Impact of reduced levels of protein C, free protein S and antithrombin in normal frozen plasma on the interpretation of patients' results.

The objective of this study was to investigate the effect of freezing of normal plasma samples on protein C, free protein S (FPS) and antithrombin levels in order to determine its potential impact on the interpretation of the results of similarly frozen patients' samples. Protein C, FPS and antithrombin levels were measured by clotting-based test, by sandwich ELISA and by chromogenic assay, respectively, in 50 normal plasma samples prior to freezing, and after 2 and 4 weeks in parallel aliquots frozen at -25°C. The mean levels of the three proteins dropped significantly after a fortnight's freezing, protein C: 130.7-122.8% (P < 0.0246); FPS: 105.9-94.1% (P < 0.0016); antithrombin: 103.2-95.8% (P < 0.0001). The corresponding inter-assay coefficient of variances of the two sets of results were 8.9, 6.6 and 9.3%. Thereafter, only FPS declined significantly (84.3%) (P < 0.0001). In two of 48 and five of 48 cases at the end of 2 and 4 weeks, respectively, the levels of FPS values went below the lower limit of the normal range established from the 50 plasma samples. Freezing of plasma at -25°C for 24 h per se did not alter the levels of protein C and antithrombin and caused only a negligible change in FPS levels. Since 6, 4 and 14% of normal plasma samples would have been labeled as antithrombin, protein C and protein S deficient, respectively, had the tests been performed after 4 weeks of freezing, it is recommended that for correct interpretation of the results, laboratories should establish their reference ranges on normal samples frozen for the same period of time as the patients' samples.