Brackett, J. W. Jr.

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Northwestern University Illinois, School of Law: Scholarly Commons

Journal of Criminal Law and CriminologyVolume 47 | Issue 6 Article 121957Acid Phosphatase Test for Seminal Stains--TheImportance of Proper Control Tests, TheJ. W. Jr. BrackettFollow this and additional works at: https://scholarlycommons.law.northwestern.edu/jclcPart of the Criminal Law Commons, Criminology Commons, and the Criminology and CriminalJustice CommonsThis Criminology is brought to you for free and open access by Northwestern University School of Law Scholarly Commons. It has been accepted forinclusion in Journal of Criminal Law and Criminology by an authorized editor of Northwestern University School of Law Scholarly Commons.Recommended CitationJ. W. Jr. Brackett, Acid Phosphatase Test for Seminal Stains--The Importance of Proper Control Tests, The, 47 J. Crim. L. Criminology& Police Sci. 717 (1956-1957)THE ACID PHOSPHATASE TEST FOR SEMINAL STAINSThe Importance of Proper Control TestsJ. W. BRACKETT, JR.The author is a criminalist in the District Attorney's Laboratory of San Jose, Cali-fornia, and a graduate of the University of California. He is active in the CaliforniaAssociation of Criminalists and has previously contributed to this iournal.-EDrroR.The acid phosphatase test is now widely used to identify stains of seminal origin.(1, 2, 3, 4) Tests for acid phosphatase which use a phenolic ester as a substrate arenon-specific; experimental interferences were studied and a simple method of detec-tion of interferences is reported.The test methods consist of reacting the suspected stain or an extract of it with asolution of suitably buffered phenolic phosphate ester. If acid phosphatase is present,the phenolic phosphate ester is hydrolyzed to phosphate ion and phenol. The phenolformed, if any, is then reacted with a third substance to form a colored product. Thepresence of this colored product indicates a positive test for the phosphatase. If thequantity of colored product is sufficient within the time and concentration limitsstated in the test, a conclusion that the stain is of seminal origin is justified. Thedifficulty with this process is that extraneous phenols will react with the chromo-genic color forming substance to produce colored substances which may interfere orproduce a false positive reaction. With some very reactive chromogenic substances,such as the diazonium ions, other classes of chemicals, such as aromatic amines andtheir salts, imidazoles, pyrroles, indoles, ammonium ion, histidine, hydrazine, hy-droxylamine, phenylalanine, tryptophane, tyrosine, arginine, also react to formcolored products under suitable conditions.Hence, with the use of this type of test the presence of a phenol, cresol, aromaticamine, or its salt in the material tested may cause an interfering or misleading re-action. This occurrence is of more than academic interest because many commercialvaginal douches, mouth and foot washes, gargles, suppositories, hemorrhoid medi-cants, anti-perspirants, contraceptive preparations, deodorizing formulas, sheepdips,poultryhouse sprays, and antiseptics contain amounts of phenolic substances suf-ficient to give interference or false positive reactions. This type of false results can bereliably detected by the use of proper controls.The control procedure is usually not detailed by the authors of the various refer-ences and texts, and in at least one published report is not considered at all. Inasmuchas the omission of, or improper use of controls may lead to a gross error in results ofthe test, it was proposed to test a number of theoretical interferences and outlinetest methods in such a manner so as to preclude as much interference as possible.PROCEDUREThe method of Seligman and Manheimer as described by Walker (4) was firstused. The substances tested were placed on cloth and the reagent added directlyto the cloth. No control tests were used.J. W. BRACKETT, JR.TABLE ISeligman-Manheimer lodified Seligman-ManheimerReagent W/O Controls Reagents W/ControlsKnown seminal stain ++ Red-brown color ++ Red-brown color1. Aniline sulfate EKC + Yellow-brown Reacted with chromogen2. Acetyl salicylic acid USP NR NR3. Thymol USP + Red-brown Reacted with chromogen4. 