Global Assays Research Articles

The Overall Haemostatic Potential (OHP) Assay Detects Abnormalities in Fibrinolytic Factors

No routine assays are available which quantify abnormalities in the fibrinolytic system. The Overall Haemostatic Potential (OHP) assay is a global assay of fibrin generation and fibrinolysis. Our aim in this study was to assess changes in fibrinolysis measured by the OHP assay in samples with variable levels of the different components of the fibrinolytic system: plasminogen, a2-antiplasmin, PAI-1 and TAFI. Plasmas deficient in these factors were obtained commercially. Samples were spiked with Lys-plasminogen, Glu-plasminogen, PAI-1, TAFI or pooled normal plasma to produce serial increases in these factors. Potato tuberose carboxypeptidase inhibitor (PTCI) was also used to inhibit TAFI in PNP and patient samples. OHP assays were performed utilizing either thrombin (0.03 IU/ml) or tissue factor (5 pM) to trigger fibrin generation, and rt-PA (350 ng/ml) to trigger fibrinolysis. Data analysis involved calculation of Spearman correlations for OHP assay parameters and concentrations of the various fibrinolytic factors tested. The OHP assay demonstrated markedly reduced fibrinolysis in plasminogen deficient plasmas, which normalised when plasminogen levels were increased to the normal range. At elevated Lys-plasminogen levels fibrin generation was impaired. Overall fibrinolytic potential was mildly increased in a2-antiplasmin deficiency. PAI-1 depleted plasma had mildly increased fibrinolysis compared with PNP but with addition of PAI-1, to concentrations of 300 ng/ml or higher, fibrinolysis was markedly impaired. TAFI deficient plasma had reduced fibrin generation but no change in fibrinolysis was seen in spiked samples, even with elevated TAFI levels. Representative hypofibrinolytic patient samples showed normalisation of fibrinolysis with TAFI inhibition by PTCI.The OHP assay shows appropriate changes in OFP with variations in the concentrations of activators and inhibitors of the fibrinolytic system. The assay provides a simple means of quantifying overall fibrinolysis and is sensitive to abnormalities in individual components of the fibrinolytic system. It should be useful for investigating fibrinolytic abnormalities in patients, where no routine assay is currently available.

Evidence of Increased Plasma Coagulative Capacity by CloFAL Assay among Pediatric Factor V Leiden and Prothrombin G20210A Heterozygotes with, Versus without, a First-or Second-Degree Family History of Venous Thromboembolism

