Legal Medicine xxx (2014) xxx–xxx

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Legal Medicine

journal homepage: www.elsevier .com/locate / legalmed

Case Report

Stability of human a-salivary amylase in aged forensic samples

http://dx.doi.org/10.1016/j.legalmed.2014.03.0041344-6223/� 2014 Elsevier Ireland Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +39 055 7946204; fax: + 39 055 7946200.E-mail address: ricciu@aou-careggi.toscana.it (U. Ricci).

Please cite this article in press as: Carboni I et al. Stability of human a-salivary amylase in aged forensic samples. Leg Med (2014), http://dx.d10.1016/j.legalmed.2014.03.004

Ilaria Carboni a, Stefano Rapi b, Ugo Ricci a,⇑a Azienda Ospedaliero-Universitaria ‘‘Careggi’’, Diagnostic Genetics Unit, Largo Brambilla, 3, I-50134 Florence, Italyb Azienda Ospedaliero-Universitaria ‘‘Careggi’’, Central Laboratory, Largo Brambilla, 3, I-50134 Florence, Italy

a r t i c l e i n f o

Article history:Received 22 November 2013Received in revised form 11 March 2014Accepted 17 March 2014Available online xxxx

Keywords:AmylaseSalivaForensic geneticsAged samplesRSID™-Saliva

a b s t r a c t

The unequivocal tissue identification in forensic casework samples is a key step for crime scene recon-struction. Just knowing the origin of a fluid can sometimes be enough to either prove or disprove a factin court. Despite the importance of this test, very few data are available in literature concerning humansaliva identification in old forensic caseworks. In this work the stability of human a-amylase activity inaged samples is described by using three different methods integrated with DNA profiling techniques.This analytical protocol was successfully applied on 26-years old samples coming from anonymous threatletters sent to prosecutors who were working on ‘‘the Monster of Florence’’, a case of serial murders hap-pened around Florence (Italy) between 1968 and 1985.

� 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Saliva is a common body fluid, easily found in many differentforensic samples like cigarette butts, stamps or balaclavas. Overthe years different methods have been routinely described in orderto identify saliva stains, mostly based on the detection of human a-amylase (1,4 a-D-glucan glucanohydrolase). a-Amylase enzymesare proteins produced and stored mainly in salivary glands andpancreas, both secretory organs; for this reason a-amylase canbe found in many body fluids as breast milk, pancreas, semenand vaginal secretions [1–3]. It is known that amylase activity insaliva stains rapidly decreases during the first hours of drying,but afterwards is rather stable throughout the years at room tem-perature in the dark [4]. Hence, amylase testing may be used to de-tect a dried saliva stain several months after deposition eventhough amylase activity varies greatly between individuals [5].

The identification of this fluid is performed using a wide rangeof tests based on the detection of the enzymatic activity or a-amy-lase presence that have been developed over the years [6]. One ofthe most accepted presumptive test uses blocked p-nitrophenyl-maltoheptaoside (BpNPG7) as substrate to detect a-amylaseactivity; it can be performed in manual [7] or automatic [8] assays.Recently, a confirmatory test based on two anti-human salivaryamylase monoclonal antibodies on an immunochromatographicstrip test was developed [9]. It detects the presence of salivary

amylase, rather than enzymatic activity. Other promising tech-niques for saliva detection are mRNA [10] and colorimetric assaykit [11], but at the moment these techniques are not used inroutine cases. Anyway, tissue identification in forensic caseworkis a crucial step for crime scene reconstruction, so it should alwaysbe performed. Even when the biological origin of a stain may seemobvious for the forensic investigator, scientific confirmation withreliable methods is necessary in court. Moreover, in forensic inves-tigation a screening method based on amylase testing could beuseful to lower the number of negative DNA samples from crimescenes [12]. It was reported that without pre-testing, over than50% of presumed crime scene saliva stains can be expected to yieldinsufficient DNA amounts [13].

