Legal Medicine xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Case Report
Stability of human a-salivary amylase in aged forensic samples Ilaria Carboni a, Stefano Rapi b, Ugo Ricci a,⇑ a b
Azienda Ospedaliero-Universitaria ‘‘Careggi’’, Diagnostic Genetics Unit, Largo Brambilla, 3, I-50134 Florence, Italy 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 2013 Received in revised form 11 March 2014 Accepted 17 March 2014 Available online xxxx Keywords: Amylase Saliva Forensic genetics Aged samples RSID™-Saliva
a b s t r a c t The unequivocal tissue identification in forensic casework samples is a key step for crime scene reconstruction. Just knowing the origin of a fluid can sometimes be enough to either prove or disprove a fact in court. Despite the importance of this test, very few data are available in literature concerning human saliva identification in old forensic caseworks. In this work the stability of human a-amylase activity in aged 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 threat letters sent to prosecutors who were working on ‘‘the Monster of Florence’’, a case of serial murders happened around Florence (Italy) between 1968 and 1985. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Saliva is a common body fluid, easily found in many different forensic samples like cigarette butts, stamps or balaclavas. Over the years different methods have been routinely described in order to identify saliva stains, mostly based on the detection of human aamylase (1,4 a-D-glucan glucanohydrolase). a-Amylase enzymes are proteins produced and stored mainly in salivary glands and pancreas, both secretory organs; for this reason a-amylase can be found in many body fluids as breast milk, pancreas, semen and vaginal secretions [1–3]. It is known that amylase activity in saliva stains rapidly decreases during the first hours of drying, but afterwards is rather stable throughout the years at room temperature in the dark [4]. Hence, amylase testing may be used to detect a dried saliva stain several months after deposition even though amylase activity varies greatly between individuals [5]. The identification of this fluid is performed using a wide range of tests based on the detection of the enzymatic activity or a-amylase presence that have been developed over the years [6]. One of the most accepted presumptive test uses blocked p-nitrophenylmaltoheptaoside (BpNPG7) as substrate to detect a-amylase activity; it can be performed in manual [7] or automatic [8] assays. Recently, a confirmatory test based on two anti-human salivary amylase monoclonal antibodies on an immunochromatographic strip test was developed [9]. It detects the presence of salivary
⇑ Corresponding author. Tel.: +39 055 7946204; fax: + 39 055 7946200. E-mail address: [email protected] (U. Ricci).
amylase, rather than enzymatic activity. Other promising techniques for saliva detection are mRNA [10] and colorimetric assay kit [11], but at the moment these techniques are not used in routine cases. Anyway, tissue identification in forensic casework is a crucial step for crime scene reconstruction, so it should always be performed. Even when the biological origin of a stain may seem obvious for the forensic investigator, scientific confirmation with reliable methods is necessary in court. Moreover, in forensic investigation a screening method based on amylase testing could be useful to lower the number of negative DNA samples from crime scenes [12]. It was reported that without pre-testing, over than 50% of presumed crime scene saliva stains can be expected to yield insufficient DNA amounts [13]. Notwithstanding the many available techniques and the importance of the test, there are very few data about saliva identification on aged forensic samples [2,14,15,4]. In this report the combined use of three independent assays on old simulated stains and in 26-year-old casework samples integrated with DNA analysis is shown. 2. Case report A prosecutor requested the analysis of three anonymous letters, in order to identify the sender by DNA profiling from the envelopes containing them. The event concerned ‘‘the Monster of Florence’’ [16] case, a serial killer that in the years between 1968 and 1985 murdered and brutally mutilated eight couples in the Florence (Italy) countryside. The gun was always the same but despite the investigations made and the many suspects the killer still remains
http://dx.doi.org/10.1016/j.legalmed.2014.03.004 1344-6223/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
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.doi.org/ 10.1016/j.legalmed.2014.03.004
2
I. Carboni et al. / Legal Medicine xxx (2014) xxx–xxx
unknown. After the last murder, three prosecutors who were working on this complex case received three letters containing death threats. The missives lay forgotten for 26 years at room temperature in an old file, until a prosecutor decided to have the three anonymous letters analysed in order to identify the sender by DNA profiling. Before approaching the real old casework samples, the analytical protocol was verified on old simulated forensic samples consisting in stamps on postcards, envelopes, cigarette butts and chewing-gums, prepared in laboratory many years before. In order to extend the protocol, ten cigarette butts collected in the streets were also analysed. 