Coagulopathy associated with massive cell salvage transfusion following aortic surgery.

Cell saver blood is used within the peri-operative setting of cardiothoracic surgery to reduce the need for transfusion of allogenic blood products. Several meta-analyses have proven a significant decrease in allogenic transfusion with the use of cell salvage techniques. Washing of red cells by the cell saver and subsequent transfusion of suspended red cells can occasionally cause coagulopathy, particularly when using high concentration heparin saline to wash the spilled blood. We present the case of a 74-year-old female who underwent complicated aortic surgery and was transfused large volumes of cell-saved blood due to post-operative bleeding, which subsequently led to coagulopathy.

Near-patient platelet function testing in patients undergoing coronary artery surgery: a pilot study.

Platelet dysfunction after cardiopulmonary bypass contributes to microvascular bleeding and is associated with blood transfusion and resternotomy. Platelet count can be readily performed, but currently there are no standardised, reproducible, rapidly available platelet function tests. We studied platelet function as measured by multiple electrode platelet aggregometery (multiplate) and light transmission aggregometry in 44 patients undergoing routine coronary artery surgery. Platelet aggregation as measured by multiplate was reduced during and after cardiopulmonary bypass compared with baseline with evidence of partial recovery by the time of transfer to ITU. In patients transfused blood, platelet aggregation measured by multiplate was reduced during chest closure with adenosine diphosphate (18 U vs 29 U, p = 0.01) and thrombin receptor agonist peptide-6 agonist (65 U vs 88 U, p = 0.01) compared with patients not transfused. This suggests that multiplate, a new point of care analyser, can detect platelet dysfunction in this setting.

A computer-based model to assess costs associated with the use of factor VIII and factor IX one-stage and chromogenic activity assays.

BACKGROUND: Measurement of coagulation factor factor VIII (FVIII) and factor IX (FIX) activity can be associated with a high level of variability using one-stage assays based on activated partial thromboplastin time (APTT). Chromogenic assays show less variability, but are less commonly used in clinical laboratories. In addition, one-stage assay accuracy using certain reagent and instrument combinations is compromised by some modified recombinant factor concentrates. Reluctance among some in the hematology laboratory community to adopt the use of chromogenic assays may be partly attributable to lack of familiarity and perceived higher associated costs. OBJECTIVES: To identify and characterize key cost parameters associated with one-stage APTT and chromogenic assays for FVIII and FIX activity using a computer-based cost analysis model. METHODS: A cost model for FVIII and FIX chromogenic assays relative to APTT assays was generated using assumptions derived from interviews with hematologists and laboratory scientists, common clinical laboratory practise, manufacturer list prices and assay kit configurations. RESULTS: Key factors that contribute to costs are factor-deficient plasma and kit reagents for one-stage and chromogenic assays, respectively. The stability of chromogenic assay kit reagents also limits the cost efficiency compared with APTT testing. Costs for chromogenic assays might be reduced by 50-75% using batch testing, aliquoting and freezing of kit reagents. CONCLUSIONS: Both batch testing and aliquoting of chromogenic kit reagents might improve cost efficiency for FVIII and FIX chromogenic assays, but would require validation. Laboratory validation and regulatory approval as well as education and training in the use of chromogenic assays might facilitate wider adoption by clinical laboratories.

The risk of a first and a recurrent venous thrombosis associated with an elevated D-dimer level and an elevated thrombin potential: results of the THE-VTE study.

BACKGROUND: D-dimer and thrombin generation have been associated with the risk of recurrent venous thrombosis. However, for both measurements, different assays are available, and in vitro thrombin generation may be affected by the problem of contact activation during blood sampling. OBJECTIVES: To determine the association between hypercoagulability and first and recurrent thrombosis by the use of different D-dimer and thrombin generation assays, to assess whether the addition of corn trypsin inhibitor (CTI) prior to blood sampling to inhibit contact activation improved the association between thrombin generation and thrombosis risk, and to calculate the DASH score with two different D-dimer assays. METHODS: A case-control study (626 patients and 361 controls) with subsequent follow-up of the cases was performed (2987 patient-years after stopping of anticoagulant therapy). Blood was drawn 2-3 months after discontinuation of anticoagulation for the first event in citrate tubes with and without CTI. RESULTS/CONCLUSIONS: An elevated D-dimer level and elevated thrombin generation were associated with an increased risk of a first event regardless of the assay used (odds ratios: 1.8-3.4). An elevated D-dimer level but not elevated thrombin generation was associated with the risk of recurrence. Patients with elevated D-dimer levels had a more than two-fold increased recurrence rate (Vidas - hazard ratio [HR] 2.3, 95% confidence interval [CI] 1.4-3.8; HemosIL - HR 2.4, 95% CI 1.5-3.9; Thrombinoscope and Technoclone assay - HR 1.3). Elimination of contact factor activation did not improve the predictive value of thrombin generation. In patients with unprovoked first events, the DASH score had a similar predictive value for deep vein thrombosis and pulmonary embolism, both when calculated with Vidas D-dimer and when calculated with HemosIL D-dimer.

