International collaborative study for the calibration of proposed International Standards for thromboplastin, rabbit, plain, and for thromboplastin, recombinant, human, plain.

Essentials Two candidate International Standards for thromboplastin (coded RBT/16 and rTF/16) are proposed. International Sensitivity Index (ISI) of proposed standards was assessed in a 20-centre study. The mean ISI for RBT/16 was 1.21 with a between-centre coefficient of variation of 4.6%. The mean ISI for rTF/16 was 1.11 with a between-centre coefficient of variation of 5.7%. SUMMARY: Background The availability of International Standards for thromboplastin is essential for the calibration of routine reagents and hence the calculation of the International Normalized Ratio (INR). Stocks of the current Fourth International Standards are running low. Candidate replacement materials have been prepared. This article describes the calibration of the proposed Fifth International Standards for thromboplastin, rabbit, plain (coded RBT/16) and for thromboplastin, recombinant, human, plain (coded rTF/16). Methods An international collaborative study was carried out for the assignment of International Sensitivity Indexes (ISIs) to the candidate materials, according to the World Health Organization (WHO) guidelines for thromboplastins and plasma used to control oral anticoagulant therapy with vitamin K antagonists. Results Results were obtained from 20 laboratories. In several cases, deviations from the ISI calibration model were observed, but the average INR deviation attributabled to the model was not greater than 10%. Only valid ISI assessments were used to calculate the mean ISI for each candidate. The mean ISI for RBT/16 was 1.21 (between-laboratory coefficient of variation [CV]: 4.6%), and the mean ISI for rTF/16 was 1.11 (between-laboratory CV: 5.7%). Conclusions The between-laboratory variation of the ISI for candidate material RBT/16 was similar to that of the Fourth International Standard (RBT/05), and the between-laboratory variation of the ISI for candidate material rTF/16 was slightly higher than that of the Fourth International Standard (rTF/09). The candidate materials have been accepted by WHO as the Fifth International Standards for thromboplastin, rabbit plain, and thromboplastin, recombinant, human, plain.

Comparison of von Willebrand factor (VWF) activity levels determined by HemosIL AcuStar assay and HemosIL LIA assay with ristocetin cofactor assay by aggregometry.

INTRODUCTION: Diagnosis of von Willebrand disease (VWD) requires quantitative as well as qualitative determination of von Willebrand factor (VWF) levels. For functional assessment of VWF, ristocetin cofactor assay by aggregometry is considered to be the gold standard. However, need for technical expertise, labour intensiveness, difficult standardization and high intra- and inter- assay variabilities are some of the limitations of this methodology. Various assays for determination of VWF adhesive function using different methodologies have been developed in recent years. AIM: To evaluate the HemosIL AcuStar chemiluminescence assay (VWF:RCo[Acu]) and the HemosIL latex immunoassay (VWF:act) as diagnostic tests for VWD and identification of type 2 VWD in comparison with the ristocetin cofactor assay performed by aggregometry (VWF:RCo[Agg]). METHODS: Results from 96 samples analysed by VWF:RCo[Acu] and 128 samples by VWF:act were compared with VWF:RCo[Agg]. Sixty of these samples (25 normal, 17 type 1 and 18 type 2) were analysed by all three assays. RESULTS: VWF:RCo[Acu] showed excellent agreement with VWF:RCo[Agg], and readily identified all type 2 VWD samples tested. VWF:act showed reasonable agreement with VWF:RCo[Agg] for most patients, but had a slightly lower sensitivity for detection of type 2 VWD. CONCLUSION: VWF:RCo[Acu] assay has the potential to replace VWF:RCo[Agg] for the diagnosis of VWD.

Fast-track rapid warfarin reversal for elective surgery: extending the efficacy profile to high-risk patients with cancer.

