Potential Laboratory Misdiagnosis of Hemophilia and von Willebrand Disorder Owing to Cold Activation of Blood Samples for Testing

Abstract

To assess the potential for misdiagnosis of von Willebrand disorder (vWD) and hemophilia A while following current National Committee for Clinical Laboratory Standards (NCCLS) guidelines and consequent to a poorly recognized cold-activation phenomenon, we processed 39 normal citrate-anticoagulated samples by standard procedures (reference) or stored at low (approximately 4 degrees C) or ambient (approximately 22 degrees C) temperature for 3.5 hours before centrifugation and processing. Samples were tested in parallel for several hemostasis factors, including von Willebrand factor (vWF). Similar results were obtained for all samples for factors II, V, VII, IX, X, XI, and XII. For factor VIII (FVIII) and vWF, only samples stored at ambient temperature had results comparable to reference sample results. In most cases, low temperature storage led to much lower results. Taking the lower reference limit as 50%, most would have been defined as "abnormal," and a misdiagnosis of vWD or hemophilia A could easily arise. ABO classification and age were associated with FVIII and vWF levels, but neither was associated conclusively with relative loss of plasma FVIII coagulant and vWF caused by the cold-activation phenomenon. We advise laboratories following current NCCLS guidelines not to store or transport whole blood samples for FVIII and vWF testing at 2 degrees C to 4 degrees C because of the risk of misdiagnosing vWD or hemophilia A. Storage and transport at ambient temperature seem acceptable and provide results comparable to freshly centrifuged samples.

The effect of time between venipuncture, processing and freezing on the measurement of coagulation factor levels

“…We demonstrate here that time to centrifugation of blood samples up to 2.5 h does not affect the levels of prothrombin, antithrombin, FVIII, FIX, and FXI. These findings are in accordance with the results of Favaloro et al [8], who reported no effect of time to centrifugation up to 3.5 h, or differences in storage temperature (room temperature vs. cold storage), for measurement of FII, FV, FVII, FIX, FX, FXI, and FXII.…”

“…The lower level of FVIII in our study after exposure to a cold environment supports the idea that cold activation of FVIII may occur, so levels are less accurate after storage in a cold environment [8,9]. The differences for fibrinogen were too small to be of relevance.…”

“…Guidelines for blood sample storage time and temperature prior to centrifugation for coagulation factors are only available for von Willebrand factor and FVIII, and are based on only a few studies [8,9]. We demonstrate here that time to centrifugation of blood samples up to 2.5 h does not affect the levels of prothrombin, antithrombin, FVIII, FIX, and FXI.…”

“…FVII, FVIII and von Willebrand factor levels may be affected by a cold environment prior to centrifugation [8][9][10]. The precentrifugation storage temperature and the duration of exposure to a low temperature prior to centrifugation did not appear to influence the measured levels of prothrombin, FV, FVII, FIX, FXI and FXII in the study of Favaloro [8].…”

“…The precentrifugation storage temperature and the duration of exposure to a low temperature prior to centrifugation did not appear to influence the measured levels of prothrombin, FV, FVII, FIX, FXI and FXII in the study of Favaloro [8]. Recommendations regarding the optimal temperature for storage prior to centrifugation of whole blood for coagulation factor assays are given in the fifth edition of the guidelines of the Clinical and Laboratory Standards Institute [11].…”