Clinical measurement of thrombin generation by calibrated automated thrombography requires contact factor inhibition.

BACKGROUND: Measurement of thrombin generation by calibrated automated thrombography (CAT) could fulfill the requirements of a global test of coagulability and is potentially applicable to routine clinical laboratory practice. The purpose of this study was to determine if corn trypsin inhibitor (CTI) could be used to abolish contact factor activation in this assay, thus allowing accurate measurement of low tissue factor (TF) concentration-triggered thrombin generation on samples taken in a routine clinical setting. METHODS: The endogenous thrombin potential (ETP) was measured by CAT. RESULTS: The study demonstrated that addition of CTI after plasma separation is not sufficient and blood must be drawn into tubes containing CTI if in-vitro contact factor-activated thrombin generation is to be abolished. Contact factor-activated thrombin generation is completely inhibited at a CTI concentration of 18.3 microg mL(-1) whole blood. Increasing the CTI concentration above this level does not lead to suppression of the TF-triggered ETP. At a TF concentration of 2 pmol, ETPs were significantly lower in the presence of CTI (P < 0.001). The difference (no CTI minus CTI) between results ranged from - 1 to 2159 nM min(-1) (median - 754). Whilst the low concentration TF-ETP assay was not optimized to distinguish degrees of coagulability between patient samples, there was a significant difference in ETP between normal and hemophilia samples and samples from patients with a clinical prothrombotic tendency. CONCLUSIONS: CTI can be applied to ETP measurement by CAT. This permits the use of CAT in a low TF-triggered thrombin generation assay without concern for the effect of interference from in-vitro contact factor activation and the optimum reagent conditions for using CAT as a global test of coagulability in clinical practice can now be defined.

High risk of recurrent venous thromboembolism in men.

BACKGROUND: We have analyzed the influence of gender on risk of recurrence after a first episode of venous thromboembolism (VTE). METHODS: The Cambridge Venous Thromboembolism Study (CVTE) is a single-center study of a cohort of unselected patients with a first episode of objectively proven VTE. RESULTS: Recurrence rates were significantly higher in men compared with women [log rank chi2=11.82; hazard ratio (HR) 2.66; 95% confidence interval (CI) 1.49, 4,77; P=0.0006]. The cumulative recurrence rate at 2 years was 19.2% in men and 7.7% in women. There was no evidence of a difference in recurrence between men with or without thrombophilia (log rank chi2=0.03; HR 1.08; 95% CI 0.49, 2.37; P=0.855). The high recurrence rate in men compared with women was still observed when only patients with idiopathic VTE were analyzed (log rank chi2=4.38; HR 2.31; 95% CI 1.027, 5.20; P=0.0363). The recurrence risk was highest in men with a first idiopathic event at 25.7% compared with 11.7% for women in the same category. CONCLUSION: The risk of recurrent VTE is higher in men than in women.

Coagulation changes following hepatic revascularization during liver transplantation.

The coagulation changes during liver transplantation have been studied in 14 selected patients. Blood usage in all cases was limited to 8.5 liters, and the preoperative coagulation results were only minimally deranged. Bleeding during the operative procedure was easily managed in all cases. Nonetheless, even in this selected group of "low risk" patients, we have demonstrated that during the anhepatic phase and particularly following hepatic revascularization there is activation of both coagulation and fibrinolysis. These findings imply that if bleeding occurs following revascularization, in addition to the use of replacement blood products, treatment should be directed at reducing the consumptive coagulopathy and inhibiting fibrinolysis. We suggest as a first step antithrombin supplementation to maintain activity above 70%, and an antifibrinolytic agent, such as aprotonin, should be considered as adjuncts to therapy at revascularization.

Changes in endothelial-related coagulation proteins in response to venous occlusion.

One hundred patients with a history of thrombophilia were divided into two groups based on fibrinolytic response to venous occlusion. Good responders with a euglobulin clot lysis time less than or equal to 105 min showed significant release of tPA, PAI and vWF. Of the poor responders with an ECLT greater than 105 min, 24% showed a subnormal increase in tPA, and a significant proportion of these also showed a reduced or absent rise in vWF. We have shown poor fibrinolytic response was related to either raised levels of PAI, poor release of both tPA and vWF, or poor release of tPA or vWF alone suggesting different mechanisms of fibrinolytic impairment. Protein S levels were not significantly changed in either group following occlusion.

