The use of acid phosphatase test papers for DNA profiling

JOURNAL OF THE FORENSIC SCIENCE SOCIETY

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Volume 45 No.2 Apr - Jun 2005ISSN 1355-0306

&justice

The Journal is alsothe official organof the California

Association ofCriminalists

Scientific and technical

Der Saure-Phosphatase-Test ist eine Routinetest fiir die Anwe-senheit von Sperma auf Spurenmaterial. Dieser Farbtest wirdauf Filterpapier durchgefiihrt, das anschlieBend asserviert wird.Auf zwei Jahre alten Saure-Phesphatase-Test-Papieren wurdehinreichend DNA-haltiges Material fiir eine STR-Typisierunggefunden. Vorzugsweise wurde es vor der PCR-Amplifikationin Sperma-arme und Sperma-angereicherte Zellfraktionen aufge-trennt. Die Auswirkungen dieser Ergebnisse auf zuriickliegendeund aktuelle Falle wird diskutiert.

The use of acid phosphatase test papersfor DNA profiling

A Reshef, M Barash, N Gallili, A Michael and P Brauner'Forensic Biology Laboratory, Israel Police, Israel Police National HQ, Jerusalem, 91906 Israel

Science & Justice 2005 45 97 - 102 Received 22 November 2004 Accepted 21 February 2005

The acid phosphatase (AP) test is a routine assay used to screencasework items for the possible presence of semen. This colourtest is carried out on filter paper which is retained after testing.Two-year-old AP test papers were found to contain sufficientDNA for short tandem repeat (STR) profiling. Prior to polymerasechain reaction (PCR) amplification, the DNA was preferentiallyseparated into sperm depleted and sperm enriched cell fractions.The implication of these findings for past and present cases isdiscussed.

Le test a I' acide phosphatase (AP) est un test de routine utilisepour rechercher rapidement la presence de semence sur desechantillons de cas reels. Ce test colore est applique sur dupapier-filtre qui est garde apres Ie test. Des papiers-tests AP,vieux de deux ans, contenaient encore suffisamment d' ADNpour determiner un profil STR (short tandem repeat). Avantl'amplification par PCR, I'ADN a ete separe en fractions pau-vre en spermatozoides et enrichie en cellules de spermatozoides,L'implication de ces observations pour des cas passes et presentsest discutee.

science&justice Volume 45 No.2 (2005) 97 -102

La fosfatasa acida (AP) es un ensayo de rutina utilizado en losobjetos a analizar la presencia de semen. Este test de color serealiza en un papel de filtro que se puede guardar despues delanalisis. Se encontr6 que papeles de test de dos afios de antigiiedadcontenian suficiente ADN como para proceder a un perfil deADN por PCR. Antes de poceder a esta amplificaci6n por PCR elADN fue preferencialmente separado en esperma enriquecido enfracciones celulares y esperma depleccionado de ellas. Se discutela implicaci6n de estos hallazgos para el pasaso y presente de loscasos.

*Author for correspondence© Israel National Police 2005Key words Acid phosphatase test papers, DNAprofiling, short tandem repeats, 8GM plus, silver staining,post-conviction DNA testing

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A Reshef, M Barash, N Gallili, A Michael and P BraunerThe use of acid phosphatase test papers for DNA profiling

IntroductionFor over 50 years, the acid phosphatase assay has been widelyused by forensic laboratories as a presumptive test for the detec-tion of semen stains [1,2]. The test is carried out on a wet piece offilter paper, which has been brought into direct physical contactwith a stain on a casework item. An amount of biological materialfrom the stain diffuses on to the paper. Subsequently, the paperis moistened with the acid phosphatase (AP) reagent. A positivecolour reaction indicates the possible presence of seminal mate-rial. Upon drying, these papers are retained as evidentiary caseresults.

We found that two-year-old AP test papers contained sufficientDNA for STR profiling by the second generation multiplex plus(SGM Plus™) and GenePrint® STR silver staining systems.By employing a preferential method to extract DNA from APpositive papers, a separation of sperm and non-sperm cells wasaccomplished in two cases.