Periprocedural management of patients on long-term warfarin therapy remains a common and important clinical issue, with little high-quality data to guide this complex process. The current accepted practice is cessation of warfarin five days preoperatively, but this is not without risk and can be complicated, particularly if bridging is required. An alternative method utilising low-dose intravenous vitamin K the day before surgery has been shown previously to be efficacious, safe and convenient in an elective surgical population receiving chronic warfarin therapy. The efficacy and utility of this 'fast-track' warfarin reversal protocol in surgical patients with cancer, who were at high risk of both thromboembolism and bleeding was investigated in a prospective, single-arm study at a dedicated cancer centre. Seventy-one patients underwent 82 episodes of fast-track warfarin reversal (3 mg intravenous vitamin K 18 to 24 hours before surgery). No patient suffered an adverse reaction to intravenous vitamin K, all but one achieved an International Normalized Ratio =1.5 on the day of surgery, and no surgery was deferred. Assays of vitamin K-dependent factor levels pre- and post-vitamin K demonstrated restoration of functional activity to within an acceptable range for surgical haemostasis. While this alternative method requires further validation in a larger prospective randomised study, we have now extended our use of fast-track warfarin reversal using vitamin K to patients with cancer, on the basis of our experience of its safety, convenience, reliability and efficacy.

Markers of coagulation activation after hepatic resection for cancer: evidence of sustained upregulation of coagulation.

We investigated the possibility that despite postoperative derangements of routine laboratory coagulation tests, markers of coagulation activation and thrombin generation would be normal or increased in patients undergoing hepatic resection for cancer In addition to the conventional coagulation tests prothrombin time and activated partial thromboplastin time, we measured select markers of coagulation activation prothrombin fragments 1 and 2 (PF1 + 2), thrombin-antithrombin complexes and plasma von Willebrand Factor antigen in 21 patients undergoing hepatic resection. The impact of hepatic resection on coagulation and fibrinolysis was studied with thromboelastography. Preoperatively, routine laboratory coagulation and liver function tests were normal in all patients. On the first postoperative day, prothrombin time was prolonged (range 16 to 22 seconds) in eight patients (38%). For these patients, thromboelastography was normal in six (75%), PF1 + 2 was elevated in four (50%), and thrombin-antithrombin complexes and von Willebrand Factor antigen were elevated in all, which was evidence of acute phase reaction, sustained coagulation factor turnover and activation. By the fifth postoperative day, despite normalisation of prothrombin time, markers of increased coagulation activity remained greater than 85% of baseline values. The findings indicate that in patients undergoing liver resection for cancer, there is significant and prolonged postoperative activation of the haemostatic system despite routine coagulation tests being normal or even prolonged. Before considering therapeutic interventions an integrated approach to interpreting haematological data with clinical correlation is essential.

Characterization of platelet aggregation induced by the human carcinosarcoma Colo 526: role of platelet activation, tumor cell cytoskeleton and tumor cell plasma membrane.

Tumor cell-platelet interactions have been shown to be involved in the process of metastasis. This study characterizes the aggregation of washed platelets induced by the human uterine carcinosarcoma Colo 526. Ultrastructural studies revealed a two-stage process in which the earliest events were the adhesion and degranulation of individual platelets in contact with the tumor cell membrane. The second stage consisted of a wave of aggregation involving all residual platelets. We found that the first stage was initiated by a factor integral to the tumor cell plasma membrane which acted independently of the tumor cell cytoskeleton or metabolic processes. This factor was found to be a glycoprotein or glycolipid with functionally important sialic acid and N-linked carbohydrate residues. The initial stage was not dependent on platelet activation as neither aspirin nor prostacyclin prevented adhesion or degranulation. The second stage was found to be dependent on platelet activation. These results suggest that platelet aggregation induced by Colo 526 involves a distinctive primary stage which is initiated by a factor on the tumor cell plasma membrane resulting in the degranulation and lysis of individual platelets. This process can occur independently of platelet activation or aggregation and thus may have some relevance to the clinical use of platelet antagonists as antimetastatic agents.

Neutralising antibodies after streptokinase treatment for myocardial infarction: a persisting puzzle.