The factor V R2 allele: risk of venous thromboembolism, factor V levels and resistance to activated protein C.

Case-control studies have yielded conflicting results regarding the relative risk of venous thromboembolism associated with the factor V R2 allele. We calculated odds ratios in 581 patients and 469 age-matched controls. The odds ratio for the R2 allele in patients relative to controls was 1.21 (95% CI 0.84 to 1.74). These results do not support the hypothesis that the R2 allele is a risk factor for venous thromboembolism. There was no relationship between factor V levels and R2 carrier status. Normalised APC sensitivity ratios were not lower in carriers of the R2 allele. In an in vitro model progressive APC resistance was observed with factor V levels of 60% and less but ratios less than 2.4 (equivalent to a normalised ratio of 0.73) did not occur until factor V levels were less than 20%. The relationship between APC resistance and factor V level was not observed in a factor VIII-independent model.

Changes in factor VIII proteins after cardiopulmonary bypass in man suggest endothelial damage.

16 patients undergoing coronary artery bypass grafting using cardiopulmonary bypass (CPB) had blood samples taken at various times before, during and up to 1 week after surgery for estimation of beta-thromboglobulin (BTG), alpha-1-antichymotrypsin (ACT), factor VIII procoagulant protein (VIII:C), von Willebrand factor antigen (vWF:Ag) and ristocetin co-factor (vWF:RiCoF). vWF:Ag and vWF:RiCoF rose during and following surgery in a different manner to ACT. At 1 week there was a significantly disproportionate rise in vWF:Ag compared to vWF:RiCoF which suggested a degree of pulmonary endothelial damage. Prostacyclin, which was administered to 8 of the patients during CPB, reduced platelet activation as measured by a reduction in the release of BTG and also attenuated the consumption of VIII:C. It had no effect on pulmonary endothelial damage as measured by the ratio of vWF:Ag to vWF:RiCoF.

Changes in the natural anticoagulants following bone marrow transplantation.

The natural anticoagulants, protein C, protein S and antithrombin, were measured in 21 patients following bone marrow transplantation (BMT). The patients were divided into two groups, those with normal protein C concentration post-BMT and those with significantly reduced protein C concentrations; 12 cases fell into the second group. In addition there was an associated fall in protein S in this group of patients. In spite of the presumed hypercoagulable state only one patient developed overt veno-occlusive disease (VOD) of the liver and no other thrombotic events occurred. The fall in protein C and protein S was coincident with the peak incidence of VOD and is likely to be a contributing factor to the genesis of this condition. Only protein S measured pre-BMT was of predictive value in identifying patients likely to develop reduced levels of these anticoagulants post-BMT. Age, sex, diagnosis, type of conditioning and the pre-BMT measurement of protein C and antithrombin had no predictive value.

Evaluation of the Ciba Corning Biotrack 512 coagulation monitor for the control of oral anticoagulation.

The Ciba Corning Biotrack 512 coagulation monitor requires a minimal degree of technical expertise to operate, and is already in use for near-patient testing. This study evaluated the monitor for possible use in decentralised control of oral anticoagulant treatment. The monitor compared well with Manchester Reagent, suggesting that it could be used in areas where this thromboplastin is used for centralised control. The inability of the monitor to allow for locally determined geometric mean normal prothrombin times in the calculation of the International Normalised Ratio (INR), and possibly the high International Sensitivity Index (ISI) of the thromboplastin used with the monitor, resulted in poor comparability with some other thromboplastins, particularly Thrombotest. These problems need to be addressed if the monitor is to be used for decentralised anticoagulant control.

Markers of thrombin and plasmin generation in patients with inherited thrombophilia.

AIM: To determine the prevalence of a biochemically detectable hypercoagulable state, defined in terms of increased thrombin or plasmin generation, in patients with phenotypically characterised thrombophilia. METHODS: Plasma concentrations of the prothrombin activation peptide F1.2 and fibrin degradation (FbDP) and fibrinogen degradation products (FgDP) were measured by enzyme immunoassay in 104 patients deficient in natural anticoagulants, and 35 unaffected relatives. RESULTS: Increased concentrations of F1.2, FbDP, and FgDP were present in 18, 25, and 19 of 104 patients, respectively. There were no correlations between F1.2, FbDP, and FgDP concentrations, or between these parameters and concentrations of natural anticoagulants except for a negative correlation between protein C concentrations and FgDP (rho = -0.46, p = 0.009). CONCLUSION: A biochemically detectable hypercoagulable state is present in some patients with asymptomatic thrombophilia. Markers of plasmin generation may be increased more frequently in thrombophilia than markers of thrombin generation. This finding should prompt the inclusion of markers of plasmin generation in prospective longitudinal cohort studies to determine the predictive value of a hypercoagulable state, defined by either excessive thrombin or plasmin generation, for the development of venous thromboembolism.