Notwithstanding the many available techniques and the impor-tance of the test, there are very few data about saliva identificationon aged forensic samples [2,14,15,4]. In this report the combineduse of three independent assays on old simulated stains and in26-year-old casework samples integrated with DNA analysis isshown.

2. Case report

A prosecutor requested the analysis of three anonymous letters,in order to identify the sender by DNA profiling from the envelopescontaining them. The event concerned ‘‘the Monster of Florence’’[16] case, a serial killer that in the years between 1968 and 1985murdered and brutally mutilated eight couples in the Florence(Italy) countryside. The gun was always the same but despite theinvestigations made and the many suspects the killer still remains

oi.org/

2 I. Carboni et al. / Legal Medicine xxx (2014) xxx–xxx

unknown. After the last murder, three prosecutors who wereworking on this complex case received three letters containingdeath threats. The missives lay forgotten for 26 years at room tem-perature in an old file, until a prosecutor decided to have the threeanonymous letters analysed in order to identify the sender by DNAprofiling. Before approaching the real old casework samples, theanalytical protocol was verified on old simulated forensic samplesconsisting in stamps on postcards, envelopes, cigarette butts andchewing-gums, prepared in laboratory many years before. In orderto extend the protocol, ten cigarette butts collected in the streetswere also analysed.

3. Materials and methods

3.1. Samples

The simulated forensic samples consisted of 10 stamps on 10postcards sent in 1974 (1S) and in the years between 1992 and1997 (from 2S to 10S); 10 envelopes sealed 10 years before con-taining 20 ll saliva (1W, 2W, 3W, 4W, 5W, 1Y, 2Y, 3Y, 4Y and5Y); three 10-year-old cigarette butts (CB1, CB2 and CB3); two7-year-old chewing-gums (CG1 and CG2); 10 cigarette butts col-lected from the streets (from ST1 to ST10). Also, five envelopedspotted each by 2 ll saliva were dried and prepared 2 days beforethe analysis, to be used as positive control. All simulated samplesprepared in laboratory were obtained after an informed consentfrom known donors and stored at room temperature. Additionally,negative controls were prepared using RSID™-Saliva extractionbuffer on a piece of sterile paper.

Real casework samples consisted of three anonymous enve-lopes (A, B and C) sent in 1986 to prosecutors who were workingon ‘‘the Monster of Florence’’ case. Three samplings from eachenvelope were performed, two coming from the closing strip (A1,A2, B1, B2, C1 and C2) and one coming from the bottom of eachenvelope (A3, B3 and C3). The samplings coming from the bottomwere used as negative control since the presence of saliva in thoseareas was considered very unlikely.

3.2. Samples preparation

Disposable scalpels and tissue forceps were used to cut andmanage all samples. Negative controls were prepared with allreagents except DNA. Each sample, consisting of 1 cm2 paper,chewing gum or cigarette butt paper was cut and placed in a1.5 ml tube with 300 ll of RSID™-Saliva extraction buffer (Galan-tos Genetics GmbH, Mainz, Germany) containing 10 mM Tris–HCland 0.1 mM EDTA at pH 8.0, vortexed and incubated for 3 h at37 �C. The whole extract was transferred in a new 1.5 ml tube con-taining a sterile spin basket and centrifuged at 8000 rpm for 3 min.The precipitate was used for DNA extraction, while the supernatantwas used for the a-amylase tests applying the protocol describedbelow.

3.3. Detection of human a-amylase

In order to detect human a-amylase three different methodswere applied: BNP-Amylase test (Sclavo-Dasit, Milano, Italy),RSID™-Saliva (Galantos Genetics GmbH, Mainz, Germany) and asemi-automated method used in clinical laboratories (SiemensHealthcare Diagnostics, Tarrytown, NY, USA). Since this last meth-od is not validated for forensic purposes, its application is at themoment limited on supplying an additional objective evaluation,unlike both RSID™-Saliva (Galantos Genetics GmbH) and BNP-amylase method (Sclavo-Dasit, Milano, Italy), which need a nakedeye interpretation by the operator.