In some instances, these AP test papers may be the sole sourceof DNA from a crime scene and therefore playa crucial role inpost-conviction DNA testing.

The casesCase 1In 2000, our laboratory received a sexual assault case in whichan assailant stalked a 16-year-old girl as she was returning home.Failing to rape his victim - no penetration took place - the attackerdeposited his semen on a grassy piece of turf (Figure 1). Shortlyafter the assault, the victim informed the police, who removed theturf and submitted it for examination, together with the referenceblood samples of the suspect and complainant.

The AP test carried out on the turf gave a positive reaction(Figure 2). Following the test, the AP paper was dried and storedat room temperature. From the grass, sperm cells were micro-scopically identified in a haematoxyl/eosin stained extract.

Blades of grass that had given an AP positive result were sam-pled. From these samples, DNA was preferentially extracted andanalyzed using the GenePrint® STR systems (Promega).

Figure 1 Piece of grassy turf on which semen was found.

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Figure 2 The result of the AP test carried out on the pieceof grassy turf. The dark area on the paper showsa positive reaction. DNA was extracted from thethree cut-out pieces of filter paper (Case 1).

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Two years later, the AP positive paper from this case, storedat room temperature, was sampled. DNA was preferentiallyextracted and analyzed using the SilverSTR® ill Multiplex(Promega).

Case 2In 2002, we received a case in which a six-year-old male child wassodomised by a 14.5-year-old male. The trousers of the child andbuccal swabs, taken from the suspect and victim, were submittedfor examination.

The AP test carried out on the child's trousers gave a positiveresult (Figure 3). Following the test, the AP paper was dried andstored at room temperature. From the trousers, sperm cells weremicroscopically identified in a haematoxyl/eosin stained extract.

Areas on the trousers that had given an AP positive result weresampled. From these samples, DNA was preferentially extractedand analyzed using the GenePrint® STR systems (Promega).

Two years later, the AP positive paper from this case, storedat room temperature, was sampled. DNA was preferentially ex-tracted and analyzed using the ABI AmpFlSTR® SGM Plus™kit (Applied Biosystems).

Materials and methodsAcid phosphatase testDampened pieces of filter paper were placed on different areasof the grassy turf (Case 1) and the trousers (Case 2) suspected tocontain seminal material. Pressure applied to the paper resultedin direct contact between the paper and the crime scene item.The moisture from the paper solubilised biological substancesfrom the stain, including seminal material, which was present inthe two cases. The papers, separated from the crime scene items,were then moistened with the Brentamine (AP) reagent, in orderto visualise the result.

science&justice Volume 45 No.2 (2005) 98 - 102

Figure 3 The result of the AP test carried out on thecomplainant's trousers. The dark area on thepaper shows a positive reaction. DNA wasextracted from the two cut-out pieces of filterpaper (Case 2).

The Brentamine reaction takes place in the presence of AP, whichis abundant in seminal fluid. Naphthol is released from sodium (¥-

naphthyl phosphate by the acid phosphatase enzyme. This resultsin the formation of a purple azo dye - a positive reaction - causedby the coupling of naphthol to the buffered Brentamine Fast BBlue salt reagent [3].

DNA extraction methodsDNA was extracted from five blades of grass (Case 1) and fromstains on the trousers (Case 2) by the preferential organic method[4]. The preferential separation resulted in two fractions; thesperm cell depleted, or S (supernatant) fraction and the spermcell, or P (pellet) fraction.

Both one and 2 cm2 pieces were cut from each of the two-year-oldAP papers (Figures 2 and 3). Preferential separation by organicDNA extraction was carried out on biological material from thepaper, as above.

Reference blood samples from the complainant and suspect(Case 1) and buccal samples from the complainant and suspect(Case 2) were extracted using the organic method [5].