Lupus anticoagulant testing with optical end point automation.

The dilute Russell viper venom time and kaolin clotting time (KCT) are very sensitive screening tests for lupus anticoagulant activity. However, due to the high turbidity of the kaolin reagent it is difficult to accommodate the KCT on the optical end point automation of today. We evaluated five recently reported screening tests (the silica clotting time, the Textarin/Ecarin ratio, the Taipan venom time, the factor V ratio, and a kaolin clotting time using low-turbidity kaolin) as potential alternatives to the KCT. The sensitivity and specificity of the silica clotting time compared well to KCT, detecting 10/12 KCT positive samples and showing equal sensitivity to dilution of lupus positive plasma. In addition, the silica clotting time allows for a confirmatory phospholipid correction procedure. False-positive results were seen in 2 of 15 warfarinised samples. A second assay utilising the ratio of extrinsic/intrinsic factor V assays was not affected by either warfarin or heparin. This assay also gave positive results with 3 of 23 samples previously screened as lupus negative but exhibiting anticardiolipin positivity. It was therefore concluded that a combination of the silica clotting time and dilute Russell viper venom time met the requirements of lupus sensitivity with demonstration of phospholipid dependence and optical end point compatibility. The factor V ratio is a useful second-line screen for both anticoagulated patients and anticardiolipin antibody-positive samples.

The effect of delayed analysis or freeze-thawing on the measurement of natural anticoagulants, resistance to activated protein C and markers of activation of the haemostatic system.

We provide a centralised thrombophilia screening service with sample transfer by courier or first class mail, a practice common to many centres. Sample quality is of prime importance, thus we have assessed the effect of delayed sample handling upon the haemostatic variables within our thrombophilia profile. This comprises screening for familial natural anticoagulant deficiency and APC resistance (1-4), and acquired lupus anticoagulant (5,6). The effect upon hypercoagulable markers were also assessed to facilitate evaluation of these in prospective clinical studies (7).

Mutational analysis of the human protein C gene.

The single gene for protein C is located at position q13-q14 on chromosome 2 (1). Two groups have described human genomic clones of protein C isolated from phage l charon libraries using cDNA for human protein C as hybridization probes (2,3). The gene is approximately 11 kb long and is composed of 9 exons and 8 introns. In common with factors VII, IX and X the exons encode specific structural domains of the protein C molecule. Exon 1 encodes the 5' untranslated region, exon 2 encodes the signal peptide and 6 amino acids of the propeptide, exon 3 encodes the remainder of the propeptide and the Gla domain (residues 1-45), exon 4 encodes the connecting segment between the Gla domain and the first EGF-like domain, exons 5 and 6 encode for the EGF-like domains (residues 49-91 and 92-137), exon 7 encodes the activation peptide (residues 157-169), the C-terminus of the light chain and the first 29 amino acids of the heavy chain. Exons 8 and 9 encode the remaining heavy chain sequence.

Monocyte procoagulant activity: development of a microtitre plate chromogenic assay.

A monocyte procoagulant assay was developed based on the original method of Surprenant and Zuckerman, which quantitates a factor Xa-specific chromogenic substrate (at 405 nm) activated via the extrinsic coagulation pathway. Normal tissue factor initiation of the pathway is replaced by tissue factor generated from monocytes, stimulated by various agents including bacterial lipopolysaccharide, and antibody/antigen complexes. Hydrolysis of the chromogenic substrate is therefore directly proportional to the degree of monocyte activation. Using a chromogenic substrate as an end-point the assay was performed in a standard microtitre plate.

Standardisation of thrombin generation test--which reference plasma for TGT? An international multicentre study.

We have previously shown that standardisation and normalization of results improve the intercentre variability of the calibrated automated thrombin generation test (TGT). We suspected that the source of reference plasma (RP) might be a contributing factor to variability and compared 5 commercial RP and a RP provided by the NIBSC, in an international, multicentre study. The detailed composition of the 6 tested plasma samples was determined in the Haemostasis Laboratory in Lyon. The lot to lot consistency, intra-assay, inter-assay variability were calculated for all tested plasmas. The RP and 3 plasma samples (a normal control, a hypocoagulable and a hypercoagulable plasmas) were tested over 6 days, in 5 European centres. Results were normalised against each of the tested RP and intercentre variability of results was compared. All laboratories used the same reagents. Before normalization, the inter-centre variability was 19.8 to 27.3%. After normalization, we observed a significantly improved inter-laboratory variation with all tested RP, despite differences between them. These results clearly demonstrate that the inter-centre variability of TGT can be significantly reduced by using a reference plasma normalization, and that certain RP have a better capacity to reduce this variability than others.