Thrombosis in Myeloproliferative Neoplasms: A Single Center Experience of Using Whole Blood Platelet Aggregation Studies for Risk Assessment and Thromboprophylaxis.

CONCLUSION: Routine use of WBPA studies enables safe and effective risk-adapted thromboprophylaxis in MPN patients, irrespective of the underlying driver mutation and their risk predicted by the IPSET- thrombosis criteria.

Serum thromboxane B2 compared to five other platelet function tests for the evaluation of aspirin effect in stable cardiovascular disease.

BACKGROUND: To assess the role of serum thromboxane B(2) (TXB(2)) measurements and the correlation between platelet function studies, in patients with stable cardiovascular disease on aspirin or clopidogrel. METHODS: 76 patients (47 on aspirin, 16 clopidogrel, 13 both) underwent assessment of TXB(2), whole blood aggregometry (WBA) after stimulation with (i) arachidonic acid (0.5mM), (ii) ADP (5 microM), (iii) collagen (1 and 5 microg/ml), PFA-100, and Cone and Plate Analyzer. Clopidogrel patients were additionally assessed by the VerifyNow System. RESULTS: TXB(2) values ranged between 0.2 and 56.2 ng/ml, with significant separation between those taking aspirin, clopidogrel and controls (0.45 ng/ml vs 6.85 ng/ml vs 12.97 ng/ml, p<0.001). There was moderate correlation between WBA-AA and TXB(2) (r=0.487, p<0.001), PFA-100((R)) (r=0.599, p<0.001), WBA-Col1 (r=0.424, p<0.001), WBA-Col1:5 (r=0.417, p<0.001), and between TXB(2) and PFA-100((R)) (r=0.509, p<0.001). The prevalence of aspirin non-responders for WBA-AA, TXB(2), PFA-100((R)), CPA and Coll1:5 was 13.1%, 8.2%, 14.8%, 9.7% and 16.4% respectively. Individual patients were not consistently classified as aspirin non-responders in all tests. Those with inadequate aspirin response on > or =3 tests had higher TXB(2) levels (mean 1.57+/-1.66, range 0.553-4.45 vs mean 0.45+/-0.18, range 0.23-1.50) (p=0.001). Clopidogrel suppressed TXB(2) (p=0.02), WBA-AA (p<0.001), WBA-Col1 (p=0.012) and WBA-ADP (p<0.001) compared to controls. TXB(2) in patients ingesting fish oil tablets was lower compared to those without (0.4 ng/ml vs 0.52 ng/ml, p=0.004). Obesity was associated with higher TXB(2) values (0.61 vs 0.41, p=0.01). CONCLUSION: Serum TXB(2) measurements are a direct measure of the pharmacological effect of aspirin, are easily performed and correlate with other measures of platelet function. Serum TXB(2) measurements could be a useful sole measure of aspirin non-response, and may be even more predictive when performed in tandem with a global measure of platelet function. Aspirin and clopidogrel both suppressed several platelet pathways.

HIT or miss? A comprehensive contemporary investigation of laboratory tests for heparin induced thrombocytopenia.

Heparin induced thrombocytopenia (HIT) is a rare but potentially fatal complication of heparin therapy, which in a proportion of patients causes platelet activation and thrombosis. Initial clinical assessment of the likelihood of HIT is facilitated by laboratory testing to confirm or exclude HIT. This prospective investigation was performed over an 18-month period, and has involved testing of over 300 test samples from over 100 consecutive patients. Clinical assessment by 4T score was supplemented by laboratory tests that comprised both immunological [lateral flow ('STiC'), chemiluminescence (AcuStar; HIT-IgG(PF4-H)), ELISA (Asserachrom HPIA IgG)] and functional assays [SRA, platelet aggregation using whole blood ('Multiplate') and platelet rich plasma ('LTA')]. We observed both false positive and false negative test findings with most assays. Overall, the whole blood aggregation method provided a reasonable alternative to SRA for identifying functional HIT. STiC, AcuStar and ELISA procedures were fairly comparable in terms of screening for HIT, although STiC and AcuStar both yielded false negatives, albeit also resulting in fewer false positives than ELISA. The 4T score had less utility in our patient cohort than we were expecting, although there was an association with the likelihood of HIT. Nevertheless, we accept that our observations are based on limited test numbers. In conclusion, no single approach (clinical or laboratory) was associated with optimal sensitivity or specificity of HIT exclusion or identification, and thus, a combination of clinical evaluation and laboratory testing will best ensure the accuracy of diagnosis.

Factor V inhibitors: rare or not so uncommon? A multi-laboratory investigation.