DNA typingDNA (Case 1) from the casework items was amplified us-ing the Promega GenePrint® STR systems, the "Silver STR®III triplex" (DI6S539, D7S820 and D13S317 loci), the "CTTtriplex" (CSFIPO, TPOX and THOlloci) and the "FFv triplex"(FI3A01, FESFPS and vWA loci). DNA from the AP paperswas amplified by the "Silver STR® III triplex" and the "CTTtriplex" only. Amplified PCR products were separated on a 4%denaturating polyacrylamide gel and silver stained [6].

science&justice Volume 45 No.2 (2005) 99 - 102


DNA (Case 2) from both the case items and the AP paper wasamplified using the AmpFlSTR ® SGM Plus ™ kit, in accordancewith the manufacturer's recommendations (PE Applied BioSys-terns, Foster City, CA). Amplified PCR products were analyzedby ABI PRISM® 3100 Genetic Analyzer. Electrophoretic sepa-ration was carried out using a 50 iuti capillary, 36 ern in length,loaded with POP-4 polymer. The running conditions were: 30 minrunning time, 5 see injection time and 60°C running temperature.Allele size was calculated using the Genescan® Analysis 3.7 soft-ware, calibrated by a known internallane size standard, ROX-500.

Figure 4 Polyacrylamide gel electrophoresis ofpreferentially separated DNA extracted from APfilter paper. Lane 1, the sperm cell fraction (P),lane 2, the sperm cell depleted fraction (8), laneL, the allelic ladders (Case 1).

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Genotyping was implemented by using Genotyper® 3.7 softw(PE Applied BioSystems).

ResultsCase 1Biological material, extracted from the blades of grass. \\':!S am-plifiable at the nine STR loci, namely, CSFIPO. TPOx.. mOl.F13AOI, FESFPS, vWA, D16S539, D7S820 and D13S3L Theresulting profile (S and P fractions) matched the suspect's orofileand differed from that of the complainant.

DNA, extracted from a 2 cm2 piece of the AP positive tesr paper.amplified at the D16S539, D7S820 and D13S31- loci. CSFlPO.TPOX and THOI failed to amplify. Biological material from theAP positive area of the two-year-old AP test paper gave the sameprofile from the P fraction as obtained from the blades of grass.

DNA from the S fraction of material extracted from the [est papergave a mixed profile, in which the suspect could have been one ofthe contributors. The complainant, however, could not have beena contributor to this mixture (Figure 4).

The alleles obtained from the biological material on the grassblades and from the two-year-old AP paper are presented inTable 1.

Biological material, extracted from a I cm2 piece of the APpositive paper, failed to amplify at the D16S539, D7S820 andD13S3I710ci.

Case 2Biological material, extracted from the trousers of the com-plainant, was amplifiable at the nine STR loci, namely, CSFIPO,TPOX, THOI, F13AOI, FESFPS, vWA, D16S539, D7S820 andD 13S3I7. From the S fraction, a mixed profile was obtained,while the P fraction profile matched that of the suspect and dif-fered from that of the complainant (results not shown).

DNA, extracted from a 2 cm2 piece of the acid phosphatase pos-itive test paper, gave a profile in the P fraction using the SGMPlus ™ kit. This profile matched the profile of the suspect in thecase (Figures 5 and 6).

the S fraction from the test paper gave a mixed profile,r.: u'1-:ir!Jme suspect's profile was a minor component. At certain

e suspect's DNA either partially amplified or failed to-~- results not shown).

Biological material, extracted from a 1 cm2 piece of the AP pos-- ive paper. failed to amplify at the SGM Plus ™ loci.

DiscussionAcid phosphatase is a test widely used by forensic laboratoriesfor detecting stains that potentially contain semen. However, apartfrom simply indicating the possible presence of semen, AP testpapers may provide significant, additional information relating tostains. Location, size and intensity of semen stains, as visualisedon the AP papers, may be critical factors in criminal investiga-tions. In light of the importance of AP papers as evidence, they aregenerally not discarded and, therefore may represent a valuablesource of inadvertently stored DNA.