BACKGROUND: The factor V (FV) Leiden and prothrombin (PT) G20210A polymorphisms in heterozygous state are present in 5% and 1–2% of Caucasians, respectively, and confer approximately 5-fold and 2-fold increases in the risk of incident venous thromboembolism (VTE). While some families who carry these genetic thrombophilia traits exhibit a prothrombotic phenotype, others have no (or only a limited) history of VTE. The ability to discern which individuals with personal and familial carriage of these genetic thrombophilias possess a clinically meaningful increase in VTE risk remains elusive, and (particularly among children) is perhaps best informed presently by family history of VTE.OBJECTIVE AND HYPOTHESES: We sought to evaluate overall plasma coagulative capacity in FV Leiden and PT G20210A heterozygotes using the Clot Formation and Lysis (CloFAL) assay, a global turbidimetric plasma assay of tissue-factor induced fibrin clot formation and tissue-type plasminogen activator enhanced fibrinolysis. We hypothesized that children heterozygous for either thrombophilia would not uniformly demonstrate hypercoagulability, but that coagulative capacity would be increased among heterozygotes who have a family history of VTE.PATIENTS AND METHODS: Children aged birth to 18 years (inclusive) enrolled in prospective inceptional cohort study of thrombosis/thrombophilia/stroke were included in the analysis if they were found to be heterozygous for FV Leiden or PT G20210A upon comprehensive thrombophilia testing and had undergone CloFAL assay testing on a research basis. Data on personal and family history of thrombotic events, thrombophilia testing, and CloFAL assay findings were analyzed. Intergroup comparisons of continuous data were performed by Mann-Whitney U test and proportions were compared between groups using chi-square or Fisher's exact test, as appropriate.RESULTS: Characteristics of the study population are shown in Table 1. Approximately 70% of patients were evaluated for a family history of VTE (with/without known thrombophilia) and nearly 50% had personal histories of VTE or arterial ischemic stroke (AIS)/recurrent transient ischemic attack (TIA); those evaluated for events were significantly older than those without events, and this difference was statistically significant among those with a positive family history fo VTE. Hypercoagulability was shown in 50% of patients and hypofibrinolysis in 13% using the CloFAL assay. Plasma coagulative capacity and maximal amplitude (MA) of the CloFAL waveform were significantly increased in patients with, versus without, family history of VTE (coagulation index, CI: 102% vs. 72% of the adult normal pooled plasma standard, respectively, p=0.04; MA: 0.415 vs. 0.322, p=0.02), and were not explained by age differences between groups. However, in this relatively small study population, the proportion of CloFAL CI results that exceeded the upper limit of normal values did not significantly differ between those with, versus without, family history of VTE. Pediatric FV Leiden or PT G20210A heterozygotes with positive family history of VTE were more likely to have multi-trait (>1) thrombophilia, in which case a trend toward increased plasma coagulability was demonstrated (CI: 139% [multi-trait] vs. 86% [isolated trait]; p=0.07); superimposed thrombophilias in this group most often consisted of elevated factor VIII activity and Lp(a) concentration.CONCLUSIONS: The present findings using the CloFAL global assay indicate that, while pediatric FV Leiden or PT G20210A heterozygotes do not uniformly exhibit hypercoagulability, plasma coagulative capacity is nevertheless significantly increased among heterozgyotes who have a family history of VTE, which may relate to the presence of superimposed thrombophilias.Table 1. Summary characteristics of the study population.N32Median age at evaluation (range)9.5 y (1–18 y)Personal history of events*14 y (1–18 y)No personal history of events*8 y (2–18 y)FV Leiden heterozygote (n)26**PT G20210A heterozygote (n)8**Personal history of VTE (n)11Personal history of AIS/recurrent TIA4Family history (1st/2nd degree) of VTE71%Multi-trait (>1) thrombophilia45%Acquired thrombophilia24%Hypercoagulability by CloFAL assay50%Hypofibrinolysis by CloFAL assay13%*VTE, AIS, or recurrent TIA**Two patients were dual heterozygotes.

In Vitro Modeling of the Influence of FVIII Activity and Heparin Induced Prolongation of APTT

Anticoagulant therapy is most commonly assessed by measuring the effect of the drug on global clotting assay, such as APTT. It is known that response of the APTT to heparin may be decreased in patients with high levels of factor VIII. In this work, we have attempted to determine in vitro conditions of experiment for obtaining relationship between different concentrations of heparin and values of APTT, and to investigate influence of factor VIII on correlation between concentrations of heparin and APTT. Measurement of the effect of heparin, added in vitro in normal coagulation control plasma (NCCP) showed that heparin in concentrations from 0.1 to 1.0 IU/mL prolonged APTT from 0.73 s to 99.26 s. Linearity of the relation of natural logarithm of APTT and concentration of added heparin in plasma for concentrations from 0.5 to 1.0 IU/mL (r = 0.995), and other characteristics of the validated method (RSD = 1.17%), made possible investigation of the influence of factor VIII addition in the solution. The addition of the Factor VIII concentrate, markedly influenced these APTT results. Increased factor VIII activity shortened the APTT, having more pronounced effect in the presence of the large amounts of heparin. Increased factor VIII was associated with downward shift in the concentration -- logAPTT response curve (y = 24644 x + 30.17 vs. y = 10.864 x + 27.256). This finding suggests the possibility for modeling of ex vivo establishment of correlation between plasma activity of FVIII and needed doses of heparin for appropriate management of heparin therapy.