3. Materials and methods 3.1. Samples The simulated forensic samples consisted of 10 stamps on 10 postcards sent in 1974 (1S) and in the years between 1992 and 1997 (from 2S to 10S); 10 envelopes sealed 10 years before containing 20 ll saliva (1W, 2W, 3W, 4W, 5W, 1Y, 2Y, 3Y, 4Y and 5Y); three 10-year-old cigarette butts (CB1, CB2 and CB3); two 7-year-old chewing-gums (CG1 and CG2); 10 cigarette butts collected from the streets (from ST1 to ST10). Also, five enveloped spotted each by 2 ll saliva were dried and prepared 2 days before the analysis, to be used as positive control. All simulated samples prepared in laboratory were obtained after an informed consent from known donors and stored at room temperature. Additionally, negative controls were prepared using RSID™-Saliva extraction buffer on a piece of sterile paper. Real casework samples consisted of three anonymous envelopes (A, B and C) sent in 1986 to prosecutors who were working on ‘‘the Monster of Florence’’ case. Three samplings from each envelope were performed, two coming from the closing strip (A1, A2, B1, B2, C1 and C2) and one coming from the bottom of each envelope (A3, B3 and C3). The samplings coming from the bottom were used as negative control since the presence of saliva in those areas was considered very unlikely. 3.2. Samples preparation Disposable scalpels and tissue forceps were used to cut and manage all samples. Negative controls were prepared with all reagents except DNA. Each sample, consisting of 1 cm2 paper, chewing gum or cigarette butt paper was cut and placed in a 1.5 ml tube with 300 ll of RSID™-Saliva extraction buffer (Galantos Genetics GmbH, Mainz, Germany) containing 10 mM Tris–HCl and 0.1 mM EDTA at pH 8.0, vortexed and incubated for 3 h at 37 °C. The whole extract was transferred in a new 1.5 ml tube containing a sterile spin basket and centrifuged at 8000 rpm for 3 min. The precipitate was used for DNA extraction, while the supernatant was used for the a-amylase tests applying the protocol described below. 3.3. Detection of human a-amylase In order to detect human a-amylase three different methods were applied: BNP-Amylase test (Sclavo-Dasit, Milano, Italy), RSID™-Saliva (Galantos Genetics GmbH, Mainz, Germany) and a semi-automated method used in clinical laboratories (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Since this last method is not validated for forensic purposes, its application is at the moment limited on supplying an additional objective evaluation, unlike both RSID™-Saliva (Galantos Genetics GmbH) and BNPamylase method (Sclavo-Dasit, Milano, Italy), which need a naked eye interpretation by the operator.
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 incubated at 37 °C for 30 min. a-Amylase hydrolyzes the 2-chloro-4-nitrophenyl-a-D-maltotrioside to release 2-chloro-4-aminophenol and forms 2-chloro-4-nitrophenyl-a-D-maltoside, maltotriose, and glucose. Positive reactions due to the presence of p-nitrophenol, stoichiometrically formed, lead to a dye tuning from colorless to yellow detectable even to the naked eye. The observed kinetic differences were also detectable even to the naked eye. Results evaluation 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-nitrophenylmaltoheptaoside as substrate, in association with a-glucosidase enzyme used to release p-nitrophenol. The terminal glucose of the substrate is chemically blocked, preventing cleavage by the indicator enzymes. Calibration system is based on the specific molar extinction coefficients of free p-nitrophenol at 410 nm, related to IFCC reference for human serum and urine and are reported as units of enzymatic activity (1 IU = 1 lmol of p-nitrophenol/min at second) [8]. System performance was verified automatically using two control levels (Multiqual Level 1 and Level 2, Bio-Rad Laboratories); the range of detection indicated by the manufacturer is 0–1500 U/L. In order to verify the methods linearity, a specific calibration curve was performed by using a 0.003 units/ml dilution of amylase standard purchased from Sigma Chemical Company A1031, Lot 080M7000 V (5000 units of activity) as suggested by Laux [18]. The calibration standard curve made with a specific human amylase standard indicates the possibility to quantify up to 5 units amylase. The R2 value of 0.9967 indicates a close fit between the standard curve regression line. The absorbance was twice measured and the instrument background was tested using negative control consisting of RSID™-Saliva extraction buffer. Activity values under 5 U/L were considered as instrument background. 100 ll of samples were loaded on Siemens system. Enzymatic activity was calculated according to the formula reported on the user’s test sheet and it was expressed as International Units per liter (U/L). 3.3.3. Rapid stain identification of human saliva (RSID™-Saliva Galantos Genetics GmbH) In order to verify the presence of salivary amylase, 40 ll of supernatant 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, otherwise the test has to be considered invalid; if saliva is present, amylase will react with strip-binding antibodies and a second band will appear, showing a double banded strip. If only the control line appears, the result is negative [9]. Results evaluation was recorded as 0 to +3 scale. 3.4. DNA analysis Sample pellets were extracted in 20 ll final volume using QIAmpÒ DNA Micro Kit (QIAGEN, Germany) according to manufacturer’s manual. DNA was quantified using QuantifilerÒ Human DNA Quantification Kit and amplified with AmpFlSTRÒ Identifiler™ Amplification Kit (Life Technologies, USA). Capillary electrophoresis was performed on 3130 Genetic Analyzer (Life Technologies, USA). All genetic profiles were compared with laboratory staff, in order to exclude samples contamination.