Acquired deficiencies of, or inhibitors to, factor V are considered rare events. We report a series of 14 acquired factor V deficiencies, 10 of which were confirmed to have inhibitors to factor V, as identified within Australia in the past 5 years following a multi-laboratory investigation. The initial index case seen by one laboratory was followed within 4 months by a separate similar case. This prompted local contact with colleagues (n = 20) working in other haemostasis referral laboratories to identify the current case series. In total, nearly one-half of all haemostasis referral laboratories contacted had seen a case within the past 5 years. Clinical features and the apparent associated risk of bleeding complications generally varied, as did laboratory findings and the likely causal event. There were three females and 11 males. Age ranged from 44 to 95 years (median, 81 years). The level of inhibitor ranged from undetectable to over 250 Bethesda units. The probable cause leading to development of the inhibitors ranged from exposure to bovine thrombin, exposure to antibiotics, surgery and malignancy. Of additional interest was the apparent association of anti-phospholipid antibodies in many of the cases. For example, in the two similar index cases, with factor V inhibitor titres > 200 Bethesda units, high levels of anti-cardiolipin antibodies (> 70 GPL units) were also detected. Although less clear because of inhibitor interference, many of the cases also showed evident co-associated lupus anticoagulant activity. In conclusion, we report a series of factor V inhibitors recently identified within our geographic region that would represent an annual incidence of around 0.29 cases per million Australians. Although considered a rare finding, there is a high likelihood that most haemostasis referral laboratories will see a case every five or so years.

Platelet function testing in uraemic patients.

INTRODUCTION: Chronic renal failure (CRF) is associated with excessive bleeding for a variety of reasons. Platelet dysfunction is probably the most consistent and important feature, particularly platelet-platelet and platelet-vessel wall interactions. The skin bleeding time (SBT) is the best established predictor of bleeding in uraemic patients but suffers from poor reproducibility and accuracy. Several newer rapid assays of platelet function are able to provide a means of assessing primary haemostasis, but have not been specifically assessed in uraemic patients. METHODS: A single centre, prospective cohort study of patients referred to a tertiary nephrology unit. Patients with both acute and chronic renal impairment were recruited. Laboratory parameters analysed included full blood count, serum creatinine and urea, calculated glomerular filtration rate (GFR) (Cockcroft formulae) APTT PT fibrinogen, SBT, whole blood platelet aggregation (WBPA), platelet function analyzer (PFA-100), thromboelastograph (TEG) and cone platelet analyzer (CPA). RESULTS: This study included 42 patients: nine with CRF (GFR < 30 ml/min) who were not receiving dialysis; 23 with CRF receiving dialysis; seven who presented in acute renal failure; and three assessed with normal renal function but with nephrotic syndrome and who presented prior to renal biopsy. Twenty-two patients were on low-dose aspirin and four patients were on clopidogrel. There was weak correlation between calculated GFR and SBT (r(2) = 0.1564) with even poorer correlation with serum creatinine and no correlation with urea levels. Overall, PFA-100 was a poor predictor of SBT. Seven of 12 patients with SBT <7 min had abnormal WBPA, 24 of 26 had abnormal WBPA if SBT >7 min. Of those with SBT <7 min, five had abnormal CPA, and >7 min, 15 of 21 had abnormal results. Nineteen patients had abnormal TEG tracings. Six patients underwent renal biopsies with one bleeding complication. CONCLUSIONS: In this pilot study we found that prolonged SBT was not predicted by serum creatinine or calculated GFR. Within the limitations of this study, an alternative in vitro test to replicate the SBT has not been identified.

Beta2-glycoprotein I protects thrombin from inhibition by heparin cofactor II: potentiation of this effect in the presence of anti-beta2-glycoprotein I autoantibodies.

OBJECTIVE: Beta2-glycoprotein I (beta2GPI) is an important autoantigen in the antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiologic functions, since it displays both anticoagulant and procoagulant properties. We have previously reported that beta2GPI can directly bind thrombin, a key serine protease in the coagulation pathway. The present study was undertaken to examine the influence of beta2GPI on thrombin inactivation by the serpin heparin cofactor II (HCII). The effect of anti-beta2GPI antibodies was also examined. METHODS: HCII inactivation of thrombin was assessed using chromogenic and various platelet functional assays. The influence of intact and proteolytically cleaved beta2GPI and anti-beta2GPI antibodies was determined in these systems. RESULTS: beta2GPI protected thrombin against inactivation by HCII/heparin. Cleavage of beta2GPI at Lys317-Thr318 abrogated its protective effect. Patient polyclonal IgG and murine monoclonal anti-beta2GPI antibodies potentiated the procoagulant influence of beta2GPI in this system. CONCLUSION: These novel findings suggest that beta2GPI may regulate thrombin inactivation by HCII/heparin. The observation that anti-beta2GPI antibodies potentiate the protective effect of beta2GPI on thrombin in this system, thereby promoting a procoagulant response, may potentially delineate one of the pathophysiologic mechanisms contributing to the prothrombotic tendency in patients with APS.