6-Chlorothymol EKC + Red-brown Reacted with chromogen5. Phenol USP ++ Red Reacted with chromogen6. Salicylic acid USP NR NR7. p-Ethylphenol EKC ++ Red Reacted with chromogen8. o-Toluidine EKC ++ Yellow-brown Reacted with chromogen9. b-Naphthylamine EKC ++ Purple Reacted with chromogen10. p-Aminobenzoic acid USP NR NR11. Menthol USP NR NR12. Eucalyptus oil USP NR NR13. Lysol USP ++ Red-brown Reacted with chromogen14. Diethyl stilbestrol USP NR NR15. Sulfanilamide USP NR NR16. p-Amino salicylic acid USP + Yellow-brown Reacted with chromogen17. Hydroquinone EKC NR NR18. Metol EKC + Yellow Reacted with chromogen19. Di-n-propyl phenol EKC + Yellow Reacted with chromogen20. Creosote USP ++ Red-brown Reacted with chromogen21. Hydroxylamine hydrochloride EKC ++ Brown Reacted with chromogen22. Ammonium chloride NR NR23. Indole (Delta) NR NR24. Hydrazine sulfate + Yellow Reacted with chromogenNR No reaction + Slight color reaction ++ Intense color reactionIn order to facilitate proper control tests, essentially the same method and reagentswere used, except the buffer solution was divided into two equal parts and the sub-strate (sodium alpha naphthyl phosphate) and one-half of the aerosol was added toone portion and the chromogen (anthroquinone-1 diazonium hydrochloride) and theremainder of the aerosol was added to the other. A portion of each of the substratesolution and chromogen solution was reserved and an equal volume of buffer addedto each to equalize control and test solution concentrations. The test solution properconsisted of a mixture of equal volumes of each of the undiluted substrate and chromo-gen solutions.Three control tests were made for each evidence test:1. A test using buffered substrate (diluted) on the stain2. A test using buffered chromogen (diluted) on the stainOne small drop of each of these two solutions was added directly to the stainat separate points3. A test using the mixed test solutionOne small drop of the mixed reagent was added to the cloth adjacent to butnot touching the stain.After 2 minutes the control tests were examined. If any of the control tests developcolor, the acid phosphatase test may give unreliable or misleading results. If thecontrol tests produced no color, a small drop of the mixed reagent was added to the[Vol. 47U2 Ho 480 00, un Z) ) r. co00 0*00+, P0 +~ C c4 .r0~ 0_or 3- &-Z. .0Z -o -0- ~~~C -d 0.P~d o*--or 30 A ~t 34to 0.CC)E! 0..0~~ C)c --, U)o 2 dud o -. 3-Pco *- pC .>uo L)u d 4 ~ .4> C0- 0 .c.00 Q 02 w 00 0 0 0m~p ch En2~ ~ 0 2UC) C)d=f o as6 ~~ 0.0 .=s 0 U30C 0 0 cd j mU2 H A H :1957] TEST FOR SEMINAL STAINS,o00So,00-0 0zC, C.)C)+C+o)U4".0o.J. W. BRACKETT, JR.stain, and the results noted after 2 minutes at room temperature. If a red colorationwas formed, sufficient acid phosphatase was present to justify a conclusion that thestain was of seminal origin.RESULTSResults on 2 mgs. each of a number of pure chemicals are shown in Table I. Tensubstances responded to the test with production of red, red-brown, or yellow-browncolor.Results on eleven commercially available proprietary drugs as received, in solutionrecommended by manufacturer for use as vaginal douche, and on the stain caused byevaporation of such a solution are shown in Table H. Six of these preparations re-sponded to the test as pure substances; two responded in dilutions recommended as adouche, and these two also gave positive tests 24 hours after the douche had beenapplied and allowed to dry. Several of the douche solutions responded with faintreactions when diluted as recommended as a vaginal douche; if the active materialconcentrations were higher, as when these substances were prepared for use as ageneral antiseptic, germicide, or with accidental increase in active material, addi-tional false positive tests could be expected.DIscussIoNMany chemical substances and some medicinal substances may interfere or maycause serious error with this type acid phosphatase test unless proper controls areused. Especially remarkable is lysol and creolin, both recommended by manufacturerfor vaginal douches, which gives a marked positive test in solution diluted 1:200with water. In all interference cases tested, proper control tests revealed the fact thatan interfering substance was present. The same principles are applicable to othertest methods for detection of enzymes which use as a test indicator the presence orabsence of a colored substance formed from a hydrolytic product of an added sub-strate and a very reactive, non-specific chromogen.REFERENCES1. FisHER, R. S., Acid Phosphatase Tests as Evidence of Rape. NEw ENGLAND J. MED. 240: 738(1949)2. KAYE, SIDNEY, Acid Phosphatase Test for Identification of Seminal Stains. J. LAE. CL. MED. 34:p 728 (1949)3. KIRK, PAuL L., CME INVESTIGATION, Physical Evidence and the Police Laboratory. p 671,Interscience Publishers, Inc., New York (1953)4. WALKER, J. T., A New Test for Seminal Stains. NEW ENGLAND J. MED. 242: 110 (1950)[Vol. 47

Acid Phosphatase Test for Seminal Stains--The Importance of Proper Control Tests, The

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Acid Phosphatase Test for Seminal Stains--The Importance of Proper Control Tests, The

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