Pediatric patients with congenital heart disease: thrombin generation measured by calibrated automated thrombography

The aim of this study was to investigate the possible suitability of the calibrated automated thrombography to determine the coagulation status of pediatric patients with congenital heart disease. Thrombin generation was measured in 60 patients with congenital heart disease using the calibrated automated thrombography and compared to data using standard coagulation parameters such as prothrombin, antithrombin, tissue factor pathway inhibitor, prothrombin fragment 1.2 (F 1.2), and activated partial thromboplastin time. A significant positive correlation was observed between prothrombin and the endogenous thrombin potential (P < 0.01; r = 0.295) as well as between prothrombin and peak height (P < 0.01; r = 0.581). A significant negative correlation was seen between tissue factor pathway inhibitor and endogenous thrombin potential (P < 0.01; r = -0.480) and between tissue factor pathway inhibitor and peak height (P < 0.01; r = -0.234). No statistically significant correlation was found between antithrombin and parameters of continuous thrombin generation. Significant correlation was seen neither between activated partial thromboplastin time and F1.2 nor between activated partial thromboplastin time and prothrombin. The data presented here indicate that calibrated automated thrombography measurements determine thrombin generation more accurately and therefore reflect better the coagulation status of pediatric patients with congenital heart disease then standard global coagulation assays such as activated partial thromboplastin time.

Screening Haemostasis – Looking for Global Assays: The Overall Haemostasis Potential (OHP) Method – A Possible Tool for Laboratory Investigation of Global Haemostasis in Both Hypo- and Hypercoagulable Conditions

A broad spectrum of global haemostatic assays has recently been developed and modified in an attempt to overcome the drawbacks of classical screening tests used for evaluation of coagulation and fibrinolysis. The Overall Haemostasis Potential (OHP) assay is one of such assays. The assay is based on repeated spectrophotometric registration of fibrin-aggregation in citrated plasma, to which small amounts of exogenous thrombin, tissue type plasminogen activator and calcium chloride have been added. The area under the fibrin aggregation curve which then develops is calculated and is the laboratory parameter used for OHP determination. The Overall Coagulation Potential (OCP) and Overall Fibrinolytic Potential (OFP) are supplementary parameters of OHP, providing details of underlying changes in coagulation and/or fibrinolysis. The sensitivity of the assay for detecting hypercoagulation in normal pregnancy, in preeclampsia, some thrombophilias, coronary heart disease, diabetes, stroke and vascular surgery has been evaluated. Since the assay can monitor haemostasis balance in the sample, it may serve as a laboratory tool to determine hypocoagulation, especially in patients with haemophilia A or B. Preliminary findings also indicate that the OHP assay may be useful in the monitoring of anticoagulant treatments. Larger controlled clinical studies are, however, mandatory before a definite conclusion about the usefulness of the method can be drawn.

Network motif-based identification of transcription factor-target gene relationships by integrating multi-source biological data

BackgroundIntegrating data from multiple global assays and curated databases is essential to understand the spatio-temporal interactions within cells. Different experiments measure cellular processes at various widths and depths, while databases contain biological information based on established facts or published data. Integrating these complementary datasets helps infer a mutually consistent transcriptional regulatory network (TRN) with strong similarity to the structure of the underlying genetic regulatory modules. Decomposing the TRN into a small set of recurring regulatory patterns, called network motifs (NM), facilitates the inference. Identifying NMs defined by specific transcription factors (TF) establishes the framework structure of a TRN and allows the inference of TF-target gene relationship. This paper introduces a computational framework for utilizing data from multiple sources to infer TF-target gene relationships on the basis of NMs. The data include time course gene expression profiles, genome-wide location analysis data, binding sequence data, and gene ontology (GO) information.ResultsThe proposed computational framework was tested using gene expression data associated with cell cycle progression in yeast. Among 800 cell cycle related genes, 85 were identified as candidate TFs and classified into four previously defined NMs. The NMs for a subset of TFs are obtained from literature. Support vector machine (SVM) classifiers were used to estimate NMs for the remaining TFs. The potential downstream target genes for the TFs were clustered into 34 biologically significant groups. The relationships between TFs and potential target gene clusters were examined by training recurrent neural networks whose topologies mimic the NMs to which the TFs are classified. The identified relationships between TFs and gene clusters were evaluated using the following biological validation and statistical analyses: (1) Gene set enrichment analysis (GSEA) to evaluate the clustering results; (2) Leave-one-out cross-validation (LOOCV) to ensure that the SVM classifiers assign TFs to NM categories with high confidence; (3) Binding site enrichment analysis (BSEA) to determine enrichment of the gene clusters for the cognate binding sites of their predicted TFs; (4) Comparison with previously reported results in the literatures to confirm the inferred regulations.ConclusionThe major contribution of this study is the development of a computational framework to assist the inference of TRN by integrating heterogeneous data from multiple sources and by decomposing a TRN into NM-based modules. The inference capability of the proposed framework is verified statistically (e.g., LOOCV) and biologically (e.g., GSEA, BSEA, and literature validation). The proposed framework is useful for inferring small NM-based modules of TF-target gene relationships that can serve as a basis for generating new testable hypotheses.

