SPERM HY-LITER™ for the identification of spermatozoa from sexual assault evidence

Abstract

Accurate microscopic identification of human spermatozoa is important in sexual assault cases. We have compared the results of examinations with (1) a fluorescent microscopy method, SPERM HY-LITER™, and (2) Baecchi's method for identification of human spermatozoa. In 35 artificial, forensic type samples, spermatozoa were identified in 45.7% with SPERM HY-LITER™ in Copenhagen, in 54.3% in the laboratory of the manufacturer of SPERM HY-LITER™, and 40.0% of the samples with Baecchi's staining method. When differences occurred between the two methods, it was significantly more often that SPERM HY-LITER™ detected spermatozoa when Baecchi's method did not (t_s = 6.567, df = 1, P = 0.048). This trend was also seen in selected compromised or degraded samples and in selected adjudicative samples. The reactions with spermatozoa from dog, horse, pig and bull were negative with SPERM HY-LITER™, whereas Baecchi's method was non-selective. Data from forensic casework samples in Copenhagen from two years (2008 and 2009) are presented. The samples from 2008 were investigated using Baecchi's method, while those from 2009 were investigated using SPERM HY-LITER™. The frequencies of positive results were similar between the two methods for the two years (27.9% and 32.1% respectively). Analysis of acid phosphatase (ACP) activity for the positive results obtained for these two years does not support the use of a negative ACP result as a prescreen for microscopic analysis for spermatozoa.

Introduction

The identification of spermatozoa and seminal fluids in sexual assault crimes is a critical aspect of forensic genetic investigations. Several chemical and cellular constituents of seminal fluid are often used in the identification of seminal fluid following sexual assault. For example, prostatic acid phosphatase (ACP), zinc, prostate specific antigen (PSA), seminogelin and MHS-5 [1], [2], [3], [4], [5], [6], [7] are often used as screening and/or presumptive tests in order to determine the location or presence of seminal fluid on various substrates. Furthermore, they are also useful in cases where the perpetrator is either oligospermatic or azoospermatic, where few or no spermatozoa can be found [8], [9]. Despite significant advances in testing procedures and methodologies, these tests still lack the specificity and stringency needed to accurately portray them as confirmatory tests. Common binding motifs among higher primates and other mammalian species have been reported, more adequately portraying these tests as presumptive screening tools [10].

The positive identification of spermatozoa in sexual assault cases is a critical step in determining the investigative strategy for the laboratory analyst. Furthermore, in most cases, the identification of spermatozoa has an important impact on the outcome of a case. Therefore, forensic genetic laboratories devote a great deal of time, efforts and resources to search for spermatozoa. Until now, search for spermatozoa has been performed by microscopic analysis using traditional histological stains like Baecchi's staining method with acid fuchsin and methyl blue, hereafter referred to as Baecchi's method, Kernechtrot-Picroindigocarmine (KPIC) and Hematoxylin-eosin (H&E) [11], [12], [13], [14], [15], [16]. These methods are based on the identification of morphological structures and staining patterns of spermatozoa (Fig. 1). However, traditional staining methods lack specificity and sensitivity, requiring extensive analyst training in order to achieve the necessary proficiency of microscopic examination of sexual assault evidence. The identification of human spermatozoa in challenging samples with low sperm count, high epithelial cell density, mixtures of cells and microorganisms from rectal samples, degradation of sperm cells with detachment of the tail from the head, etc. can be extremely difficult, often resulting in negative and/or inconclusive results. Furthermore, the examination may be very time consuming. Given the limitations of microscopic examination of samples stained with usual histological stains, improvements in specificity and effectiveness of methods for identifying human spermatozoa from sexual assault evidence would be highly advantageous.

SPERM HY-LITER™ was developed and validated for the microscopic analysis of human spermatozoa from sexual assault evidence [17]. The specificity of this method is obtained through an Alexa 488 fluorescently (green flourescein isothiocyanate – FITC) tagged monoclonal antibody, which specifically targets an antigen on the nuclear membrane of sperm cells. In conjunction with the Alexa 488 tag, the blue nuclear stain, 4′,6-diamidino-2-phenylindole (DAPI), is also incorporated as part of the staining (Fig. 2). As a result, all cells containing DNA rich nuclei can be visualized through the selective use of a DAPI compatible fluorescent filter. This makes it possible to visualize the various cells without the need for selective degradation of epithelial/vaginal cells by proteinase K treatment prior to microscopic analysis. Due to the intense fluorescence, as few as one sperm cell can be identified among a dense sample of vaginal epithelial cells common in sexual assault type swabs.

Although the advantages of the fluorescent microscopy for identifying cellular components are widely recognized by the scientific community, its application in forensic science has been limited to date. The objective of the current study, therefore, was to compare the success of identifying human spermatozoa with the fluorescent SPERM HY-LITER™ method with that of Baecchi's method that is a traditional, histological investigation used in many laboratories. Here, we present the results of our internal validation study performed at the Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark.