A comprehensive study of ovine haemostasis to assess suitability to model human coagulation.

INTRODUCTION: Similarities in size, anatomy and physiology have supported the use of sheep to model a wide range of human diseases, including coagulopathy. However, coagulation studies involving sheep are limited by the absence of high quality data defining normal ovine coagulation and fibrinolysis. MATERIALS AND METHODS: Full blood examination, routine and specialised coagulation tests, rotational thromboelastometry and whole blood platelet aggregometry was performed on 50 healthy Samm & Border Leicester Cross ewes and compared to corresponding human ranges. Intraspecies breed and gender variability was investigated by comparison to a smaller population of 13 healthy Merino wethers. RESULTS: Ovine coagulation was similar to human according to routine coagulation methods (PT, aPTT, TCT, Fib(C)) and some specialised coagulation tests (vWF, AT, Plasmin Inh). Despite these similarities, ovine secondary haemostasis demonstrated substantial differences to that of human. Rapid initiation of the contact activation pathway, high levels of FVIII, low Protein C, greater overall clot firmness and a reduced capacity for clot lysis was documented in sheep. In addition, ADP and collagen agonists precipitated a reduced primary haemostatic response in sheep relative to human. Intraspecies differences in whole blood platelet aggregometry between the cohorts of sheep indicate the need for breed-specific normal ranges. CONCLUSIONS: The application of a board spectrum of coagulation assays has enabled elucidation of the similarities as well as differences between ovine and human coagulation. The new knowledge generated from this study will guide the design of future translational coagulation studies in ovine models.

Using HitAlert flow cytometry to detect heparin-induced thrombocytopenia antibodies in a tertiary care hospital.

We aimed to assess the utility of HitAlert flow cytometry as a diagnostic functional heparin-induced thrombocytopenia (HIT) assay in a tertiary care hospital. The 4Ts score was used to assess pretest probability of HIT in 37 patients. Serum was analysed for HIT antibodies by the flow cytometry HitAlert assay. Results were compared with an antigenic assay, the particle gel immunoassay, PaGIA ID PF4/Hep Ab assay; and two functional assays, the Multiplate whole blood impedance aggregometry assay (WBIA), and the serotonin release assay (SRA). Flow cytometry was positive in 14 out of 37 patients, including zero out of eight, five out of 19 and nine out of 10 in the low, intermediate and high-risk groups by 4Ts score, respectively. Using the SRA as a 'gold standard', flow cytometry has a sensitivity of 81% and a specificity of 100% for the diagnosis of HIT. The other functional assay (WBIA) had similar sensitivity (81%) and specificity (90%) to flow cytometry. In contrast, the PaGIA maintained a high sensitivity of 100% but a specificity of only 20%. The improved specificity of flow cytometry over the antigenic assay was most marked in the 4T intermediate-risk group in which similar results were obtained between all three functional assays. We demonstrate that compared with an immunological assay (PaGIA), flow cytometry can improve the specificity of laboratory diagnosis of HIT without loss of sensitivity using SRA as a standard. Flow cytometry may have a role in the first-line laboratory diagnosis of HIT, especially when combined with an immunological assay such as PaGIA.

The utility of thromboelastography in monitoring low molecular weight heparin therapy in the coronary care unit.

Low molecular weight heparins (LMWHs) are used for prevention and management of vascular thrombosis. In general, monitoring of anticoagulant activity is not required, however, certain populations may be susceptible to overdosing or underdosing. As anti-activated factor X(anti-Xa) activity testing is not readily available, it was our aim to investigate the usefulness of thromboelastography (TEG; Haemoscope Corporation, Skokie, Illinois, USA) for the assessment of coagulation in patients on LMWH. All patients admitted to the coronary care unit on therapeutic dose of enoxaparin were included (1 mg/kg twice daily). Blood samples were collected 4 h after the morning dose of enoxaparin once the participant had received at least three doses. When anti-Xa activity was classified as low (0-0.5), correct (0.5-1.0) or high (>1.0), the distribution of reaction time (R) and dose per kg showed little association with anti-Xa activity. The difference between mean R for the high anti-Xa group and the correct anti-Xa group was statistically nonsignificant using two-sample t-test (P = 0.26). A linear regression model showed no evidence of association between dose per kg and anti-Xa (P = 0.95). However, there was evidence of positive association between dose per kg and R (P = 0.011) wherein a 10% increase in dose per kg was associated with an increase in R of 2.7 (95% confidence interval 0.6-4.7). There was no evidence of association between R and anti-Xa (P = 0.38). TEG was unable to be used to predict anti-Xa activity. However, TEG R was prolonged in more than 90% patients and correlated with dose of enoxaparin. As enoxaparin dose correlated poorly with anti-Xa activity, a more global test might be necessary to adjust dosing of LMWH in sick, hospitalized patients.

Centrifugation protocols: tests to determine optimal lithium heparin and citrate plasma sample quality.

BACKGROUND: Currently, no clear guidelines exist for the most appropriate tests to determine sample quality from centrifugation protocols for plasma sample types with both lithium heparin in gel barrier tubes for biochemistry testing and citrate tubes for coagulation testing. METHODS: Blood was collected from 14 participants in four lithium heparin and one serum tube with gel barrier. The plasma tubes were centrifuged at four different centrifuge settings and analysed for potassium (K(+)), lactate dehydrogenase (LD), glucose and phosphorus (Pi) at zero time, poststorage at six hours at 21 °C and six days at 2-8°C. At the same time, three citrate tubes were collected and centrifuged at three different centrifuge settings and analysed immediately for prothrombin time/international normalized ratio, activated partial thromboplastin time, derived fibrinogen and surface-activated clotting time (SACT). RESULTS: The biochemistry analytes indicate plasma is less stable than serum. Plasma sample quality is higher with longer centrifugation time, and much higher g force. Blood cells present in the plasma lyse with time or are damaged when transferred in the reaction vessels, causing an increase in the K(+), LD and Pi above outlined limits. The cells remain active and consume glucose even in cold storage. The SACT is the only coagulation parameter that was affected by platelets >10 × 10(9)/L in the citrate plasma. CONCLUSION: In addition to the platelet count, a limited but sensitive number of assays (K(+), LD, glucose and Pi for biochemistry, and SACT for coagulation) can be used to determine appropriate centrifuge settings to consistently obtain the highest quality lithium heparin and citrate plasma samples. The findings will aid laboratories to balance the need to provide the most accurate results in the best turnaround time.