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.doi.org/ 10.1016/j.legalmed.2014.03.004
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4. Results Table 1 shows the results on simulated saliva samples, analysed before processing casework samples. Amylase activity was not confirmed in two samples (2W and CB3) using BNP-amylase method. This result could be explained because of sampling, of partial or total enzyme degradation, of unfolded status of the protein, of active site modification or of erroneous results interpretation due to the yellowish colored samples (cigarette butts). Anyway, in 2W and CB3 samples amylase activity was confirmed by RSID™Saliva and Siemens clinical chemistry system: 2W and CB3 samples gave respectively 29 and 25 U/L values. RSID™-Saliva detected the presence of the enzyme in all simulated forensic samples. Positive and negative controls resulted as expected. After DNA extraction, Real-Time quantification highlighted human DNA presence in the saliva samples from the envelopes, the old smoked cigarette butts and the two chewing-gums (Table 1). DNA typing using a forensic validated kit confirmed donors’ DNA profile. In the stamps on envelopes and in the cigarette butts collected in the street, positive results were obtained both for amylase presence and activity, with different degrees of positivity (Table 1). However, in five stamps, including the 39-year-old one, and in one cigarette butt collected in the street, no human DNA was detected in real time. The same protocol was finally applied to 26 years old casework samples. All samples coming from the closing strip gave positive results for a-amylase presence and activity (Table 2). B1 sample failed for amylase activity presence when tested with BNP-amylase method but the amylase activity and presence was confirmed by using automated clinical system and RSID™-Saliva. The samples
Table 1 Results summary from old simulated forensic samples using three different methods to detect amylase and DNA quantification. Sample
BNP (a-amylase)
RSID™ Saliva
Siemens [U/l]
[DNA] ng
1W 2W 3W 4W 5W 1Y 2Y 3Y 4Y 5Y 1S 2S 3S 4S 5S 6S 7S 8S 9S 10S CB1 CB2 CB3 CG1 CG2 ST1 ST2 ST3 ST4 ST5 ST6 ST7
+ – + + + ++ ++ ++ +++ +++ + ++ ++ + ++ ++ + ++ + ++ ++ +++ – ++ ++ +++ ++ +++ ++ ++ ++ ++
++ + + + + ++ ++ ++ +++ +++ ++ + + ++ ++ +++ + ++ +++ + ++ +++ + ++ ++ ++ ++ +++ ++ + ++ ++
56 29 43 154 58 85 117 106 49 233 645 633 664 204 184 2085 2250 2035 2355 768 111 216 25 137 98 171 188 1677 1050 194 277 87
1.12 0.58 0.86 3.08 1.16 1.70 2.34 4.12 0.98 4.66 0 0 0.24 0 0.11 0 0.08 0.13 0.55 0 2.22 4.32 0.50 2.74 1.96 1.40 1.30 10.90 3.81 5.74 0 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.
Table 2 Summary of results for a-amylase tests and DNA quantification in 26 years old envelopes. Sample
BNP-amylase
RSID™ Saliva
Siemens [U/l]
[DNA] ng
A1 A2 A3 B1 B2 B3 C1 C2 C3
+ ++ – – ++ – + + –
++ ++ – + ++ – + + –
54 87 4 32 86 4 41 65 4
1.6 2.4 0 1.6 2.0 0 1.8 1.7 0
A1, A2, B1, B2, C1and C2: samples from envelopes A, B and C; A3, B3, C3: negative controls from the bottom of the envelopes.