Monitoring Inhibitor Patients With the Right Assays

The inhibitor titer is the most important clinical measurement in inhibitor patients, and the Nijmegen method is preferable to the original and well-established Bethesda assay for this purpose; however, both methods have high inter-laboratory variability. Monitoring inhibitor patients after treatment with bypassing agents is difficult. Treatment with recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark) can be monitored with assays for FVII clotting activity or with a specific assay for FVIIa; while the latter is more reproducible, its relevance to the clinical response of individual patients remains unclear. Recent years have also witnessed a revival of global assays, which contribute useful additional information and which may be more relevant to the hemostatic state of individual inhibitor patients. One such assay currently undergoing a renaissance is the thrombin generation test (TGT). The TGT has many methodological variations--several of which have been studied in our laboratory--and shows the most promise for use in treatment monitoring. This article reviews the assays most appropriate for monitoring inhibitor patients and discusses some of the most recent developments in the use of global assays in this indication.

State-of-the-art thrombosis prevention and treatment

Thrombosis is the leading cause of death in developed countries, with arterial thrombosis presenting as myocardial infarction, stroke or peripheral arterial occlusion. Venous thromboembolism (VTE) is a major cause of mortality worldwide, despite some variations in prevalence of the disease [1]. While the incidence of VTE increases with aging, it is the leading cause of vascular morbidity in young adults. A multitude of risk factors can contribute to presentation of VTE [2]. This includes transient risk factors such as trauma, surgery, immobilization, long-haul flights, acute medical illness, hormonal therapy and pregnancy. Long-standing risk factors include chronic conditions such as congestive heart failure, chronic lung disease, inflammatory bowel disease and nephrotic syndrome. The discovery of thrombophilic risk factors opened a new era of genetic assessment of the predisposition for VTE with potential ability for better definition of the risk associated with transient risk factors. For example, hormonal therapy in women with thrombophilic risk factors, such as Factor V Leiden and the prothrombin gene polymorphism, multiply the risk for VTE [3]. While testing for thrombophilia is appealing, wider application has been hampered by equivocal cost-effectiveness analysis. A screening test for thrombophilia would be useful, and global assays such as the Protein C Global have been suggested in this regard [4]. In general, VTE is preventable by application of prophylactic measures. Applying early mobilization and physical measures such as elastic stockings and external pneumatic compression devices is useful. The current pharmacological approach is based on unfractionated, low-molecular-weight heparins (LMWH) and fondaparinux. Vitamin K

Can We Predict Bleeding?

The prior identification of subjects who are likely to bleed excessively when subjected to operative surgery and other invasive procedures is desirable. Frequently, reliance is placed on laboratory-based screening tests of blood coagulation for this purpose. However, published evidence does not support this approach as the tests are not fit for purpose, and their sensitivity and specificity are low. Some more global assays may have use in the diagnostic workup in subjects with hemorrhage, but none has been established to date as an efficient method for prediction of bleeding in unselected populations. There is renewed interest in the use of the clinical history for the prediction of bleeding. Recent reports suggest that when a structured questionnaire is employed to derive a bleeding score, the positive predictive value of the approach for the detection of bleeding disorders is high.

