BACKGROUND Positive control materials for clinical diagnostic molecular genetic testing are in critically short supply. High-quality DNA that closely resembles DNA isolated from patient specimens can be obtained from Epstein-Barr virus (EBV)-transformed peripheral blood lymphocyte cell lines. Here we report the development of a process to (a) recover residual blood samples with clinically important mutations detected during routine medical care, (b) select samples likely to provide viable lymphocytes for EBV transformation, (c) establish stable cell lines and confirm the reported mutation(s), and (d) validate the cell lines for use as positive controls in clinical molecular genetic testing applications. METHODS A network of 32 genetic testing laboratories was established to obtain anonymous, residual clinical samples for transformation and to validate resulting cell lines for use as positive controls. Three panel meetings with experts in molecular genetic testing were held to evaluate results and formulate a process that could function in the context of current common practices in molecular diagnostic testing. RESULTS Thirteen laboratories submitted a total of 113 residual clinical blood samples with mutations for 14 genetic disorders. Forty-one EBV-transformed cell lines were established. Thirty-five individual point and deletion mutations were shown to be stable after 20 population doublings in culture. Thirty-three cell lines were characterized for specific mutations and validated for use as positive controls in clinical diagnostic applications. CONCLUSIONS A process for producing and validating positive control cell lines from residual clinical blood samples has been developed. Sustainable implementation of the process could help alleviate the current shortage of positive control materials.

Context.-Coagulation and bleeding problems are associated with substantial morbidity and mortality, and inappropriate testing practices may lead to bleeding or thrombotic complications.Objective.-To evaluate practices reported by hospital coagulation laboratories in the United States and to determine if the number of beds in a hospital was associated with different practices.Design.-From a sampling frame of institutions listed in the 1999 directory of the American Hospital, Association, stratified into hospitals with 200 or more beds ("large hospitals") and those with fewer than 200 beds ("small hospitals"), we randomly selected 425 large hospitals (sampling rate, 25.6%) and 375 small hospitals (sampling rate, 8.8%) and sent a survey to them between June and October 2001. Of these, 321 large hospitals (75.5%) and 311 small hospitals (82.9%) responded.Results.-An estimated 97.1% of respondents reported performing some coagulation laboratory tests. Of these, 71.6% reported using 3.2% sodium citrate as the specimen anticoagulant to determine prothrombin time (81.3% of large vs 67.7% of small hospitals, P <.001). Of the same respondents, 45.3% reported selecting thromboplastins insensitive to heparin in the therapeutic range when measuring prothrombin time (59.4% of large vs 39.8% of small hospitals, P <.001), and 58.8% reported having a therapeutic range for heparin (72.9% of large vs 53.2% of small hospitals, P <.001). An estimated 96.3% of respondents assayed specimens for activated partial thromboplastin time within 4 hours after phlebotomy, and 89.4% of respondents centrifuged specimens within 1 hour of collection. An estimated 12.1% reported monitoring low-molecular-weight heparin therapy, and to do so, 79% used an assay for activated partial thromboplastin time (58% of large vs 96% of small hospitals, P =.001), whereas 38% used an antifactor Xa assay (65% of large vs 18% of small hospitals, P =.001).Conclusions.-Substantial variability in certain laboratory practices was evident. Where significant differences existed between the hospital groups, usually large hospitals adhered to accepted practice guidelines to a greater extent. Some reported practices are not consistent with current recommendations, showing a need to understand the reasons for noncompliance so that better adherence to accepted standards of laboratory practice can be promoted.

Positive control materials for clinical molecular genetic testing applications are currently in critically short supply or non-existent for many genetically based diseases of public health importance. Here we demonstrate that anonymous, residual, clinical blood samples are potential sources of viable lymphocytes for establishing Epstein-Barr virus (EBV)-transformed blood lymphocyte cell lines. We attempted to transform 34 residual blood samples, and analyzed transformation success with respect to sample age, anticoagulant, storage temperature, volume, hemolysis, and patient age and sex. In univariate analysis, sample age was significantly associated with transformation success (P = 0.002). The success rate was 67% (6 of 9) for samples 1 to 7 days old, 38% (3 of 8) for samples 8 to 14 days old and 0% for samples 15 to 21 (0 of 11) days old. When we controlled for sample age in multivariate logistic regression, anticoagulant and storage temperature approached significance (P = 0.070 and 0.087, respectively; samples in acid citrate dextrose (ACD) and refrigerated samples were more likely to transform). Based on these findings, we suggest that samples collected in either ACD or ethylene diamine tetraacetic acid, and up to 14 days old (refrigerated) or 7 days old (stored ambient), are reasonable candidates for EBV transformation. The transformation rate for samples that met these criteria was 63% (10 of 16). Implementation of this process could help alleviate the shortage of positive control materials for clinical molecular genetic testing.