect the H erythrocyte antigen The results of this study demonstrate that (no agglutination noted). However, the detec­ forensic serologists and crime scene personnel tion of the H erythrocyte antigen was unaffected should seriously consider the decision to use by luminol in the treated group 0 samples (+4 luminol reagents as a blood screening test. agglutination for all samples). The ease of application of luminol reagents ABO serum antibody detection via the Lattes and its resulting chemiluminescence on dried crust reverse testing technique in aged stains bloodstains make luminol extremely appealing to was completely unaffected by either luminol crime scene workers. It must be remembered treatment. that when used improperly, luminol can sabotage The major difference between aged and fresh a criminal investigation just as easily as it can luminol treated samples was noted in the blood enhance it when used properly. enzyme PGM. Although some PGM subtyping Historically, luminol was meant to be used could be performed on fresh luminol treated sparingly as a preliminary blood screening test stains, once the stains were aged, luminol ~reat­ when there was reason to believe that blood may ment rendered PGM subtyping undetectable. As have been present, yet was not visible. with the fresh luminol treated stains, no PGM The typical application of luminol is as an subtyping enzyme activity was noted in aged aerosol spray, although the choice of luminol luminol treated samples in the following systems: recipe is at the discretion of the analyst. Al­ EsD, GLO, ADA, AK and Peptidase A. The kaline luminol stored in an amber-darkened EAP system was inconclusive in that migration container maintains a relatively long shelf life. from origin was absent. When alkaline luminol is mixed with a hydroxyl 20 ion source, the shelf life of the mixture is ap­ the forensic serologist. The luminol reagent proximately 1 hour. Therefore, it is suggested severely compromises this ability and greatly that separate luminol reagents be included in diminishes the profile comparison possibilities. crime scene kits for fresh mixing in appropriate The luminol spray regimen is appropriate for circumstances. a determination of invisible blood traces on large In this series of experiments, NaB02 and areas such as carpets, walls, flooring or the car­ HP2 were the chosen hydroxyl ion sources. The peted interior of a vehicle when no blood is ob­ NaB02 did not readily enter into solution and vious. In these cases, if blood is present, it is constantly clogged the aerosol sprayer after mix­ there in such low concentrations as to usually ture with alkaline luminol. The HP2 solution preclude further ABO or enzyme analysis. readily mixed with alkaline luminol and was Thus, nothing is lost or compromised by luminol much easier to apply to the stains. spray application. What is gained is the ability As previously discussed, luminol can be to screen a large item or area quickly, easily and applied via direct spray or indirect swab/drop­ efficiently for the possible presence of blood. per bottle method with no loss in sensitivity to Luminol spray application may develop a stain blood. If the spray regimen is implemented, pattern which could be of interest to investi­ the following suggestions are offered: gators or could suggest a mechanism by which the crime took place. 1) The luminol mixture should be sprayed in a Of paramount importance is the understand­ darkened, well ventilated room. Luminol has ing that luminol remains a preliminary blood been shown to be moderately toxic to the screening test wh!ch alone is insufficient to Hver and kidneys (Schneider 1970); therefore conclusively establish the presence of blood. human exposure to the spray should be The appropriate use of iuminol at a crime scene limited. should be discussed and evaluated on a case­ by-case basis. Luminol is a serologically de­ 2) Known blood and a copper penny should be structive reagent when used improperly. If included as positive controls when using the preliminary screening tests must be employed luminol spray as indkators of the success and at a crime scene, the following guideline should degree of relative intensity of the chemilu­ be observed: With visible blood, preserve the minescence reaction. stain, package it appropriately and send it to a crime laboratory for analysis. If no visible 3) False positive results may be obtained with blood is present, consider the use of luminol. luminol. A metal staple or carpet tack in a rug or a rusted metal vehicle interior will glow after treatment with luminol, simulating a positive blood reaction. REFERENCES