collected from the bottom of the envelopes gave negative results. DNA coming from envelopes ranged from 1.6 to 2.4 ng of total human DNA in 20 ll TE buffer; the presence of inhibitors was excluded by IPC probe contained in QuantifilerÒ. All DNA profiles obtained using AmpFlSTRÒ IdentifilerÒ Plus Kit were attributed to the same unknown donor (data not shown). 5. Discussion Saliva stains are one of the most common biological fluid found in crime scenes. Although many protocols have been developed and have been described over the years and despite the importance of the identification of a biological sample before the DNA test, the detection of a body fluid is an underestimated analytical step. However, sometimes just knowing the biological origin of a sample can be enough to influence the outcome of a case. When this kind of analyses are omitted or incorrectly performed, the evidence in court can fail, even when positive results are obtained by DNA analysis; for this reason the laboratory data have to be performed with reliable and robust protocols, in order to be supported in court discussion [19]. Biological identification tests have some degree of subjectivity [20], because a human ruling is often required to confirm or exclude body fluid presence in a sample. In order to minimize erroneous interpretations, both presumptive and confirmatory tests should always be performed before proceeding with DNA typing. The introduction of an automated analysis method, not validated for forensic purposes, has confirmed amylase activity, just supporting data already obtained with Amylase and RSID™-Saliva tests, widely accepted by forensic community. These methods that can be used independently depending on workflow strategy, consistently showed the presence of amylase activity, even many years after deposition. Though amylase test is sensitive and reliable, eye interpretation is required in order to evaluate samples positivity or negativity. The evaluation is easy in solutions coming from uncolored substrates, but it could become more difficult when very low saliva quantity is present in the solution or when the substrate is yellowish-colored, as in cigarette butts. For this reason amylase test is very useful for a primary sample screening, while RSID™- Saliva test should be used in a second step to confirm the salivary a-amylase presence. The clinical chemistry system is used in this work only as a supporting test to confirm amylase activity. Further validation studies should be performed before considering the possibility to routinely introduce this automated system in forensic practice. In this work, amylase presence and activity has been confirmed on a wide variety of old samples containing saliva (envelopes, postage stamps on postcards, cigarette butts and chewing-gums). However, amylase activity levels and DNA concentration in saliva
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.doi.org/ 10.1016/j.legalmed.2014.03.004
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varied greatly between samples; in fact, there was no proportional correlation between presumptive tests intensity and DNA quantification. For example, in sample 2Y the observed value of amylase activity was 117 U/l, and the final DNA was 2.34 ng, while in sample 3Y the final DNA quantity was approximately double despite the same observed value of the amylase activity. In the sample 6S, the amylase activity value was 2085 U/l, but no human DNA was identified in the sample. This can be explained considering that amylase is an extracellular enzyme produced by salivary glands, while DNA amount depends on shedding buccal cells in the oral cavity or exudates [5], therefore a quantity correlation is not always to be expected. The encouraging results concerning enzymatic stability in old samples led up to analyze the real casework case, successfully demonstrating saliva presence in 26 years old threatening missives stored for years at room temperature. Thus, a reliable degree of confidence before proceeding with DNA analysis was supplied. DNA extraction, quantification and PCR amplification confirmed human DNA presence. The genetic profile was reproducible and this will be useful to throw light on this complex cold case, still unresolved. Acknowledgements The authors are grateful to Dr. Paolo Canessa, Court of Florence Public Prosecutor, for his cooperation. We also thank Sigma Chemical Company for providing the standard Amylase, Marco Meloni for technical assistance and the Association of Forensic Identification (A.I.Fo., www.aifo-italia.it) for the financial support. References [1] Hochmeister MN, Schlatter P, Rudin O, Dimhofer R. High levels of a-amylase in seminal fluid may represent a simple artefact in the collection process. J Forensic Sci 1997;42:535–6.
