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Presumptive tests: A substitute for Benzidine in blood samples recognition

  • Cláudia Gomes
    Correspondence
    Corresponding author.
    Affiliations
    Laboratory of Forensic and Population Genetics, Toxicology and Health Legislation Department, Medicine School, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain
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  • César López-Matayoshi
    Affiliations
    Laboratory of Forensic and Population Genetics, Toxicology and Health Legislation Department, Medicine School, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain

    Pharmaceutical Sciences Section, Department of Cellular and Molecular Sciences, Faculty of Sciences and Philosophy, Universidad Peruana Cayetano Heredia, Lima, Peru
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  • Sara Palomo-Díez
    Affiliations
    Laboratory of Forensic and Population Genetics, Toxicology and Health Legislation Department, Medicine School, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain
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  • Ana María López-Parra
    Affiliations
    Laboratory of Forensic and Population Genetics, Toxicology and Health Legislation Department, Medicine School, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain
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  • Pedro Cuesta-Alvaro
    Affiliations
    Computing Services, Research Support, Complutense University of Madrid, Avda de la Complutense s/n, 28040 Madrid, Spain
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  • Carlos Baeza-Richer
    Affiliations
    Laboratory of Forensic and Population Genetics, Toxicology and Health Legislation Department, Medicine School, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain
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  • Juan F. Gibaja
    Affiliations
    Consejo Superior de Investigaciones Científicas (IMF-CSIC), Archaeology of Social Dynamics, Egipcíaques, 15. 08001 Barcelona, Spain
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  • Eduardo Arroyo-Pardo
    Affiliations
    Laboratory of Forensic and Population Genetics, Toxicology and Health Legislation Department, Medicine School, Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain
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Published:September 26, 2017DOI:https://doi.org/10.1016/j.fsigss.2017.09.213

      Abstract

      The nature of the sample in a forensic case is one of the most important factors, since it determines the posterior analysis, helping to define or discard its identity (like blood versus semen).
      A presumptive test is a qualitative analysis that allows to identify, or confirm, the presence of a substance in a sample. These determinations usually occur, after a chemical reaction, and a specific colour is produced. A false positive is another substance reacting the same way, producing the expected result.
      The aim of this work was to evaluate the most effective presumptive test (with fewer false-positives) when analysing products that could look and behave like blood during a forensic screening assay. Eight different products were tested, like Betadine®, and four reagents were considered: Tetramethylbenzidine, O-toluidine, Leuchomalachite green and BlueStar® Forensic (BlueStar). Each product was tested with the reagents five times − mixed with human blood (3:1), with three different animal blood (3:1), and then unmixed.
      Our results indicated that Leuchomalachite green is the most suitable presumptive test, since it was the reagent with less false positives.