Please cite this article in press as: Carboni I et al. Stability of human a-saliv10.1016/j.legalmed.2014.03.004

3.3.1. BNP-amylase test (Sclavo-Dasit, Milano, Italy)In order to test amylase activity, 50 ll supernatant was mixed

with 50 ll amylase CNPG3 reagent as described [17] and incubatedat 37 �C for 30 min. a-Amylase hydrolyzes the 2-chloro-4-nitro-phenyl-a-D-maltotrioside to release 2-chloro-4-aminophenol andforms 2-chloro-4-nitrophenyl-a-D-maltoside, maltotriose, and glu-cose. Positive reactions due to the presence of p-nitrophenol, stoi-chiometrically formed, lead to a dye tuning from colorless toyellow detectable even to the naked eye. The observed kinetic dif-ferences were also detectable even to the naked eye. Results eval-uation was recorded in 0 to +3 scale.

3.3.2. Clinical chemistry system (Siemens Healthcare Diagnostics,Tarrytown, NY, USA)

The AMYLAS method uses ethylidene blocked p-nitrophenyl-maltoheptaoside as substrate, in association with a-glucosidaseenzyme used to release p-nitrophenol. The terminal glucose ofthe substrate is chemically blocked, preventing cleavage by theindicator enzymes.

Calibration system is based on the specific molar extinctioncoefficients of free p-nitrophenol at 410 nm, related to IFCC refer-ence for human serum and urine and are reported as units of enzy-matic activity (1 IU = 1 lmol of p-nitrophenol/min at second) [8].

System performance was verified automatically using two con-trol levels (Multiqual Level 1 and Level 2, Bio-Rad Laboratories);the range of detection indicated by the manufacturer is0–1500 U/L. In order to verify the methods linearity, a specific cal-ibration curve was performed by using a 0.003 units/ml dilution ofamylase standard purchased from Sigma Chemical CompanyA1031, Lot 080M7000 V (5000 units of activity) as suggested byLaux [18]. The calibration standard curve made with a specific hu-man amylase standard indicates the possibility to quantify up to 5units amylase. The R2 value of 0.9967 indicates a close fit betweenthe standard curve regression line. The absorbance was twice mea-sured and the instrument background was tested using negativecontrol consisting of RSID™-Saliva extraction buffer. Activityvalues under 5 U/L were considered as instrument background.100 ll of samples were loaded on Siemens system. Enzymaticactivity was calculated according to the formula reported on theuser’s test sheet and it was expressed as International Units per li-ter (U/L).

3.3.3. Rapid stain identification of human saliva (RSID™-SalivaGalantos Genetics GmbH)

In order to verify the presence of salivary amylase, 40 ll ofsupernatant were mixed with 60 ll of TBS+ running buffer (0.01%Twee-20, 0.01% sodium azide, 0.5% BSA [Fraction V], 0.05 M Tris/Cl, 0.0027 M KCl, 0.137 NaCl). The extract was added on RSID™-Saliva strip and results were scored and recorded after 10 min.The strip has a control line that has to appear in any case, other-wise the test has to be considered invalid; if saliva is present, amy-lase will react with strip-binding antibodies and a second band willappear, showing a double banded strip. If only the control lineappears, the result is negative [9]. Results evaluation was recordedas 0 to +3 scale.

3.4. DNA analysis

Sample pellets were extracted in 20 ll final volume usingQIAmp� DNA Micro Kit (QIAGEN, Germany) according to manufac-turer’s manual. DNA was quantified using Quantifiler� HumanDNA Quantification Kit and amplified with AmpFlSTR� Identifiler™Amplification Kit (Life Technologies, USA). Capillary electrophore-sis was performed on 3130 Genetic Analyzer (Life Technologies,USA). All genetic profiles were compared with laboratory staff, inorder to exclude samples contamination.

ary amylase in aged forensic samples. Leg Med (2014), http://dx.doi.org/

Table 2Summary of results for a-amylase tests and DNA quantification in 26 years oldenvelopes.

