Ronda A. Crist

Comparison of Five Thrombin Time Reagents

The thrombin time (TT) assay is a core test in clinical laboratories that perform coagulation testing. This assay screens for abnormalities in the conversion of fibrinogen to fibrin. The time necessary for fibrinogen to clot is affected by hypofibrinogenemia, dysfibrinogenemia, and the presence of inhibitors of the fibrinogen-to-fibrin reaction (heparin, hirudin, fibrin degradation products, and paraproteins) (1). The TT is used primarily to evaluate plasma specimens with prolonged activated partial thromboplastin time (APTT) values and, to a lesser extent, prolonged prothrombin time (PT) values for heparin or other thrombin inhibitors. The test is also useful to detect quantitative and qualitative fibrinogen abnormalities. Important criteria for selecting a TT reagent are high sensitivity to heparin, sensitivity to hypofibrinogenemia, and acceptable precision. We compared five commercial TT reagents on our automated coagulation analyzer and evaluated their sensitivity to fibrinogen and heparin concentration, their precision, and their accuracy. Information on each of the five TT reagents evaluated is provided in Table 1⇓ of the data supplement (available with the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue1/). Each commercial reagent was reconstituted according to the manufacturer’s instructions and used to measure TT in singleton measurements on the STA-R coagulation analyzer (Diagnostica Stago) according to the manufacturer’s specifications and laboratory standards. Clotting times for the TT assay were initiated when the instrument pipetted 100 μL of patient plasma, which then incubated for 120 s. The addition of 100 μL of thrombin reagent activated the final measurement of analyte. The Diagnostica Stago and BioPool reagents required a longer incubation time of 240s. Fibrinogen was measured on the STA Compact (Diagnostica Stago) with use of the STA-Fibrinogen reagent (Diagnostica Stago) containing human thrombin of 100 000 IU/L (100 IU/mL). The assay was initiated when the plasma sample was diluted 1:20 (5 μL + 95 …

Effects of EDTA on routine and specialized coagulation testing and an easy method to distinguish EDTA-treated from citrated plasma samples

Apoptosis deregulation is important for cancer development, chemotherapy response, and prognosis. Survivin and X-linked inhibitor of apoptosis protein (XIAP) are 2 members of the inhibitor of apoptosis proteins family (IAP). We used semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to determine the levels of expression of survivin and XIAP in 30 patients with de novo acute myeloid leukemia (AML) and 20 age- and sex-matched healthy volunteers. Survivin and XIAP overexpression were detected in 36.7% and 43.3% of cases, respectively. Patients with overexpression of either survivin or XIAP showed unfavorable response to chemotherapy in 81.2% and 91.7%, respectively. Also, these cases showed shorter median survival time (30 days) compared to patients with normal expression of either survivin or XIAP (150 days and 180 days). Patients with overexpression of both survivin and XIAP showed unfavorable response to induction therapy in 100% of the patients and the shortest median survival (30 days). These findings suggest that survivin and XIAP may have a role in leukemogenesis and provide prognostic information.Coagulation testing is performed with citrate-treated plasma. Samples submitted in other anticoagulants, such as EDTA, should not be tested. We aimed to evaluate the effects of EDTA on routine and specialized coagulation tests and to establish sodium tetraphenylborate testing as a quick and reliable method to identify EDTA-treated plasma samples. We performed the following measurements on citrateand EDTA-treated plasma samples from 10 healthy volunteers: sodium tetraphenylborate testing, prothrombin time (PT), partial thromboplastin time (PTT), potassium concentration, and functional assays for factors II, V, VII, VIII, IX, X, XI, XII, proteins C and S, and antithrombin. Mean values for citrate- and EDTA-treated plasma were most different for PT, PTT, factors V and VIII, and proteins C and S. Sodium tetraphenylborate testing correctly classified 100% of citratetreated and EDTA-treated samples. We confirm that EDTA has effects on coagulation assays. Sodium tetraphenylborate testing is a quick, simple, and inexpensive method for coagulation laboratories to identify samples erroneously submitted in EDTA.

Discard tubes are not necessary when drawing samples for specialized coagulation testing.