[2] Auvdel MJ. Amylase levels in semen and saliva stains. J Forensic Sci 1986;31:426–31. [3] Sensabaugh GF. Isozymes in forensic science. In: Rattizzi MN, Scandalios JG, Whitt GS, editors. Isozymes: current topics in biological and medical research. New York: Alan R Liss Inc.; 1982. p. 247–60. [4] Tsutsumi H, Higashide K, Mizuno Y, Tamaki K, Katsumata Y. Identification of saliva stains by determination of the specific activity of amylase. Forensic Sci Int 1991;50:37–42. [5] Clem D, Maidment J, Ringham M. A study into the measurement of alphaamylase activity using phadebas, iodine and gel-diffusion procedures. Nutr Food Sci 2001;21:141–6. [6] Virkler K, Lednev IK. Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Sci Int 2009;188:1–17. [7] Barni F, Berti A, Rapone C, Lago G. a-Amylase kinetic test in bodily single and mixed stains. J Forensic Sci 2006;51:1389–96. [8] Da Vela G, Bertino MG, Fusini F, Ricci U. Evaluation of an automated system for amylase detection in forensic samples. Int Congr Ser 2006;1288:636–8. [9] Old JB, Schweers BA, Boonlayangoor PW. Developmental validation of RSID™Saliva: a lateral flow immunochromatographic strip test for the forensic detection of saliva. J Forensic Sci 2009;54:866–73. [10] Bauer M. RNA in forensic science. Forensic Sci Int Genet 2007;1:69–74. [11] Li R. Forensic biology. Boca Raton: CRC Press; 2008. [12] Hedman J, Dalin E, Rasmusson B, Ansell R. Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs. Forensic Sci Int Genet 2001;5:194–8. [13] Bond JW, Hammond C. The value of DNA material recovered from crime scenes. J Forensic Sci 2008. 797-01. [14] De Leo D, Tagliaro F, Maschio S, Marigo M. A sensitive and simple assay of saliva on stamps. Z Rechtsmed 1985;95:27–33. [15] Zubakov D, Kokshoorn M, Kloosterman A, Kayser M. New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16year-old stains. Int J Legal Med 2009;123:71–4. [16] Moody J, Italy. The Monster of Florence. Time Magazine US Tuesday, June 21, 2005. . [17] Rapone C, D’Errico G, Bruno M, Serafini M, Berti A, Lago G. DNA typing after aamylase tests. Int Congr Ser 2003;1239:911–5. [18] Laux DL. Development of biological standard for the quality assurance of presumptive testing reagents. Sci Justice 2011;51:143–5. [19] Budowle B, Bottrell MC, Bunch SG, Fram R, Harrison D, Meagher S, et al. A perspective on errors, bias, and interpretation in the forensic sciences and direction for continuing advancement. J Forensic Sci 2009;54. 798-09. [20] Saviano J. The pursuit of objectivity in the examination of forensic evidence. J Forensic Identif 2006;56:877–84.
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.doi.org/ 10.1016/j.legalmed.2014.03.004
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Case Report
Stability of human a-salivary amylase in aged forensic samples Ilaria Carboni a, Stefano Rapi b, Ugo Ricci a,⇑ a b
Azienda Ospedaliero-Universitaria ‘‘Careggi’’, Diagnostic Genetics Unit, Largo Brambilla, 3, I-50134 Florence, Italy 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 2013 Received in revised form 11 March 2014 Accepted 17 March 2014 Available online xxxx Keywords: Amylase Saliva Forensic genetics Aged samples RSID™-Saliva
a b s t r a c t The unequivocal tissue identification in forensic casework samples is a key step for crime scene reconstruction. Just knowing the origin of a fluid can sometimes be enough to either prove or disprove a fact in court. Despite the importance of this test, very few data are available in literature concerning human saliva identification in old forensic caseworks. In this work the stability of human a-amylase activity in aged 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 threat letters sent to prosecutors who were working on ‘‘the Monster of Florence’’, a case of serial murders happened around Florence (Italy) between 1968 and 1985. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Saliva is a common body fluid, easily found in many different forensic samples like cigarette butts, stamps or balaclavas. Over the years different methods have been routinely described in order to identify saliva stains, mostly based on the detection of human aamylase (1,4 a-D-glucan glucanohydrolase). a-Amylase enzymes are proteins produced and stored mainly in salivary glands and pancreas, both secretory organs; for this reason a-amylase can be found in many body fluids as breast milk, pancreas, semen and vaginal secretions [1–3]. It is known that amylase activity in saliva stains rapidly decreases during the first hours of drying, but afterwards is rather stable throughout the years at room temperature in the dark [4]. Hence, amylase testing may be used to detect a dried saliva stain several months after deposition even though amylase activity varies greatly between individuals [5]. The identification of this fluid is performed using a wide range of tests based on the detection of the enzymatic activity or a-amylase presence that have been developed over the years [6]. One of the most accepted presumptive test uses blocked p-nitrophenylmaltoheptaoside (BpNPG7) as substrate to detect a-amylase activity; it can be performed in manual [7] or automatic [8] assays. Recently, a confirmatory test based on two anti-human salivary amylase monoclonal antibodies on an immunochromatographic strip test was developed [9]. It detects the presence of salivary
⇑ Corresponding author. Tel.: +39 055 7946204; fax: + 39 055 7946200. E-mail address: [email protected] (U. Ricci).