We have demonstrated that, after a period of two years, DNA canbe profiled from biological material extracted from AP test papers.Results were obtained on DNA amplified by the SilverSTR® IIIMultiplex systems and by SGM Plus" kit. By preferentiallyseparating the biological material from the AP papers in twocases, we obtained a DNA profile in the P fraction that matchedthe profile of the suspect in each case, respectively.

The importance of DNA extraction by the preferential separationwas evident from the results obtained on the AP test paper in bothcases. In Case 1, the S fraction (Table 1) contained a mixed DNAprofile. The suspect could have been one of the contributors to thismixture, whereas the complainant was excluded as a contributor.(This was not a surprising result, as this was a case of sexualassault without penetration, in which semen was deposited ongrass in a public place.) The P fraction (Table 1), on the otherhand, contained a profile that matched the profile of the suspectwithout additional bands.

In Case 2, the S fraction contained a mixed profile in which thesuspect was the minor contributor. This resulted in both allele andloci dropouts. The P fraction DNA, however, amplified at all loci,

Table 1 Comparison of the alleles obtained from material sampled from grass blades at the time of the incident and APpapers sampled two years after testing.

Loci: 0165539 075820 0135317Blades of grass

Sperm cell depleted fraction S (supematant) 12, 12 10,9 12,8Sperm cell fraction P (pellet) 12, 12 10, 9 12,8

AP test paperSperm cell depleted fraction S (supematant) 12, 11 11, 10, 9, 8 12, 11, 8Sperm cell fraction P (pellet) 12, 12 10,9 12,8

Suspect's profile 12, 12 10,9 12, 8Complainant's profile 13,9 10,10 11, 9

Page 100 science&justice Volume 45 No.2 (2005) 100 -102


Figure 5 Electropherogram of the 8GM Plus™ profile of the AP test sperm cell fraction (P) in Case 2., Iii • i i i i I i I

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with a resulting profile that matched that of the suspect (Figures 5and 6).

scene samples and laboratory prepared slides containing biolog-ical material was proposed. In addition, biological samples, un-suitable for testing by previous techniques, were recommendedto be re-tested by the currently available DNA tests.DNA profiling of biological material from past casework items

may be an individual's sale means of proving his innocence. TheU.S. Department of Justice has recommended DNA testing, orre-testing of biological evidence, in cases in which exclusionaryresults may exonerate a petitioner [7]. Re-evaluation of crime

However, destruction, loss or improper storage of casework itemsmay preclude DNA profiling. Similarly, stains, entirely consumedin tests other than DNA, would be unavailable for profiling.

Figure 6 Electropherogram of the 8GM Plus™ profile of the suspect in Case 2.I I i I i I j

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science&justice Volume 45 No.2 (2005) 101 -102 Page 101


Moreover, there may be cases in which biological mate .the stains was profiled by an obsolete DNA technology.variable number tandem repeats (VNTR). Such cases w,tain results that are not comparable to results of currentmethods.

Biological samples plated on old microscope slides hdemonstrated to be a good source of material for D~-\ ~[8]. Similarly, we have found that AP test papers ~_ """"-""-:;alternative source of long-term, stored DNA thatestimable value in providing new evidence in oldpapers, like slides, are probably best archived inambient temperature.

Furthermore, in cases in which extracted D~-\ Iinot peR amplifiable, a profile may still be obtainableterial that was absorbed onto an AP test paper. The -time of the dampened filter paper with the stain 1Il3. _

the amount of peR inhibitors - such as non-fast eoloer - "lifted"on to the test paper. In this manner. the chances of successful PCRamplification maybe increased.

Finally, in the two cases in this report, we found a piece of APpaper smaller than 2 cm2 did not contain a sufficient quantityof DNA for peR amplification. Methods developed to profilevery small amounts of DNA [9] may overcome this difficulty.Additionally, Y-STR analysis may also be a successful method

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of i?=-'=-;.: ~logical material from AP papers originating fromperpetrator of a crime.

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The use of acid phosphatase test papers for DNA profiling

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