Sample BNP-amylase RSID™ Saliva Siemens [U/l] [DNA] ng

A1 + ++ 54 1.6A2 ++ ++ 87 2.4A3 – – 4 0B1 – + 32 1.6B2 ++ ++ 86 2.0B3 – – 4 0C1 + + 41 1.8C2 + + 65 1.7C3 – – 4 0

A1, A2, B1, B2, C1and C2: samples from envelopes A, B and C; A3, B3, C3: negativecontrols from the bottom of the envelopes.

I. Carboni et al. / Legal Medicine xxx (2014) xxx–xxx 3

4. Results

Table 1 shows the results on simulated saliva samples, analysedbefore processing casework samples. Amylase activity was notconfirmed in two samples (2W and CB3) using BNP-amylase meth-od. This result could be explained because of sampling, of partial ortotal enzyme degradation, of unfolded status of the protein, ofactive site modification or of erroneous results interpretation dueto the yellowish colored samples (cigarette butts). Anyway, in2W and CB3 samples amylase activity was confirmed by RSID™-Saliva and Siemens clinical chemistry system: 2W and CB3 sam-ples gave respectively 29 and 25 U/L values. RSID™-Saliva detectedthe presence of the enzyme in all simulated forensic samples. Po-sitive and negative controls resulted as expected. After DNA extrac-tion, Real-Time quantification highlighted human DNA presence inthe saliva samples from the envelopes, the old smoked cigarettebutts and the two chewing-gums (Table 1). DNA typing using aforensic validated kit confirmed donors’ DNA profile. In the stampson envelopes and in the cigarette butts collected in the street, po-sitive results were obtained both for amylase presence and activity,with different degrees of positivity (Table 1). However, in fivestamps, including the 39-year-old one, and in one cigarette buttcollected in the street, no human DNA was detected in real time.

The same protocol was finally applied to 26 years old caseworksamples. All samples coming from the closing strip gave positiveresults for a-amylase presence and activity (Table 2). B1 samplefailed for amylase activity presence when tested with BNP-amylasemethod but the amylase activity and presence was confirmed byusing automated clinical system and RSID™-Saliva. The samples

Table 1Results summary from old simulated forensic samples using three different methodsto detect amylase and DNA quantification.

Sample BNP (a-amylase) RSID™ Saliva Siemens [U/l] [DNA] ng

1W + ++ 56 1.122W – + 29 0.583W + + 43 0.864W + + 154 3.085W + + 58 1.161Y ++ ++ 85 1.702Y ++ ++ 117 2.343Y ++ ++ 106 4.124Y +++ +++ 49 0.985Y +++ +++ 233 4.661S + ++ 645 02S ++ + 633 03S ++ + 664 0.244S + ++ 204 05S ++ ++ 184 0.116S ++ +++ 2085 07S + + 2250 0.088S ++ ++ 2035 0.139S + +++ 2355 0.5510S ++ + 768 0CB1 ++ ++ 111 2.22CB2 +++ +++ 216 4.32CB3 – + 25 0.50CG1 ++ ++ 137 2.74CG2 ++ ++ 98 1.96ST1 +++ ++ 171 1.40ST2 ++ ++ 188 1.30ST3 +++ +++ 1677 10.90ST4 ++ ++ 1050 3.81ST5 ++ + 194 5.74ST6 ++ ++ 277 0ST7 ++ ++ 87 1.74

W and Y = envelopes; S = stamps on postcards.CB = 10-years-old cigarette butts.CG = chewing-gums.ST = cigarette butts on the street.