OBJECTIVE: To determine the development of titres of streptokinase (SK) neutralising antibodies after a single dose of SK, to establish when titres decrease to levels at which a second dose might be effective. DESIGN: Analyses of blood samples taken from patients at intervals after SK administration. SETTING: Australian public hospital. PATIENTS: 104 patients with acute myocardial infarction who were treated with SK and 27 controls who were not. OUTCOME MEASURE: SK neutralising antibodies were measured once in each of the 27 controls and on 166 occasions in the 104 treated patients. RESULTS: Titres of SK neutralising antibodies rose after SK administration but returned to control levels by 2 years. CONCLUSIONS: SK might be effective again as a thrombolytic agent as early as 2 years after a single dose. These results are at variance with most previously published data and the reasons for this are not clear. Data evaluating patency rates after standard doses of streptokinase in patients with increased titres of neutralising antibodies are necessary before re-exposure to streptokinase can be recommended.

An evaluation of 200 consecutive patients with spontaneous or recurrent thrombosis for primary hypercoagulable states.

Two hundred consecutive patients who were referred for evaluation of spontaneous or recurrent thrombosis were investigated for possible hypercoagulable states to determine the relative frequencies of these conditions in the Australian population and to identify features that would indicate which patients should be investigated with the expensive battery of tests for hypercoagulable states. Thirty-two percent were found to have prolongation of the postvenous occlusion euglobulin clot lysis time (PVO-ELT), 32% were found to have elevated levels of plasminogen activator inhibitor-1 (PAI) and 66% were found to have reduced release of tissue plasminogen activator (tPA). Antiphospholipid antibodies were found in 12%. Hereditary antithrombin III deficiency was found in 2%. Hereditary deficiency of the naturally-occurring anti-coagulant factors protein C and protein S was found in 2%. Age, sex, site of thrombosis (venous or arterial), or presence of a family history was not helpful in predicting a group more likely to have abnormal investigation results. Reduced fibrinolytic activity and the presence of antiphospholipid antibodies are the most common findings in patients with thromboembolic disease. Further prospective studies are required to assess the natural history and appropriate management of patients with these abnormalities.

Effects of calcium- and ETA-receptor antagonists on endothelin-induced vasoconstriction and levels of endothelin in the human internal mammary artery.

1. The effects of the ETA receptor antagonist BQ123 and dihydropyridine calcium antagonists on the vasoconstrictor effect of endothelin-1 (ET-1) were studied in human isolated internal mammary artery (IMA). The effect of the calcium antagonist, nisoldipine, on ET-1 levels has also been examined in cultured IMA endothelial cells (IMAEC). 2. The results showed that BQ123 and the calcium antagonists nisoldipine, isradipine, nitrendipine and nifedipine fully relaxed IMA precontracted with 3 nmol/L ET-1 with the EC50 values of 7.18 +/- 0.09 (-log mol/L) for BQ123, and 7.68 +/- 0.07, 7.02 +/- 0.12, 6.96 +/- 0.08 and 6.89 +/- 0.09 for the calcium antagonists, respectively. 3. Pretreatment of IMA with 10, 30, 100 and 300 nmol/L nisoldipine significantly depressed the maximal response (Max; 88.3 +/- 5.1, 75.2 +/- 4.9, 59.3 +/- 5.6 and 56.2 +/- 4.8% of maximal noradrenaline response versus 99.1 +/- 13.2% in control, P < 0.01) of IMA to ET-1 without a significant change in the EC50 values. 4. Pretreatment of IMA with 300 nmol/L BQ123 significantly increased both the EC50 (7.97 +/- 0.09 vs 8.36 +/- 0.08 in the control, P < 0.05) and the Max (138.1 +/- 10.2% vs the control, P < 0.01) of IMA to ET-1. 5. Incubation of IMAEC with nisoldipine for 7 h resulted in a dose-dependent (10(-8)-10(-5) mol/L) reduction up to 93.1% in ET levels in the conditioned media.(ABSTRACT TRUNCATED AT 250 WORDS)

Hematologic abnormalities occur in both cortical and lacunar infarction.