Characterization of a novel obesity phenotype caused by interspecific hybridization

Interspecific hybridization in mammals causes hybrid dysgenesis effects, such as sterility and abnormal placentation. Here, we describe a novel obesity syndrome caused by interspecific hybridization in the genus Mus and show that this obesity, appearing sporadically in F1 littermates derived from inbred strains, has an epigenetic basis. Mus hybrids from various strains of M. musculus and M. spretus were generated and the sporadic obese phenotype was confirmed through assessment of physiological and biochemical parameters in littermates. To understand the underlying mechanisms, large-scale and candidate gene expression assays, global DNA methylation assays and allelic expression analysis were performed. Studies showed that obese hybrids are similar to other known models of obesity. While increased axial growth indicated a defect in POMC pathway, comparison of global gene expression patterns in brain of obese F1 and obese Pomc mutant mice showed little similarity. In F1 obese mice many genes involved in the maintenance of epigenetic states, as well as several imprinted genes, were differentially expressed. Global DNA methylation analysis in brain showed that increased methylation levels were associated with obesity. The imprinted gene Gnasxl, known to be important in lipid homeostasis, was found over expressed in the obese hybrids. Allelic expression and methylation analysis of Gnasxl showed that alterations of epigenetic marks underlying F1 obesity are probably many and multi-factorial. Conclusions: This model of obesity, which is both spontaneous and epigenetic, may be a useful tool to address the epigenetic aspects of clinical obesity.

Do we need thrombin generation assays for monitoring anticoagulation?

Thromb Haemost 2008; 100: 179–180 Protamine sulphate neutralizes the anticoagulant effect of unfractionated heparin (UFH), and this effect can easily be demonstrated by a variety of assay systems, including the traditional activated partial thromboplastin time (aPTT), as well as anti-factor Xa assays, or thrombin generation tests such as the Calibrated Automated Thrombogram (CAT) (1). Protamine also neutralizes the anticoagulant effect of low-molecular-weight heparins (LMWH), but to a lesser and variable extent (2). In this issue of Thrombosis and Haemostasis Gatt et al. (3) compared the effect of protamin sulphate and other drugs on the effect of UFH, LMWH, danaparoid and fondaparinux on aPTT, anti-factor Xa-level, and a thrombin generation test. Reversal of anticoagulation is an interesting model for testing the performance of monitoring assays for anticoagulant drugs. One important finding of the present paper by Gatt et al. is that protamine has little effect on apparent anti-factor Xa-activity levels of LMWH, but a considerable effect on the CAT results. This suggests that the success of protamine administration cannot be monitored by measuring anti-factor Xa activity.This is in some contrast to other investigations showing that anti-factor Xa activity of LMWH is affected by protamine (4, 5). Finally, protamine has no effect on the anticoagulant effect of danaparoid and fondaparinux, and this was evident in both anti-factor Xa-activity and CAT assays. Reversal of danaparoid and fondaparinux, and the conventional LMWH is seldom necessary, but these drugs display a considerably prolonged half-life in patients with impaired renal function and severe bleeding may occur if the dose is not adjusted appropriately (6). Monitoring of the anticoagulant effect in patients with impaired renal function and in critically ill patients in general is essential for prevention of bleeding complications, and an assay system that gives a more global image of haemostatic function than an anti-factor Xa assay might be advantageous (7). Recombinant factor VIIa (rFVIIa, NovoSeven®) has been suggested for treatment of patients with severe anticoagulant-induced bleeding (8), and several case reports have been published on the use of rFVIIa in bleeding fondaparinux-treated patients (9, 10). As shown by Gatt et al. (3), the anti-factor Xa levels are not influenced by rFVIIa, whereas the CAT results reflect the procoagulant effect of rFVIIa. FEIBA® is an alternative to rFVIIa in the treatment of haemophilia patients with inhibitors, and may also be helpful in anticoagulant-induced bleeding. In the present experiments of Gatt et al., FEIBA acted quite similar to rFVIIa, although rFVIIa seemed to be more efficient. Another interesting result is that fresh frozen plasma reduces the anti-factor Xa-activity of UFH, LMWH, danaparoid, and fondaparinux, but has little effect on the anticoagulant effect measured by CAT. The main effect of fresh frozen plasma is dilution, and it might be that the threshold concentration of the anticoagulants for an effect on the assay systems is different. Additional experiments might be needed to determine the threshold concentration of LMWH, danaparoid and fondaparinux for the CAT. From the present results, and experiments by other investigators it seems that thrombin generation tests more reliably reflect the anticoagulant effect of these drugs (11). Anticoagulant drugs delay clot formation and traditional methods for the monitoring of anticoagulation are based precisely on this effect.After addition of an activator, clot formation occurs when only about 5% of the prothrombin has been activated to form thrombin. There are many theories why additional thrombin is formed after clot formation.The additional thrombin may not only improve clot stability by activating factor XIII and TAFI, induce cellular invasion or activation of endothelial cells adjacent to the clot in vivo, but also serves to induce the anticoagulant protein C-dependent feedback control of thrombin generation. Thrombin generation assays not only detect delayed thrombin generation (leading to a prolonged clotting time), but also reduced thrombin activity (leading to changes in structural and functional properties of the clot). Both may be relevant for the therapeutic effect of anticoagulant drugs. The results of Gatt et al. (3) indicate that the CAT may be a prototype for global coagulation assays used for monitoring of anticoagulant therapy. New anticoagulants are entering the scene and one of the major advantages of these drugs is the reliable anticoagulant effect with little intraand inter-individual varia-

