This study reports on the frozen stability of all commonly measured coagulation proteins in normal citrated plasma: activated partial thromboplastin time, prothrombin time (%), thrombin time and fibrinogen (Clauss); clotting assays for factors II, V, VII, VIII, IX, X, XI and XII; functional assays for protein C (clotting), protein S (clotting), antithrombin (chromogenic) and plasminogen (chromogenic); and immunological assays for von Willebrand factor and D-dimer. All these factors listed are stable for up to 3 months if frozen at -24 degrees C or lower. At -74 degrees C, all these factors (allowing for 10% variation) were stable for at least 18 months, most were stable for 24 months. The number of proteins showing > 5% variation over baseline after 6 months storage indicates that some decay does occur even at -74 degrees C. There was no clear advantage in snap freezing at -74 degrees C and then storing at -24 degrees C over both freezing and storing at -24 degrees C; therefore, the freezing process itself is not responsible for the loss of stability. The best stability, especially at -24 degrees C, was obtained when small samples (1 ml) were stored in screw-cap tubes with a minimum dead space. The decrease in stability of the coagulation proteins directly correlates with the effect of temperature and time.
"In citrated plasma, AT activity in plasma from healthy individuals has been shown to be stable in unseparated citrated Vacutainer sampling tubes for up to 72 h at room temperature (Hickey et al., 2009/2010), but this may not be the case for some type II AT defects, such as AT Wibble, where thermostability is impaired (Beauchamp et al., 1998). AT has been shown to be stable for up to 2 years in plasma anticoagulated with 1/10th part 0.129 M sodium citrate (higher than that used in the UK) when stored in tightly stoppered vials at a mean temperature of )24 °C where the temperature never rose above )20 °C; however, the authors recommended that plasma stored for longer than 3 months should be kept at )74 °C (Woodhams et al., 2001). Interestingly , we have observed erroneously low AT levels in citrated plasma samples that had been frozen in a solid plastic block and stored in a self-defrosting )20 °C freezer for no more than 1 week (unpublished observation). "
[Show abstract][Hide abstract] ABSTRACT: Antithrombin (AT) deficiency is associated with an increased risk of deep vein thrombosis and pulmonary embolism which are major causes of morbidity and death. The incidence of deficiency in healthy populations has been reported to vary from 1/600 to 1/5000, with the variation being due to the different populations studied and detection methods used. When reduced activity levels are identified it is important to measure the AT antigen levels to differentiate type I from type II disorders, as type II defects have varying thrombotic risk.
Functional AT assays detect the ability of AT to inactivate thrombin or factor Xa, and AT antigen assays detect the quantity of AT in plasma. In functional assays, reducing the incubation time of sample with enzyme/heparin reagent may increase sensitivity to type II defects. An excess of antigen over activity level suggests the presence of functionally defective AT, which can be characterized further by assaying AT in the absence of heparin, electrophoresis to investigate the ability of heparin to bind to AT, and gene sequencing.
Many patients with AT deficiency have a type II defect and these defects may not be detected by all routine diagnostic assays. Assays using human thrombin may lack specificity and assays that use factor Xa may fail to detect the common variant, AT Cambridge II, which can be detected by assays using bovine thrombin, especially if activity is compared to antigen by ratio. Factor Xa based assays may be particularly sensitive to certain heparin binding defects, and sensitivity of assays to both heparin binding and reactive site defects can be improved by shortening the incubation time with enzyme.
uAT activity assays are essential for the detection of AT deficiency because type II defects are relatively common in patients with heritable deficiency. No one functional assay can be assumed to detect all forms of AT deficiency, and assays can sometimes be improved by reducing reaction time of AT with thrombin or factor Xa.
International journal of laboratory hematology 03/2011; 33(3):227-37. DOI:10.1111/j.1751-553X.2011.01307.x · 1.82 Impact Factor
"If more prolonged storage is required, then 4°C is a suitable temperature for Clauss fibrinogen determination, and storage at 4°C for up to 48 h did not affect assay results in one study (Rosenson et al, 1998). Plasma can be stored deep frozen for longer periods (Cushman et al, 1995), preferably at )70°C, where it is stable for at least 18 months (Blanco et al, 2000). Storage of samples from patients with DIC or those receiving thrombolytic therapy may lead to in vitro proteolysis of fibrinogen unless suitable protease inhibitors are included in the anticoagulant . "
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