BACKGROUND AND PURPOSE: Primary hematologic abnormalities are a rare but established cause of ischemic stroke. In addition, activation of hemostatic parameters is often present during the acute phase of stroke. However, it is uncertain whether these abnormalities occur in both cortical and lacunar infarction; this study aimed to further assess this issue. METHODS: Hematologic parameters (prothrombin, activated partial thromboplastin, thrombin clotting, and euglobulin lysis times; and fibrinogen, fibrinopeptide A, antithrombin III, protein C, protein S, and plasminogen levels) were measured in 19 patients within 48 hours of the onset of acute cerebral infarction. These patients included 10 with cortical infarcts and 9 with lacunar infarcts, as determined by standard clinical and radiological criteria. RESULTS: Five patients with lacunar infarction and 7 patients with cortical infarction demonstrated raised fibrinopeptide A levels, indicating enhanced thrombin activity. Fibrinolysis, assessed by the euglobulin lysis time, was impaired in 6 of 9 patients with lacunar infarction and in 2 of 10 patients with cortical infarction. Lupus anticoagulants were detected in 3 patients with lacunar infarction and in 1 patient with cortical infarction. Three patients in each group displayed decreased antithrombin III function, and 1 patient with a lacunar infarction had a low protein C level. CONCLUSIONS: Primary hematologic disorders and secondary hemostatic derangements may occur in patients with either cortical or lacunar infarction.

Reduction of endothelin levels by the dihydropyridine calcium antagonist nisoldipine and a 'natural factor' in cultured human endothelial cells.

OBJECTIVE: Endothelin is thought to be related to cardiovascular disease. The purpose of this study was to determine whether endothelin levels could be reduced by a calcium antagonist and a 'natural factor'. DESIGN: Since calcium ionophores can induce endothelin-1 messenger RNA synthesis in cultured endothelial cells, the calcium antagonist nisoldipine was used in this study to determine whether it could reduce endothelin levels. It has been reported that coculture of endothelial cells and smooth muscle cells from different species and different parts of the body can reduce endothelin levels. This study was also designed to determine whether coculture of the two cell types from the same species and the same section of an artery could reduce endothelin levels. METHODS: Cultured endothelial cells from human umbilical artery (HUAEC) and umbilical vein (HUVEC) were treated with increasing concentrations of nisoldipine. HUAEC were cocultured with human umbilical artery smooth muscle cells (HUASMC). Endothelin levels were measured by a radioimmunoassay. RESULTS: Incubation of the HUAEC with nisoldipine for either 7 or 24 h resulted in a dose-dependent (10(-8)-10(-5) mol/l) reduction in endothelin levels in the conditioned media. Endothelin levels in cell lysates were not detectable in either the absence or the presence of nisoldipine. This suggests that the reduction of endothelin levels in the media could be due to inhibition of endothelin synthesis. Under the same conditions, incubation of HUVEC with the same concentrations of nisoldipine produced a similar concentration-dependent reduction in endothelin levels. Endothelin levels were undetectable in the conditioned media from HUASMC. Coculture of HUAEC with HUASMC significantly reduced endothelin levels (P < 0.01) compared with HUAEC cultured alone. CONCLUSIONS: Endothelin levels can be reduced by the calcium antagonist nisoldipine and a 'natural factor' associated with smooth muscle cells.

The effects of leukaemia inhibitory factor on platelet function.

Leukaemia inhibitory factor (LIF) is able to promote megakaryocytopoiesis in vitro and elevate platelet counts in vivo, and is a potential new therapeutic agent for the treatment of thrombocytopenia. To determine whether platelets released under conditions of LIF-stimulated megakaryocytopoiesis have intact function, we compared aggregation responses of platelets from mice with constitutively elevated LIF levels (FD/LIF mice) and mice injected with recombinant murine LIF (rmLIF mice) with their respective control mice. We report that ex vivo platelet aggregability and thromboxane B2 release were intact in the LIF-treated mice, and were significantly enhanced in some situations. LIF-treated mice also had significantly increased platelet counts (FD/LIF mice: 1302 +/- 173 x 10(9)/l compared to 1012 +/- 99 x 10(9)/l for FD mice; rmLIF mice: 1460 +/- 193 x 10(9)/l compared to 985 +/- 67 x 10(9)/l for FCS/NS mice), increased platelet volumes and elevated plasma fibrinogen and calcium levels. The platelet hyperreactivity seen in the LIF-treated mice is likely to reflect the larger platelet volumes and/or the effect of plasma components such as fibrinogen, elevated levels of which were due to the concomitant action of LIF as a stimulant of acute phase protein synthesis.