Evaluating the Role of a New Dilute Prothrombin Time Assay ACTICLOT dPT in Lupus Anticoagulant Testing.

The current ISTH recommendations for Lupus Anticoagulant (LA) testing include screening with two or more sensitive assays with different assay principles. Many laboratories utilize a single Global assay such as the Hexagonal Phase Neutralization (STACLOT-LA) to satisfy the diagnostic criteria of a prolonged screen, inhibitor on mixing study, and demonstration of phospholipid dependence. Recent data presented at the SSC meeting of the ISTH suggests that laboratories may be relying too heavily on global assay to make the determination of LA. The aim of this study was to evaluate LA positivity in a pre-selected cohort of LA positive plasmas in which the diagnosis was based only upon a single positive result from a hexagonal phase global assay using a new dilute Prothrombin time test ACTICLOT dPT™Methods: 100 LA positive plasmas characterized as STACLOT-LA positive and dRVVT negative were collected. A STACLOT-LA delta > 8 sec was considered positive. The sample mean delta was 13.3 (SD +/− 5.5). ACTICLOT dPT screen and confirm was performed according to package insert instructions on a Beckman Coulter ACL automated coagulation analyzer. Additionally, 1:1 immediate and 1 hour incubated mixing studies were performed. The samples were also characterized for antiphospholipid antibodies using IMUCLONE aCL-HS and IMUCLONE anti-B2GPI IgM and IgG ELISAs. Thirty dRVVT Positive LA controls were also tested using the same ELISAs. Results are tabulated below. In graphs that will be presented, the STACLOT-LA Positive/dRVVT Negative samples have a stronger association with IgM antiphospholipid antibodies, whereas the dRVVT positive control cases demonstrate a stronger association with IgG antiphospholipid antibodies.Conclusions: The hexagonal phase positive/dRVVT negative samples examined in this study (n=100) were characterized as “weak” positive LA by hexagonal phase assay and contained mostly low titer IgM antiphospholipid antibodies by ELISA. These results suggest that many of the LA plasmas pre-selected in this cohort by hexagonal phase alone are likely to be transient LA positives. About 25% of the samples were considered LA positive using the ACTICLOT dPT screening, confirmatory and mixing protocols. The diagnosis of LA in the remaining samples may still be equivocal based upon the hexagonal phase results alone. Adding the dPT test which functions through the extrinsic pathway may improve current LA screening panels comprised of dRVVT and hexagonal phase assays. The third LA test may also identify certain LA samples that are not picked up by the dRVVT or hexagonal phase assays. Additional studies (in progress) of other LA cohorts such as STACLOT-LA Negative/dRVVT positive and prospective evaluations will help determine if single global assays overestimate LA positivity.dPT Screen, Mix, and Confirm Results (n=100)Prolonged screenProlonged screenProlonged screenProlonged screenInhibitor pattern on 1:1 mixCorrection pattern on mixing studyInhibitor pattern on mixing studyCorrection pattern on mixing studyPositive confirmNegative confirmNegative confirmPositive confirm23 %21%5 %1 %

