Effect of freezing method and storage at-20 °C and-70 °C on prothrombin time, aPTT and plasma fibrinogen levels

Clinical and epidemiological trials often involve central laboratory analyses of coagulation tests, including fibrinogen, which requires freezing of the plasma samples. Although rapid freezing by immersion of sample tubes in liquid nitrogen and storage at -70 degrees C is recommended, plasma samples are often transferred directly to the storage compartments, and stored at -20 degrees C. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen using a kinetic fibrinogen assay, PT-derived fibrinogen, and an immunoassay were measured in fresh plasma samples from 16 healthy blood donors. In addition, four sets of aliquots were prepared. Set A was transferred directly to a -20 degrees C storage compartment, set B was first snap-frozen in liquid nitrogen and then transferred to the -20 degrees C compartment. Set C was transferred directly to a -70 degrees C freezer, set D was first snap-frozen in liquid nitrogen and then stored at -70 degrees C. Aliquots were thawed after one, two, three and four months storage and laboratory assays repeated. PT and aPTT were strongly influenced by freezing and storage. In contrast, freezing had little effect on fibrinogen levels. Differences were below three percent for all variants. Changes were smaller for samples stored at -70 degrees C compared to -20 degrees C, and for snap-frozen compared to not snap-frozen samples. Frozen and thawed samples generated slightly higher fibrinogen levels compared to fresh samples. Prothrombin time and aPTT should be measured in fresh samples, since freezing has an inconstant and unpredictable effect on the results. In contrast, freezing and storage has little effect on results of fibrinogen assays. A limitation of the study is that only samples from healthy blood donors were used. Plasma samples with abnormal fibrinogen concentration, or with abnormal concentrations of coagulation factors might behave differently. (C) 2008 Elsevier Ltd. All rights reserved.