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Chemiluminescence of Forensic Casework: A Comparison of the Hybridization and Detection Methods of Three Independent Laboratories

Tim Stacy, BS¹, Elizabeth A. Johnson, PhD², John Krebsbach, MS³, Sara Bowne, BS² and Robin Cotton, PhD^1
1Cellmark Diagnostics, Germantown, MD
²Harris County Medical Examiner's Office, Houston, TX
³Albuquerque Police Department, Albuquerque, NM

With the high interest in the implementation of chemiluminescent detection methods in forensic casework, many questions have arisen regarding specific techniques for hybridization and detection. Problems involving high background on lumigraphs and/or lack of sensitivity must be addressed in each laboratory prior to implementation of chemiluminescent detection procedures. Additional questions may arise as to which commercially available products and protocols produce the best overall results.

We will present data from three independent laboratories employing different hybridization and detection methods which have produced comparable results using alkaline phosphatase conjugated oligo NICE™ probes. The hybridization methods used by the above mentioned laboratories are as follows:

Each laboratory produced 3 membranes of serially diluted Hae III digested K562. Each laboratory then sent one membrane to the other participating laboratories. These membranes were then hybridized, with the above mentioned probes, according to the respective laboratory's hybridization and detection protocol. Lumigraphs were produced and compared by probe and exposure times against the hybridization conditions used by each laboratory. Selected lumigraphs and a summary of the sensitivity and background level generated by each method will be presented. Results show that numerous procedural variations exist which will produce lumigraphs with high sensitivity and low background signal.

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