A New Enzyme Immuno-Assay That Only Detects IgG Antibodies to Heparin-PF4 Complexes (Zymutest HIA IgG®) Improves the Specificity of the Diagnosis of HIT without Loss of Sensitivity.

Heparin-induced thrombocytopenia (HIT) is associated in most patients with the development of antibodies to heparin-modified platelet factor 4 (PF4). Commercial immuno-assays frequently detect these antibodies after cardiac surgery but only few patients develop clinical HIT. Therefore, platelet activation tests such as serotonin release assay (SRA) are necessary to ensure the diagnosis of HIT with high specificity. Another approach to increase diagnosis specificity could be to detect IgG antibodies which are of major clinical relevance since they are the only class able to directly activate platelets in the presence of heparin. Therefore, we evaluated the performances of a new commercial immuno-assay specific to IgG for the diagnosis of HIT Abs (Zymutest HIA IgG®, Hyphen Biomed, Neuville sur Oise, France). Samples from 101 patients with suspected HIT were analysed. 40 cases had developed significant levels of Abs to PF4 measured with PVS/PF4 ELISA (HAT45®,GTI, Brookfiled, WI, USA) and the diagnosis of HIT had been confirmed since SRA was positive. Every sample was then tested with a global assay named Zymutest HIA G/A/M® as followed: each diluted plasma (200 μl) was incubated for 1 hour with 50 μl of platelet lysate providing PF4 into wells previously coated with unfractionated heparin. After washings and incubation (1 hour) with anti IgG/A/M-HRP immunoconjugate, the enzyme activity was developed and absorbance was read at 450nm. In case of positive result (A450 ≥ 0.5), the isotype distribution was analysed with a specific and standardized assay using monospecific anti-IgG-, anti-IgA- and anti IgM-HRP conjugates (Zymutest HIA-IgG® or -IgA® or -IgM®). A450 values ≥ 0.5 were also considered as positive. GTI assay that detected IgG/A/M Abs to PVS/PF4 complexes in the 40 patients with HIT (Ss 100%) was also positive in 30 of the 61 cases with no HIT (Sp 50.8%). Comparatively, Zymutest HIA® global assay was positive in 39 of the 40 patients with HIT (Ss 97.5%) and in 14 of 61 cases without HIT (Sp 77%). On the other hand, significant levels of heparin-dependent IgG antibodies were also measured in these 39 HIT patients using Zymutest HIA IgG® assay (mean A450: 1.81; range A450: 0.5 – 2.76), and only in 6 patients without HIT (Sp: 90%). However, IgG levels measured in patients without HIT were significantly lower (mean A450: 0.60; range A450: 0.08 – 2.20) than in those with HIT (p < 0.0001). In addition, IgA or IgM heparin-dependent antibodies were only present, i.e. without IgG, in samples from patients for whom the diagnosis of HIT had been ruled out (n = 3). In conclusion, this study supports that the detection of significant levels of IgG heparin-dependent antibodies improves the diagnosis specificity in patients with a suspicion of HIT without loss of sensitivity. Nonetheless, whether IgG-specific immunoassays could avoid to perform platelet activation tests in patients with a strong pre-test probability of HIT warrant further study.