amylase, rather than enzymatic activity. Other promising techniques for saliva detection are mRNA [10] and colorimetric assay kit [11], but at the moment these techniques are not used in routine cases. Anyway, tissue identification in forensic casework is a crucial step for crime scene reconstruction, so it should always be performed. Even when the biological origin of a stain may seem obvious for the forensic investigator, scientific confirmation with reliable methods is necessary in court. Moreover, in forensic investigation a screening method based on amylase testing could be useful to lower the number of negative DNA samples from crime scenes [12]. It was reported that without pre-testing, over than 50% of presumed crime scene saliva stains can be expected to yield insufficient DNA amounts [13]. Notwithstanding the many available techniques and the importance of the test, there are very few data about saliva identification on aged forensic samples [2,14,15,4]. In this report the combined use of three independent assays on old simulated stains and in 26-year-old casework samples integrated with DNA analysis is shown. 2. Case report A prosecutor requested the analysis of three anonymous letters, in order to identify the sender by DNA profiling from the envelopes containing them. The event concerned ‘‘the Monster of Florence’’ [16] case, a serial killer that in the years between 1968 and 1985 murdered and brutally mutilated eight couples in the Florence (Italy) countryside. The gun was always the same but despite the investigations made and the many suspects the killer still remains
http://dx.doi.org/10.1016/j.legalmed.2014.03.004 1344-6223/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
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.doi.org/ 10.1016/j.legalmed.2014.03.004
2
I. Carboni et al. / Legal Medicine xxx (2014) xxx–xxx
unknown. After the last murder, three prosecutors who were working on this complex case received three letters containing death threats. The missives lay forgotten for 26 years at room temperature in an old file, until a prosecutor decided to have the three anonymous letters analysed in order to identify the sender by DNA profiling. Before approaching the real old casework samples, the analytical protocol was verified on old simulated forensic samples consisting in stamps on postcards, envelopes, cigarette butts and chewing-gums, prepared in laboratory many years before. In order to extend the protocol, ten cigarette butts collected in the streets were also analysed. 3. Materials and methods 3.1. Samples The simulated forensic samples consisted of 10 stamps on 10 postcards sent in 1974 (1S) and in the years between 1992 and 1997 (from 2S to 10S); 10 envelopes sealed 10 years before containing 20 ll saliva (1W, 2W, 3W, 4W, 5W, 1Y, 2Y, 3Y, 4Y and 5Y); three 10-year-old cigarette butts (CB1, CB2 and CB3); two 7-year-old chewing-gums (CG1 and CG2); 10 cigarette butts collected from the streets (from ST1 to ST10). Also, five enveloped spotted each by 2 ll saliva were dried and prepared 2 days before the analysis, to be used as positive control. All simulated samples prepared in laboratory were obtained after an informed consent from known donors and stored at room temperature. Additionally, negative controls were prepared using RSID™-Saliva extraction buffer on a piece of sterile paper. Real casework samples consisted of three anonymous envelopes (A, B and C) sent in 1986 to prosecutors who were working on ‘‘the Monster of Florence’’ case. Three samplings from each envelope were performed, two coming from the closing strip (A1, A2, B1, B2, C1 and C2) and one coming from the bottom of each envelope (A3, B3 and C3). The samplings coming from the bottom were used as negative control since the presence of saliva in those areas was considered very unlikely. 3.2. Samples preparation Disposable scalpels and tissue forceps were used to cut and manage all samples. Negative controls were prepared with all reagents except DNA. Each sample, consisting of 1 cm2 paper, chewing gum or cigarette butt paper was cut and placed in a 1.5 ml tube with 300 ll of RSID™-Saliva extraction buffer (Galantos Genetics GmbH, Mainz, Germany) containing 10 mM Tris–HCl and 0.1 mM EDTA at pH 8.0, vortexed and incubated for 3 h at 37 °C. The whole extract was transferred in a new 1.5 ml tube containing a sterile spin basket and centrifuged at 8000 rpm for 3 min. The precipitate was used for DNA extraction, while the supernatant was used for the a-amylase tests applying the protocol described below. 3.3. Detection of human a-amylase In order to detect human a-amylase three different methods were applied: BNP-Amylase test (Sclavo-Dasit, Milano, Italy), RSID™-Saliva (Galantos Genetics GmbH, Mainz, Germany) and a semi-automated method used in clinical laboratories (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Since this last method is not validated for forensic purposes, its application is at the moment limited on supplying an additional objective evaluation, unlike both RSID™-Saliva (Galantos Genetics GmbH) and BNPamylase method (Sclavo-Dasit, Milano, Italy), which need a naked eye interpretation by the operator.
