Effects of preanalytical frozen storage time and temperature on screening coagulation tests and factors VIII and IX activity

Abstract

Preanalytical quality control of blood samples is critical for tests of coagulation function and coagulation factor activity. Preanalytical storage time and temperature are the main variables. We investigated the effects of preanalytical frozen storage time and temperature on activated partial thromboplastin time (APTT), fibrinogen (Fbg), prothrombin time (PT)/international normalized ratio (INR), thrombin time (TT), factor VIII activity (FVIII:C), and factor IX activity (FIX:C) in frozen plasma. Samples (n = 144) were randomly and equally divided into four groups (storage at −80 °C or −20 °C) and analysed by CS5100 or CA7000 coagulation analysers. Baseline values and results after storage for 15 days, 1 month, 3 months, 6 months, and 1 year were measured after thawing. Mean percent changes and scatter plots were used to determine clinically relevant differences. The stabilities of coagulation tests and coagulation factor activities measured by the CS5100 system were consistent with those measured by the CA7000 system. At −80 °C, assessment samples of PT/INR, Fbg, and TT can be safely stored for 1 year, APTT for 6 months, and FVIII:C and FIX:C for 1 month. At −20 °C, samples of Fbg and TT can be stored for 1 year, PT/INR and FIX:C for 1 month, and APTT and FVIII:C for 15 days.

Effects of Freeze-Thaw Times on Screening Coagulation Tests and Factors VIII and IX Activities in Citrate-Anticoagulated Plasma at -20°C and -80°C

“…Differences between the values for PT/INR, APTT, Fbg, TT, FVIII:C, and FIX:C before and after freeze-thawing were calculated by the following equation: (value after freeze-thawing X times -value at baseline) x 100% / value at baseline. In accordance with our previous studies [14,15] and that by van Geest-Daalderop et al [7], a clinically relevant difference was defined as a mean change of > 10%. If the number of individuals with > 10% changes was less than 25% of the total 38 samples (i.e., less than 10 samples), the effect was termed moderate.…”

“…Accurate detection of screening coagulation tests, FVIII:C, and FIX:C can be affected by sample collection, transportation, storage temperature, and time [1,7,8]. In our previous studies [14,15], we found that along with prolonged time from sample collection to testing, screening coagulation test results increased or decreased and FVIII:C and FIX:C gradually decreased after both short-term storage (24 hours, 4°C or 25°C) and long-term storage (1 year, -80°C or -20°C). If the required test items cannot be detected in samples at the same time or need to be re-tested, or if precious samples need to be re-stored in biobanks, the thawed samples need to be re-frozen.…”

“…Moreover, the establishment of biobanks to facilitate the development of drugs and diagnostic tests has led to the development of biobank networks that collect and preserve large numbers of high-quality clinical biospecimens [12,13]. Although our preliminary tests established the shortterm/long-term storage times and optimal temperatures for screening coagulation tests on FVIII:C and FIX:C freeze-thaw cycles and temperatures are lacking for these items in citrate-anticoagulated plasma samples [14][15][16]. Previous studies have focused on freeze-thaw studies of coagulation factor assays in fresh-frozen plasma (FFP) for transfusion [17][18][19][20].…”

Recommendations for the measurement of thrombin generation: Communication from the ISTH SSC Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies

“…It is not recommended to store plasma at 4°C as cold activation might occur. 45,47 Thawing of frozen samples should be done in a water bath at 37°C. Water baths are preferred over heating blocks to ensure an evenly heat distribution.…”

Effect of Repeated Freeze-Thaw on Serum Biomarkers Associated with Eye Disease

“…In particular, cryopreserved serum samples from the same biobank might undergo various sample handling procedures that result in repeated freeze-thawing. Although previously published studies have shown that storage temperature, storage time, freeze-thaw cycles, and other factors could affect the serum or plasma levels of several biomarkers [ 19 – 22 ], there is still limited guidance for the optimal selection of cryopreservation of these samples for clinical monitoring and clinical studies. Repeated freeze-thaw is the almost inevitable for cryopreserved samples, as multiple testing or re-analysis leads to repeated freeze-thaw.…”