An evaluation of the Sysmex NE-8000 hematology analyzer.

The Sysmex NE-8000 is a new, fully automated hematology analyzer capable of providing a five-part white blood cell differential count and identifying abnormal specimens. This instrument was evaluated on 5,000 consecutive blood specimens and compared to the Coulter S Plus-IV analyzer and manual differential cell counts to determine the efficacy of its five-cell differential and screening capabilities. There was a high correlation between the commercial counters for the standard parameters, white blood cell count, red blood cell count, hemoglobin level, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, and platelet count (r greater than 0.95), except for the mean corpuscular hemoglobin concentration (r = 0.51), for which the NE-8000 was considered the more accurate measurement. Precision and linearity studies were excellent. The white blood cell count, red blood cell count, hemoglobin level, and platelet count were reproducible on specimens stored at 4 degrees C or room temperature for 72 hours and the differential counts were reproducible for 12 hours. The correlations between automated and manual counts for neutrophils, eosinophils, basophils, and lymphocytes were excellent: r = 0.912, 0.945, 0.332, and 0.964, respectively. The monocyte correlation improved with software modification from 0.306 to 0.801. The NE-8000 gave accurate and reproducible differential counts for neutrophils, lymphocytes, and monocytes on specimens with white blood cell counts as low as 0.6 x 10(9)/1. The ability of the instrument to 'flag' abnormal specimens was excellent. The false-positive rate on normal samples was 1.8%, and the false-negative rate on known abnormal samples was 0.3%, due only to nonrecognition of a mild left shift. The identification of specific abnormalities was less precise. The NE-8000 is a powerful hematology analyzer that can perform a five-part white blood cell differential count accurately for a wide range of WBCs and reliably indicate abnormal specimens. It is an excellent screening tool for distinguishing between normal and abnormal specimens and identifying those that require microscopy. Its reliability significantly reduces the need for manual film examination.

High gradient magnetic separation of yeast.

High gradient magnetic separation (HGMS) is used to separate nonmagnetic microorganisms from solution by a technique known as seeding. Fine magnetic particles are adhered to the cells' surfaces, making them magnetic and amenable to magnetic separation. Attachment of the sub-micron, acicular gamma-Fe(2)O(3) seed to the yeast surface occurs irrespective of the solution pH and surface charge and is essentially irreversible. A model is developed to predict the separation of yeast in a high gradient magnetic separator. The effective capture radius is assumed to be proportional to the derived magnetic parameter gamma for the case where the dominant competing force to magnetic attraction is the magnetic floc's inertia. Using this parameter, yeast separation in an HGMS unit is predicted. The measured separation of Saccharomyces cerevisiae at differing magnetic seed concentrations and two flow rates supports the above model.

Bleeding due to an acquired inhibitor of platelet associated factor V.

Factor V inhibitors are uncommon, bleeding manifestations variable and recommendations for management are unclear. We present a patient with non-Hodgkins lymphoma who developed gastrointestinal bleeding and was found to have a Factor V inhibitor. The inhibitor was active against both plasma Factor V and platelet associated Factor V, and was associated with a five-fold increase in platelet associated IgG. Fresh frozen plasma was ineffective in preventing bleeding. Resolution of bleeding was associated with a fall in the levels of the inhibitor and of platelet associated IgG. The patient had no further bleeding episodes nor evidence of progression of his lymphoma, but six months later died as a result of metastatic adenocarcinoma.