Advancing the Microscopic Analysis of Sexual Assault Evidence by Integrating Fluorescence Microscopy and Immunoassays

Forensic Microscopy Fall 2011

Ciaran F. A. Phillip

Introduction

• Visual detection of sperm is valuable to sexual assault case

• Provides DNA profile of contributor(s)

• Undeniable evidence

• Sperm are important!

The Problem

• Detection dependent on what can be seen

• Phase contrast enhances detail for all biological material

• Staining is non-specific and brightfield image is low contrast

• Monopolizes time of microscopist and not automatable

The Solution

• Enhance visualization of sperm with a sensitive and specific technique

• John C. Herr’s SpermPaints® and IFI’s SPERM HY-LITER™

• SpermPaints® not widely accepted

• SPERM HY-LITER™ seeking approval

The Process• Kevin Miller et. al. J Forensic Sci 2011

• Monoclonal mouse antibodies to bind surface protein unique to human sperm head

• Antibodies tagged with fluorescent Alexa 488 dye

• 4,6-diamidino-2-phenylindole (DAPI) dye with albumin

• FITC filter to visualize Alexa and different filter for DAPI

The Results

• Single human sperm surrounded by human epithelial cells. 200x magnification

Miller et. al. J Forensic Sci 2011

The Results

• Mixture of human and chimpanzee sperm at 200x magnification

Miller et. al. J Forensic Sci 2011

The Conclusion

• Visualization dramatically enhanced

• Staining is specific for human sperm

• Sensitive detection

• Sperm are important!

The Issues

Advantages• Viable without some

specialized kit components

• No interference with other staining techniques

• Results are reproducible

Disadvantages• Only available as kit

• Loss of specificity for extreme heat fixed specimens

• Alcohol based lubricants increase background fluorescence

References

1. Miller KW, Old J, Fischer BR, Schweers B, Stipinaite S, Reich K. Developmental Validation of the SPERM HY-LITER™ Kit for the Identification of Human Spermatozoa in Forensic Samples. J Forensic Sci 2011; 56(4): 853-865.

2. Herr J. SpermPaint optimization and validation. Washington, DC: United States Department of Justice, 2007 Oct.; Report No.: 220289.

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