B-324 Prevalence of high phosphatidylethanol (PEth) concentrations in a blood transfusion product

Background Recent publications have demonstrated the possibility of false-positive 16:0/18:1 phosphatidylethanol (PEth) testing results due to the presence of PEth in blood transfusion products. PEth biomarker results are used to evaluate individuals for ethanol abstinence in a variety of clinical settings. PEth positivity in individuals expected to abstain from alcohol can have serious legal and clinical consequences. Little is known about the prevalence of donated blood products with a high enough PEth concentration to cause a false-positive result in a a patient who receives a transfusion. The Blood Donor History Questionnaire does not ask about ethanol use. Packed red blood cells (pRBC) are the most commonly stored and used blood component in blood banks. To assess the prevalence of clinically significant PEth concentrations, 400 residual pRBC segments samples were analyzed and the concentration of PEth was quantified. Current clinical interpretation is that >20 ng/mL PEth concentration indicates moderate alcohol consumption. Methods A liquid-chromatography tandem mass spectrometry (LC-MS/MS) method was validated using a Waters TQS micro triple quadrupole in whole blood or pRBC. In brief, PEth and the internal standard PEth 16:0/18:1-D5 were obtained from Cerilliant. Calibrators and QC materials were made from separate, recently expired (<2 weeks), and PEth negative units of pRBCs. Chromatography mobile phase A was 2 mM ammonium acetate and mobile phase B was methanol/acetone (95/5). pRBC lysing buffer was 100 µL of H2O:ACN (80:20 v/v) and elution buffer is 5% IPA in ethyl acetate. The reconstitution solvent consisted of isopropanol. Analysis is conducted using a Phenominex Luna Phenyl Hexyl column. The mass spectrometer was operated in selected reaction monitoring (SRM) mode and used MassLynx Software for acquisition and analysis. The detector signal was integrated using a linear calibration curve and 1/x weighting. Compounds were identified by retention time, relative retention time to a deuterated internal standard, and SRM ion pair ratios. The LC-MS/MS validation assessed linearity, bias, recovery, precision, interferences, matrix effects, and carry-over. 400 deidentified, previously transfused pRBC unit tube segments were analyzed according to the protocol developed during validation. Using calculations corrected for average hematocrit, a single unit transfusion of pRBC containing a PEth concentration of >350 ng/mL could potentially result in a patient PEth result >20 ng/mL. Thus, 350 ng/mL in a pRBC segment was used as the criteria for clinical significance. Results Out of 400 tested pRBC segments, 185(46%) were PEth positive above the assay LOQ of 10 ng/mL. Out of the 400 pRBC segments, 33 (8%) had a PEth concentration >350 ng/mL. A pRBC unit value of >350 ng/mL could potentially result in a PEth result >20 ng/mL from a single unit transfusion. Conclusion Of the tested pRBC segments, 8% contained sufficient PEth concentration to potentially yield a positive PEth (>20 ng/mL) if a single unit of pRBC is transfused. This limitation may lead to a prejudicial test interpretation that an individual is not compliant with alcohol abstinence. Vulnerable patient populations that may be affected by this potential false positive include transplant patients, pediatric patients, incarcerated/paroled individuals, and drug treatment patients.