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 incubated at 37 °C for 30 min. a-Amylase hydrolyzes the 2-chloro-4-nitrophenyl-a-D-maltotrioside to release 2-chloro-4-aminophenol and forms 2-chloro-4-nitrophenyl-a-D-maltoside, maltotriose, and glucose. Positive reactions due to the presence of p-nitrophenol, stoichiometrically formed, lead to a dye tuning from colorless to yellow detectable even to the naked eye. The observed kinetic differences were also detectable even to the naked eye. Results evaluation 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-nitrophenylmaltoheptaoside as substrate, in association with a-glucosidase enzyme used to release p-nitrophenol. The terminal glucose of the substrate is chemically blocked, preventing cleavage by the indicator enzymes. Calibration system is based on the specific molar extinction coefficients of free p-nitrophenol at 410 nm, related to IFCC reference for human serum and urine and are reported as units of enzymatic activity (1 IU = 1 lmol of p-nitrophenol/min at second) [8]. System performance was verified automatically using two control levels (Multiqual Level 1 and Level 2, Bio-Rad Laboratories); the range of detection indicated by the manufacturer is 0–1500 U/L. In order to verify the methods linearity, a specific calibration curve was performed by using a 0.003 units/ml dilution of amylase standard purchased from Sigma Chemical Company A1031, Lot 080M7000 V (5000 units of activity) as suggested by Laux [18]. The calibration standard curve made with a specific human amylase standard indicates the possibility to quantify up to 5 units amylase. The R2 value of 0.9967 indicates a close fit between the standard curve regression line. The absorbance was twice measured and the instrument background was tested using negative control consisting of RSID™-Saliva extraction buffer. Activity values under 5 U/L were considered as instrument background. 100 ll of samples were loaded on Siemens system. Enzymatic activity was calculated according to the formula reported on the user’s test sheet and it was expressed as International Units per liter (U/L). 3.3.3. Rapid stain identification of human saliva (RSID™-Saliva Galantos Genetics GmbH) In order to verify the presence of salivary amylase, 40 ll of supernatant 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, otherwise the test has to be considered invalid; if saliva is present, amylase will react with strip-binding antibodies and a second band will appear, showing a double banded strip. If only the control line appears, the result is negative [9]. Results evaluation was recorded as 0 to +3 scale. 3.4. DNA analysis Sample pellets were extracted in 20 ll final volume using QIAmpÒ DNA Micro Kit (QIAGEN, Germany) according to manufacturer’s manual. DNA was quantified using QuantifilerÒ Human DNA Quantification Kit and amplified with AmpFlSTRÒ Identifiler™ Amplification Kit (Life Technologies, USA). Capillary electrophoresis was performed on 3130 Genetic Analyzer (Life Technologies, USA). All genetic profiles were compared with laboratory staff, in order to exclude samples contamination.
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.doi.org/ 10.1016/j.legalmed.2014.03.004
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4. Results Table 1 shows the results on simulated saliva samples, analysed before processing casework samples. Amylase activity was not confirmed in two samples (2W and CB3) using BNP-amylase method. This result could be explained because of sampling, of partial or total enzyme degradation, of unfolded status of the protein, of active site modification or of erroneous results interpretation due to the yellowish colored samples (cigarette butts). Anyway, in 2W and CB3 samples amylase activity was confirmed by RSID™Saliva and Siemens clinical chemistry system: 2W and CB3 samples gave respectively 29 and 25 U/L values. RSID™-Saliva detected the presence of the enzyme in all simulated forensic samples. Positive and negative controls resulted as expected. After DNA extraction, Real-Time quantification highlighted human DNA presence in the saliva samples from the envelopes, the old smoked cigarette butts and the two chewing-gums (Table 1). DNA typing using a forensic validated kit confirmed donors’ DNA profile. In the stamps on envelopes and in the cigarette butts collected in the street, positive results were obtained both for amylase presence and activity, with different degrees of positivity (Table 1). However, in five stamps, including the 39-year-old one, and in one cigarette butt collected in the street, no human DNA was detected in real time. The same protocol was finally applied to 26 years old casework samples. All samples coming from the closing strip gave positive results for a-amylase presence and activity (Table 2). B1 sample failed for amylase activity presence when tested with BNP-amylase method but the amylase activity and presence was confirmed by using automated clinical system and RSID™-Saliva. The samples
Table 1 Results summary from old simulated forensic samples using three different methods to detect amylase and DNA quantification. Sample
BNP (a-amylase)
RSID™ Saliva
Siemens [U/l]
[DNA] ng
1W 2W 3W 4W 5W 1Y 2Y 3Y 4Y 5Y 1S 2S 3S 4S 5S 6S 7S 8S 9S 10S CB1 CB2 CB3 CG1 CG2 ST1 ST2 ST3 ST4 ST5 ST6 ST7
+ – + + + ++ ++ ++ +++ +++ + ++ ++ + ++ ++ + ++ + ++ ++ +++ – ++ ++ +++ ++ +++ ++ ++ ++ ++
++ + + + + ++ ++ ++ +++ +++ ++ + + ++ ++ +++ + ++ +++ + ++ +++ + ++ ++ ++ ++ +++ ++ + ++ ++
56 29 43 154 58 85 117 106 49 233 645 633 664 204 184 2085 2250 2035 2355 768 111 216 25 137 98 171 188 1677 1050 194 277 87
1.12 0.58 0.86 3.08 1.16 1.70 2.34 4.12 0.98 4.66 0 0 0.24 0 0.11 0 0.08 0.13 0.55 0 2.22 4.32 0.50 2.74 1.96 1.40 1.30 10.90 3.81 5.74 0 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.