Discard tubes have traditionally been obtained when drawing samples for coagulation testing to avoid potential tissue factor activation of coagulation in the first tube that may lead to inaccurate results. Although discard tubes are no longer required for prothrombin time and partial thromboplastin time, the practice is still recommended for other coagulation studies due to lack of sufficient evidence that discard tubes are not needed. The objective of the present study was to determine whether the first citrate tube drawn can be used for special coagulation testing. We performed testing for fibrinogen, D-dimer, factors VIII, IX, XI, proteins C and S, and antithrombin on 30 healthy individuals and factors II, VII, IX, X, and proteins C and S on a second group of 30 healthy individuals and 30 individuals receiving warfarin. Testing was performed on two consecutive samples to evaluate the level of agreement between the two tubes. Paired t-testing showed no statistically significant differences between tube 1 and tube 2 for any of the tests performed. Most data pairs (tube 1, tube 2) agreed within 10% difference or less, and no positive or negative biases were observed. To our knowledge, this study is the first to evaluate the need for discard tubes in a variety of coagulation tests using both normal and abnormal samples. Our data suggest that discard tubes are not necessary when drawing samples for specialized coagulation testing.

Elevated Factor IX Activity Is Associated With an Increased Odds Ratio for Both Arterial and Venous Thrombotic Events

OBJECTIVES Elevations of factor IX (FIX) are thought to contribute to thrombotic risk, but this has not been well characterized. We retrospectively sought to determine whether elevated FIX levels are a risk factor for thrombosis in 81 adult subjects younger than 65 years (mean, 47 years) who were referred for evaluation of a hypercoagulable state. METHODS Patients were classified by arterial transient ischemic attack/stroke (TIA/stroke, n = 62) or venous thromboembolism (VTE, n = 19) events. FIX activity testing was performed on all 81 subjects and a reference group of 40 healthy individuals. RESULTS Thirteen (21%) of 62 subjects with TIA/stroke and 5 (26%) of 19 subjects with VTE had elevated FIX activity. Odds ratios for TIA/stroke and VTE in subjects with elevated FIX activity were 3.7 (95% confidence interval [CI], 0.76-17.65) and 6.8 (95% CI, 1.18-39.07), respectively. CONCLUSIONS Our findings suggest an association between elevated FIX levels and both arterial and venous thrombotic events.

A high-performance liquid chromatography method for the serotonin release assay is equivalent to the radioactive method

The serotonin release assay (SRA) is considered the gold standard laboratory test for heparin‐induced thrombocytopenia (HIT). The historic SRA method uses platelets loaded with radiolabeled serotonin to evaluate platelet activation by HIT immune complexes. However, a nonradioactive method is desirable. We report the performance characteristics of a high‐performance liquid chromatography (HPLC) SRA method.

A Comparison of Two Commercial ADAMTS13 Activity Assays With a Reference Laboratory Method

Background: ADAMTS13 is the enzyme responsible for cleaving ultra-large von Willebrand factor (vFW) multimers. Commercial kits have been recently developed that use a fluorescence resonance energy transfer (FRET) methodology to measure ADAMTS13 activity. Methods: In this study, we compared the results from 2 recently released commercial kits (GTI and Technoclone) with results obtained from a reference laboratory also using FRET technology to assay samples from patients referred for a possible diagnosis of thrombotic thrombocytopenic purpura (TTP). Results: A total of 29 samples were assayed and compared. Six samples had less than 5% of normal ADAMTS13 activity identified by the reference laboratory. The GTI kit identified only 4 of these 6 samples, while the Technoclone kit identified all 6. Conclusion: The results of this study demonstrate that commercial ADAMTS13 activity assays currently available are clinically useful in diagnosing TTP.

Evaluation of a new commercial method for von Willebrand factor multimeric analysis

Evaluation of von Willebrand factor (VWF) multimeric distribution is useful for subclassification of von Willebrand disease (VWD). Multimer analysis has historically been a manual, labor‐intensive laboratory‐developed test. The first commercial method for multimeric analysis was recently developed that utilizes a single instrument for gel electrophoresis, staining, and densitometry. The current study was undertaken to evaluate the performance characteristics of the new commercial method.