Kevin D. Ballard¹, Ralph S. Orkiszewski¹, Marc S. Taylor², Elizabeth A. Johnson³ and Larry Ragle⁴.
¹Baylor College of Medicine, Houston, TX
²Technical Associates, Altadena, CA
³Harris County Medical Examiner’s Office, Houston, TX
⁴Center for Forensic Sciences, Laguna Beach, CA

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During forensic cases, the question occasionally arises as to whether a reference blood sample from a suspected criminal has been deliberately planted on evidence by someone in a position of public trust, in an effort to secure a criminal conviction of the suspect. Reference samples from suspects most commonly take the form of a blood sample anticoagulated with a salt form of ethylenediaminetetraacetic acid (EDTA), usually employing a commercially available purple (lavender) topped blood collection tube. Thus, one means of testing whether evidence planting has occurred would be the quantitative determination of EDTA relative to the amount of blood present in an evidentiary bloodstain. Methodology to address this issue has been developed in these laboratories. Initial screening for EDTA is accomplished through capillary GC-MS, with definitive identification and quantification through GC-MS/MS. The method employs the tetramethyl ester derivative of EDTA, with [¹³C₄]EDTA (synthesized in these laboratories) as an internal standard. The commercially available [²H₁₂]EDTA is unsuitable as an internal standard for these methods due to extensive and variable isotope exchange during the harsh derivatization conditions employed. EDTA is extracted from forensic bloodstains in a manner such that the cellular material is preserved, thus enabling quantification of EDTA and DNA typing from the same sample. The method is sufficiently sensitive to detect the equivalent of 1 nanoliter of EDTA-anticoagulated blood. Mixtures of unpreserved and purple top blood are readily accommodated, even in a 50:1 (respectively) ratio mixture. The method overcomes the potential interference from environmental contaminants containing metallic ions such as iron or copper, which can exert a strong negative interference on FAB and electrospray based methods through highly stable chelation with EDTA. The present method has shown that EDTA is selectively excluded from red cells, and no detectable blood levels of EDTA have been found in an ethnically diverse group of normal individuals. These latter findings confirm a previous elegant pharmacokinetic study that employed radiolabeled EDTA [1]. A series of 29 simulated forensic stains, with and without simulated evidence tampering, was analyzed in a blind fashion by both methods. In all cases, the assays accurately quantified the EDTA present in the samples, with no false positives and no false negatives. DNA typing of selected samples demonstrated that the combination of bacterial degradation of an original stain, followed by deliberated spiking of the collected stain with EDTA-anticoagulated blood from another individual, can lead to completely deceptive DNA typing results. The methods described here can provide unequivocal evidence for the presence and quantity of EDTA in forensic stains where evidence tampering involving EDTA-anticoagulated blood has occurred.