Please cite this article in press as: Carboni I et al. Stability of human a-saliv10.1016/j.legalmed.2014.03.004

collected from the bottom of the envelopes gave negative results.DNA coming from envelopes ranged from 1.6 to 2.4 ng of total hu-man DNA in 20 ll TE buffer; the presence of inhibitors was ex-cluded by IPC probe contained in Quantifiler�. All DNA profilesobtained using AmpFlSTR� Identifiler� Plus Kit were attributed tothe same unknown donor (data not shown).

5. Discussion

Saliva stains are one of the most common biological fluid foundin crime scenes. Although many protocols have been developedand have been described over the years and despite the importanceof the identification of a biological sample before the DNA test, thedetection of a body fluid is an underestimated analytical step.However, sometimes just knowing the biological origin of a samplecan be enough to influence the outcome of a case. When this kindof analyses are omitted or incorrectly performed, the evidence incourt can fail, even when positive results are obtained by DNAanalysis; for this reason the laboratory data have to be performedwith reliable and robust protocols, in order to be supported incourt discussion [19].

Biological identification tests have some degree of subjectivity[20], because a human ruling is often required to confirm orexclude body fluid presence in a sample. In order to minimize erro-neous interpretations, both presumptive and confirmatory testsshould always be performed before proceeding with DNA typing.The introduction of an automated analysis method, not validatedfor forensic purposes, has confirmed amylase activity, just support-ing data already obtained with Amylase and RSID™-Saliva tests,widely accepted by forensic community. These methods that canbe used independently depending on workflow strategy, consis-tently showed the presence of amylase activity, even many yearsafter deposition.

Though amylase test is sensitive and reliable, eye interpretationis required in order to evaluate samples positivity or negativity.The evaluation is easy in solutions coming from uncolored sub-strates, but it could become more difficult when very low salivaquantity is present in the solution or when the substrate is yellow-ish-colored, as in cigarette butts. For this reason amylase test isvery useful for a primary sample screening, while RSID™- Salivatest should be used in a second step to confirm the salivary a-amy-lase presence. The clinical chemistry system is used in this workonly as a supporting test to confirm amylase activity. Furthervalidation studies should be performed before considering the pos-sibility to routinely introduce this automated system in forensicpractice.

In this work, amylase presence and activity has been confirmedon a wide variety of old samples containing saliva (envelopes, post-age stamps on postcards, cigarette butts and chewing-gums). How-ever, amylase activity levels and DNA concentration in saliva

ary amylase in aged forensic samples. Leg Med (2014), http://dx.doi.org/

4 I. Carboni et al. / Legal Medicine xxx (2014) xxx–xxx

varied greatly between samples; in fact, there was no proportionalcorrelation between presumptive tests intensity and DNA quantifi-cation. For example, in sample 2Y the observed value of amylaseactivity was 117 U/l, and the final DNA was 2.34 ng, while in sam-ple 3Y the final DNA quantity was approximately double despitethe same observed value of the amylase activity. In the sample6S, the amylase activity value was 2085 U/l, but no human DNAwas identified in the sample. This can be explained consideringthat amylase is an extracellular enzyme produced by salivaryglands, while DNA amount depends on shedding buccal cells inthe oral cavity or exudates [5], therefore a quantity correlation isnot always to be expected.

The encouraging results concerning enzymatic stability in oldsamples led up to analyze the real casework case, successfullydemonstrating saliva presence in 26 years old threatening missivesstored for years at room temperature. Thus, a reliable degree ofconfidence before proceeding with DNA analysis was supplied.DNA extraction, quantification and PCR amplification confirmedhuman DNA presence. The genetic profile was reproducible andthis will be useful to throw light on this complex cold case, stillunresolved.

Acknowledgements

The authors are grateful to Dr. Paolo Canessa, Court of FlorencePublic Prosecutor, for his cooperation. We also thank Sigma Chem-ical Company for providing the standard Amylase, Marco Melonifor technical assistance and the Association of Forensic Identifica-tion (A.I.Fo., www.aifo-italia.it) for the financial support.

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