Table 2 Summary of results for a-amylase tests and DNA quantification in 26 years old envelopes. Sample
BNP-amylase
RSID™ Saliva
Siemens [U/l]
[DNA] ng
A1 A2 A3 B1 B2 B3 C1 C2 C3
+ ++ – – ++ – + + –
++ ++ – + ++ – + + –
54 87 4 32 86 4 41 65 4
1.6 2.4 0 1.6 2.0 0 1.8 1.7 0
A1, A2, B1, B2, C1and C2: samples from envelopes A, B and C; A3, B3, C3: negative controls from the bottom of the envelopes.
collected from the bottom of the envelopes gave negative results. DNA coming from envelopes ranged from 1.6 to 2.4 ng of total human DNA in 20 ll TE buffer; the presence of inhibitors was excluded by IPC probe contained in QuantifilerÒ. All DNA profiles obtained using AmpFlSTRÒ IdentifilerÒ Plus Kit were attributed to the same unknown donor (data not shown). 5. Discussion Saliva stains are one of the most common biological fluid found in crime scenes. Although many protocols have been developed and have been described over the years and despite the importance of the identification of a biological sample before the DNA test, the detection of a body fluid is an underestimated analytical step. However, sometimes just knowing the biological origin of a sample can be enough to influence the outcome of a case. When this kind of analyses are omitted or incorrectly performed, the evidence in court can fail, even when positive results are obtained by DNA analysis; for this reason the laboratory data have to be performed with reliable and robust protocols, in order to be supported in court discussion [19]. Biological identification tests have some degree of subjectivity [20], because a human ruling is often required to confirm or exclude body fluid presence in a sample. In order to minimize erroneous interpretations, both presumptive and confirmatory tests should always be performed before proceeding with DNA typing. The introduction of an automated analysis method, not validated for forensic purposes, has confirmed amylase activity, just supporting data already obtained with Amylase and RSID™-Saliva tests, widely accepted by forensic community. These methods that can be used independently depending on workflow strategy, consistently showed the presence of amylase activity, even many years after deposition. Though amylase test is sensitive and reliable, eye interpretation is required in order to evaluate samples positivity or negativity. The evaluation is easy in solutions coming from uncolored substrates, but it could become more difficult when very low saliva quantity is present in the solution or when the substrate is yellowish-colored, as in cigarette butts. For this reason amylase test is very useful for a primary sample screening, while RSID™- Saliva test should be used in a second step to confirm the salivary a-amylase presence. The clinical chemistry system is used in this work only as a supporting test to confirm amylase activity. Further validation studies should be performed before considering the possibility to routinely introduce this automated system in forensic practice. In this work, amylase presence and activity has been confirmed on a wide variety of old samples containing saliva (envelopes, postage stamps on postcards, cigarette butts and chewing-gums). However, amylase activity levels and DNA concentration in saliva
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.doi.org/ 10.1016/j.legalmed.2014.03.004
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varied greatly between samples; in fact, there was no proportional correlation between presumptive tests intensity and DNA quantification. For example, in sample 2Y the observed value of amylase activity was 117 U/l, and the final DNA was 2.34 ng, while in sample 3Y the final DNA quantity was approximately double despite the same observed value of the amylase activity. In the sample 6S, the amylase activity value was 2085 U/l, but no human DNA was identified in the sample. This can be explained considering that amylase is an extracellular enzyme produced by salivary glands, while DNA amount depends on shedding buccal cells in the oral cavity or exudates [5], therefore a quantity correlation is not always to be expected. The encouraging results concerning enzymatic stability in old samples led up to analyze the real casework case, successfully demonstrating saliva presence in 26 years old threatening missives stored for years at room temperature. Thus, a reliable degree of confidence before proceeding with DNA analysis was supplied. DNA extraction, quantification and PCR amplification confirmed human DNA presence. The genetic profile was reproducible and this will be useful to throw light on this complex cold case, still unresolved. Acknowledgements The authors are grateful to Dr. Paolo Canessa, Court of Florence Public Prosecutor, for his cooperation. We also thank Sigma Chemical Company for providing the standard Amylase, Marco Meloni for technical assistance and the Association of Forensic Identification (A.I.Fo., www.aifo-italia.it) for the financial support. References [1] Hochmeister MN, Schlatter P, Rudin O, Dimhofer R. High levels of a-amylase in seminal fluid may represent a simple artefact in the collection process. J Forensic Sci 1997;42:535–6.
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