4) A camera should be available to immediately Albrecht, H. O. (1928). Uber die chemilumi­ photograph any observed chemiluminescence. nescenz des aminophthalsaurehydrazids, Z. Respraying with luminol will restore any Physiol. Chern. 136:321. faded glow (Thornton and Murdock 1966; Zweidinger et al. 1973). Alper, C. and Johnson, A. ( 1969). Immunofix­ ation electrophoresis - A technique for the If the swab regimen is implemented, it should study of protein polymorphisms, Vox. Sang. duplicate that of the phenolphthalein test (that 17:445-452. is, a dampened swab is touched to a suspected stain and the swab then treated with luminol). Brundrett, R. B., Roswell, D. F. and White, E. H. Any swabbed area which results in a chemilu­ (1972). Yields of chemically produced excited minescent glow should be well marked and states, J. Am. Chern. Soc. 94:7536-7541. preserved for future analysis using standard techniques. Budowle, B. (1984a). Phosphoglucomutase-l The results of this study have shown that sub typing of human bloodstains on ultrathin luminol routinely denatures most blood enzymes layer polyacrylamide gels, Electrophoresis after a short exposure in neat bloodstains. Only 5:165-167. limited serum proteins could be determined in luminol treated samples. The ability to deter­ Budowle, B. (1984b). Rapid electrofocusing of mine as complete a biochemical profile as pos­ erythrocyte acid phosphatase, Electrophoresis sible on a blood sample is the ultimate goal of 5:254-255. 21 Budowle. B. (1984c). Typing of esterase D by Kraut. R. and Meyer. H. (1941). 1st der isoelectdc focusing, Electrophoresis 5:314-316. Nachweis von blutspuren durch 3 amino­ phthalsaurehydrazid ein kennzeichnemdes Budowle B. (1985). An agarose gel method for verfahren? Angew. Chemie 54:213-215. typing phosphoglucomutase-I, esterase D or glyoxalase I, J. Forensic Sci. 30:1216-1220. Lattes, L. (1927). Praktische erfahrungen uber blutgruppenbestimmung in flecken, Dtsch. Z. Budowle. B. and Chow. G. H. (1985). Discontin­ Gesamte Gerichtt. Med. 9:402-419. uous polyacrylamide gel electrophoresis for typing haptoglobin in bloodstains, J. Forensic Lattes. L. (1928). Blutgruppendiagnose von Sci. 30:893-897. blutflecken, Ukr. Zentralbl. Blutgruppen Forsch. 2:36-46. Budowle. B. and Gambel. A. M. (1988). A hy­ brid ampholyte focusing technique for esterase Lytle, L. and Hedgecock, D. G. (1978). Chemi­ D subtyping of evidentiary material, J. Forensic luminescence in the visualization of forensic Sci. 33:738-743. bloodstains, J. Forensi~ Sci. 23:550-562.

Budowle. B .• Murch. R. S .• Davidson. L. C .• Murch. R. S .. Gambel, A. M. and Kearney. J. J. Gambel. A. M. and Kearney. J. J. (1986). (1986). A double origin electrophoretic method Subtyping phosphoglucomutase-l in semen for the simultaneous separation of adenosine stains and bloodstains: A report on the method, deaminase, adenylate kinase and carbonic J. Forensic Sci. 31:1341-1348. anhydrase II, J. Forensic Sci. 3.1.:1349-1356.