      Keywords

      1. Introduction

      The correct identification of a body fluid allows crucial laboratory tests, like the determination of a genetic profile, a fundamental step in a wide range of investigations [
      • Cox M.
      A study of the sensitivity and specificity of four presumptive tests for blood.
      ,
      • Virkler K.
      • Lednev I.K.
      Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene?.
      ]. One of the most common samples found at crime scenes is the blood stain [
      • Cox M.
      A study of the sensitivity and specificity of four presumptive tests for blood.
      ,
      • Virkler K.
      • Lednev I.K.
      Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene?.
      ].
      In order to identify blood, the expert can use different techniques for preliminary identification (screening or presumptive tests). These determinations occur after a chemical reaction, and a specific colour is produced [
      • Delman G.
      • Manti V.
      • van Ruth S.M.
      • van Leeuwen T.
      • Aalders M.
      Identification and age estimation of blood stains on colored backgrounds by near infrared spectroscopy.
      ].
      Several presumptive tests have been described for blood stains recognition [
      • Cox M.
      A study of the sensitivity and specificity of four presumptive tests for blood.
      ,
      • Virkler K.
      • Lednev I.K.
      Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene?.
      ,
      • Delman G.
      • Manti V.
      • van Ruth S.M.
      • van Leeuwen T.
      • Aalders M.
      Identification and age estimation of blood stains on colored backgrounds by near infrared spectroscopy.
      ], and one of the most used is Benzidine and its derivative Tetramethylbenzidine (TMB) [
      • Tobe S.S.
      • Watson N.
      • Daéid N.N.
      Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular-weight DNA.
      ]. Both reagents have similar sensitivity, specificity, and stability [
      • Garner D.D.
      • Cano K.M.
      • Peimer R.S.
      • Yeshion T.E.
      An evaluation of tetramethylbenzidine as a presumptive test for blood.
      ]. Indeed, Culliford et al., [
      • Culliford B.J.
      • Nickolls L.C.
      The Benzidine test.
      ] indicate its large number of false positives, for example, due to blood contamination, chemical oxidants, catalysts, or even vegetable peroxidases.
      Therefore, the aim of this work was to determine the best reagent for presumptive blood test recognition, with fewer false positives, in order to substitute (Tetramethyl)benzidine as the first option in blood recognition. To perform so, four reagents will be tested (Leuchomalaquite Green (LMG), BlueStar® Forensic (BF), and two benzidine derivate, ortho-Tolidine (OT), and Tetramethylbenzidine (TMB), on previously described false positives [
      • Delman G.
      • Manti V.
      • van Ruth S.M.
      • van Leeuwen T.
      • Aalders M.
      Identification and age estimation of blood stains on colored backgrounds by near infrared spectroscopy.
      ,
      • Tobe S.S.
      • Watson N.
      • Daéid N.N.
      Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular-weight DNA.
      ].

      2. Materials and methods

      2.1 Samples

      Blood human samples were collected from an anonymous and voluntary donor, used for all experiments and positive controls.
      Blood animal samples were collected from three death animals: chicken, pig and horse.

      2.2 False positives

      Products found to give false positives previously [
      • Delman G.
      • Manti V.
      • van Ruth S.M.
      • van Leeuwen T.
      • Aalders M.
      Identification and age estimation of blood stains on colored backgrounds by near infrared spectroscopy.
      ,
      • Tobe S.S.
      • Watson N.
      • Daéid N.N.
      Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular-weight DNA.
      ], or with an external appearance similar to a possible blood stain, were tested. Eight commercial products were selected, five whose stain might look like a blood stain (Wine, Coffee, Betadine®, Ketchup, Nail polish) and three cleaning products (Dish Detergent, Bleach, Stain Remover).

      2.3 Reagents

      OT, TMB, and LMG were prepared according to Cox 1991 [
      • Cox M.
      A study of the sensitivity and specificity of four presumptive tests for blood.
      ], and BF, according to the manual instructions.
      For OT and TMB a turquoise blue colour formation is considered a positive reaction; for LMG a bluish green colour, and for BF a fluorescent blue colour, better observed in a dark room. It was considered a negative result, when the obtained colour was different from the expected one.
      To perform each presumptive blood tests recognition, excepting for BF reaction, it is necessary to add H2O2 (3%), in the same proportion as the reagent previously added.
      The adopted procedure was to perform the presumptive tests in a proportion 3:1 (reagent and H2O2 (3%): sample)

      2.4 Controls

      Positive controls were obtained by applying the reagent in the human blood stain. Performing the same procedure, the negative control was obtained with sterile water.

      2.5 Specificity tests

      Three distinct analyses were performed in glass support as follows:
       Test a) Different animal blood were mixed with each reagent.
       Test b) Eight products (“false positives”) were separately mixed with each presumptive test.
       Test c) In order to precise the reagents specificity, each false positive was mixed with a positive control sample (1:1). A similar analysis to the previous ones was undertaken, adding each reagent to the sample.
      The mixture of the compounds must produce a quick and observable colour change, comparatively to the initial blend colour (see 2.3). The consideration that a reaction was positive or negative was a result of four independent observations. Each essay a), b) and c) was repeated five times, in order to obtain a significant result (p < 0,05).

