Italian experience in the assessment of performance of clinical laboratories for DNA analyses to detect three thrombophilic mutations

The International Coagulation Survey (ICS) organized by the CISMEL (Italian Committee for Standardization of Laboratory Methods) is an External Quality Assurance Scheme (EQAS) aimed at assessing the laboratory performance of basic coagulation and thrombophilia tests. Recently, DNA analyses have been introduced with the aim of assessing the performance of clinical laboratories in detecting the three most important thrombophilic mutations factor V Leiden, prothrombin and MTHFR. We report results of three consecutive surveys with an average of 50 participants. After informed consent 4/6 DNA samples (400 ng/µl) were prepared at the organizing center from patients with known genotypes for the three mutations (wild-type, heterozygotes, or homozygotes). Samples were coded, aliquoted and stored at 4°C until shipment. Ten µl aliquots for each of the 4/6 DNA samples were sent to the participants who were asked to perform DNA analyses with their own methods. Results were centralized at the organizing center and analyzed for diagnostic accuracy. Overall, the average percentage (range in the three surveys) rate of misclassification was 2.2% (1.9–2.3%), 3.4% (1.2–4.6%) and 1.6% (0.7–2.4%) for factor V Leiden, prothrombin and MTHFR, respectively. The majority of laboratories, that misclassified DNA samples in the 1st (7/8), 2nd (6/8) and 3rd (2/5) survey, used either single PCR or multiplex PCR restriction enzyme analyses and most were home-made methods. The results indicate the need for regular standardization programs and EQAS for DNA analyses.