Gaennsslen. R. (1983). Sourcebook in Forensic Ouchterlony. O. (1948). Antigen-antibody Serology, Immunology and Biochemistry. reactions in gels, Arkh. Kemi. Mineral. Geol. United States Department of Justice, 26B:14. Washington, D. C. Ouchter[ony. O. (J949a). Antigen-antibody Gleu. K. and Pfannstiel. K. {1936}. Uber 3- reactions in gels, Acta. Pathol. Microbiol. Scand. aminophthalsaurehydrazid, J. Prakt. Chern. 26:507-515. 146:137. Ouchterlony O. (1949b). Antigen-antibody Gundermann. K. D. (1965). Chemiluminescence reactions in gels II. Factors determining the in organic compounds, Angew. Chemie (inter­ site of the precipitate, Arkh. Kemi. 1:43-48. national edition) 4:566-573. Ouchterlony. O. (1949c). Antigen-antibody Huntress. E .• Stanley. L. and Parker. A. (1934). reactions in gels III. The time factor, Arkh. The preparation of 3 aminophthalhydrazide for Kemi 1:55-59. use in the demonstration of chemiluminescence, J. Am. Chern. Soc. 56:241-242. Ouchterlony. O. (1968). Handbook of Immuno­ diffusion and Immunological Electrophoresis. Kastle. J. H. (1909). Chemical Tests for Blood. Ann Arbor Science Publishers, Inc., Ann Arbor, United States Hygienic Laboratory Bulletin 51. Michigan. United States Public Health and Marine Hospital Service, U. S. Government Printing Service, Outterridge. R. A. (1962). Absorption-elution Washington, D.C. method of grouping bloodstains, Nature 195:818-~19. Kastle. J. and Shedd. O. (1901). Phenol­ phthalein as a reagent for the oxidizing Outterridge. R. A. (1965a). The biological ferments, Am. Chern. J. 26:526-539. individuality of dried human bloodstains, J. Forensic Sci. Soc. 5:22-51. Kind. S. (1960a). Absorption-elution grouping of dried blood smears, Nature 185:397-398. Outterridge. R. A. (1965b). Recent Advances in the Grouping of Dried Blood and Secretion Kind. S. (1960b). Absorption-elution grouping Stains. In: Methods of Forensic Science, Vol. of dried bloodstains on fabrics, Nature 4, pp. 299-332. Edited by A. S. Curry. 187:789-790. Interscience, New York.

22 Parkin. B. H. (1978). The typing of peptidase Thornton. J. I. and Murdock. J. E. (1966). A in bloodstains, J. Forensic Sci. Soc. 18:65-67. Photography of the luminol reaction in crime scenes, Criminologist 10: 15-19. Proescher. F. and Moody. A. M. (1939). Detection of blood by means of chemilumi­ Thornton. J. I. and Maloney. R. S. (1985). The nescence, J. Lab. Clin. Med. 24:1183-1189. chemistry of the luminol reaction - Where to from here?, CAC Newsletter, September, Prom.'!.. H. (1943). The preparation of precip­ pp.9-17. itating sera for the identification of animal species, J. Pathol. Bacteriol. 55:419-426. Thornton. J. I .. Guarino. K .. Rios. F. G. and Cashman. P. J. (1986). Enhancement of the Roswell. D. F. and White. E. H. (1978). The luminol test by means of light amplification, chemiluminescence of luminol and related J. Forensic Sci. 31:254-257 hydrazides, Methods Lnzymol. 57:409-499. Wei. C. C. and White. E. H. (1971). An efficient Schmitz. A. (1902). Uber das hydrazid der chemiluminescent hydrazide: Benzo (ghi) pery­ trimensinsaure und der hemimellitsaure, Inaug. lene-l, 2-dicarboxylic acid hydrazide, Tetra­ disseration Heidelberg cited in Curtius T. and hedron Letters 39:3559-3562. Semper A. (1913) Ber. Btsch. Chern. Ges. 46:1162. Wildes. P. D. and White. E. H. (1973). Dif­ ferences between excited states produced Schneider. H. W. (1970). A new long lasting chemically and photochemically. Ion pairs of luminol chemiluminescent cold light, J. Chern. excited states derived from luminol, J. Am. Educ. 47:519-522. Chern. Soc. 95:2610-2617.

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Takayama. M. (1912). A method for identify­ Zweidlinger. R. A .• Lytle. L. T. and Pitt. C. G. ing blood by hemochromgen crystallization, (1973). Photography of bloodstains visualized Kokubyo Gakkai Zasshi 306:463-481. by luminol, J. Forensic Sci. 18:296-302.

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