      3. Results

      Considering the positive control analysis, all blood samples reacted positively within a few seconds with all of the tested reagents. All negative controls did not yield colour changes.
      For Test a), a total of 60 analyses were performed, and 160 analyses were accomplished for each one of the Tests b) and c).
      On the other hand, by performing five essays for each presumptive test, it was possible to obtain significant results, with p = 0, 0114. This value was obtained when comparing 5 positive results versus 0 positive results.
      Considering a) and c) essays, all of them produced positive results for all of the four reagents. Table 1 shows the results for Test b).
      Table 1Specificity results for Test b), considering eight commercial products as false positives for blood recognition, after the reaction with four presumptive blood tests (O-Tolidine (OT), Tetramethylbenzidine (TMB), and Leuchomalachite Green (LMG), and BlueStar® Forensic (BF)). (+) positive result; (−) negative result; (?) doubtful result.
      Reagent ProductOT + H2O2 (3%)TMB + H2O2 (3%)LMG + H2O2 (3%)BF
      Betadine®++++++++++
      Wine+++++
      Coffee
      Ketchup®+++++++
      Nail Polish++++++++++
      Dish Detergent??++++++++++
      Stain remover?????+????+++++
      Bleach+++++++++++

      4. Discussion

      Regarding the specificity of presumptive tests for human blood, all reacted positively when in contact with this sample, as well as, when testing human blood mixed with the false positives (Test c)). However, the same result was obtained with other animals’ blood (Test a)), demonstrating the low specificity of these presumptive tests for human blood.
      Observing the results for Test b), both LMG and BF has the same efficiency when dealing with false positives, whose aspect could be confused with a blood stain. Nonetheless, the behavior of these reagents is different when tested with cleaning products (Dish detergent, Stain remover and Bleach), where LMG is better than BF.
      Concerning OT and TMB, these were the presumptive tests with more false positives detected. The similarity between the structures of these benzidine’s derivatives [
      National Center for Biotechnology Information. PubChem Compound Database; CID = 7111.
      ,
      National Center for Biotechnology Information. PubChem Compound Database; CID = 41206.
      ] can justify that, most of the times, both reagents share the same false positives. However, OT was the only one able to detect “stain remover” as a false positive.
      The chemical structure of LMV [
      National Center for Biotechnology Information. PubChem Compound Database. CID = 67215.
      ] is quite different from benzidine and its derivatives [
      National Center for Biotechnology Information. PubChem Compound Database; CID = 7111.
      ,
      National Center for Biotechnology Information. PubChem Compound Database; CID = 41206.
      ]. This implicates that, for a colour change, different conditions are needed, since for the reaction takes place, the sample must interact and modify the reagent structure [
      National Center for Biotechnology Information. PubChem Compound Database; CID = 7111.
      ,
      National Center for Biotechnology Information. PubChem Compound Database; CID = 41206.
      ,
      National Center for Biotechnology Information. PubChem Compound Database. CID = 67215.
      ]. Such structure differences support the results for LMG, when compared with the other presumptive tests.
      Thus, it was possible to conclude that LMG is the best reagent to substitute (Tetramethyl)benzidine in a blood stain recognition, since it was the presumptive test with fewer false positives.
      For further research, it would be important to perform similar tests with different trademarks, as well as, with different reagents concentrations. Also, it would be interesting to analyze the behavior of each reagent, supposed to react in the presence of peroxidase, in a cadaver’s fluid, since this enzyme is expected to be in lower concentrations.

      Conflict of interest statement

      None.

      Acknowledgment

      This work was supported by G/6401400/8000 research project (Santander-Universidad Complutense de Madrid, Spain) for C.Gomes.

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