Influence of factor IX on overall plasma coagulability and fibrinolytic potential as measured by global assay: monitoring in haemophilia B

We sought to determine the influence of factor IX (FIX) deficiency upon overall coagulative and fibrinolytic capacities in plasma using the clot formation and lysis (CloFAL) assay, and to investigate the role of this global assay as an adjunctive monitoring tool in haemophilia B. CloFAL assay parameters were measured in vitro in platelet-poor plasma in relation to FIX activity and antigen (FIX:Ag), and were determined ex vivo among FIX-deficient patients (n = 41) in comparison to healthy individuals (n = 48). Supplementation of FIX-deficient plasma with FIX in vitro demonstrated a non-linear concentration dependence of FIX upon overall plasma coagulability. Ex vivo, coagulability was significantly decreased in FIX-deficient vs. healthy subjects among adults [median coagulation index (CI): 4% vs. 104% respectively; P < 0.001] and children (median CI: 9% vs. 63%; P < 0.001). Fibrinolytic capacity was increased in adult FIX-deficient vs. healthy subjects (median fibrinolytic index: 216% vs. 125%, respectively, P < 0.001), and was supported by a trend in shortened euglobulin lysis time (ELT). Severe haemophilia B patients showed heterogeneity in aberrant CloFAL assay waveforms, influenced partly by FIX:Ag levels. Patients with relatively preserved FIX:Ag (i.e. dysfunctional FIX) exhibited a shorter time to maximal amplitude in clot formation than those with type I deficiency. During patient treatment monitoring, markedly hypocoagulable CloFAL assay waveforms normalized following 100% correction with infused FIX. The CloFAL global assay detects FIX deficiency, demonstrates differences in coagulability between dysfunctional FIX and type I deficiency, and appears useful as an adjunctive test to routine FIX measurement in monitoring haemophilia B treatment.

Approach to the diagnosis and management of mild bleeding disorders.

Symptoms suggestive of the presence of a mild bleeding tendency are commonplace. Whilst the majority with such symptoms are healthy, it is important to identify those with bleeding disorders in order to manage symptoms, to minimize risk from invasive procedures and to avoid unnecessary exposure to blood products. Thorough clinical assessment remains the cornerstone of the diagnostic strategy for mild bleeding disorders, although the sensitivity and specificity of the clinical history and examination are limited. When clinical suspicion is aroused the use of a staged protocol of laboratory investigations is appropriate, but the limitations of currently available tests of primary hemostasis and blood coagulation must be recognized if diagnostic errors are to be avoided. Whilst there is considerable current interest in global assays of hemostasis and coagulation, none has yet been demonstrated conclusively to be more effective than the more standard approach. Iatrogenic bleeding has increasing prominence in clinical practise. The expanding use of anticoagulants and platelet inhibitor drugs has resulted in an increased proportion of the population being at risk of abnormal bleeding. Knowledge of the levels of risk associated with particular drugs and combinations, and the advantages and hazards of interruption of drug use for planned interventional procedures, are essential in order to reduce the incidence of iatrogenic bleeding. Prevention and treatment of hemorrhage in subjects with mild bleeding disorders includes the application of general measures, including attention to surgical technique, measures specific to the precise diagnosis, and less specific treatments that enhance hemostasis and coagulation or inhibit fibrinolysis. The last of these includes the widely prescribed drugs desmopressin, aprotinin, epsilon aminocaproic acid and tranexamic acid. Data are now available on their efficacy and safety in a range of clinical situations.

Determination of the global fibrinolytic state

Fibrinolysis consists of a plasmatic part and a cellular part. A rapid global assay for plasmatic fibrinolysis is the fibrinolysis parameters assay (FIPA). Cellular fibrinolysis is measured by testing the clot lysis capacity using the microtitre plate clot lysis assay with polymorphonuclear neutrophils (CLA-PMN). Individual citrated plasma or pooled normal plasma (50 microl) of 232 patients was recalcified, incubated for 90 min at 37 degrees C, oxidized with 0 or 1.5 mmol/l (final concentration) chloramine-T, and supplemented with 50 microl respective polymorphonuclear neutrophil plasma. The turbidity of the clots was measured at 405 nm after 12 h and 60 h (37 degrees C). Plasma (50 microl) was also incubated with 5 microl of 100 IU/ml urokinase, 6 mmol/l tranexamic acid, 6% human albumin for 10 min (37 degrees C). Then 100 microl of 0.5 mmol/l Val-Leu-Lys-pNA in 2.45 mol/l arginine, pH 8.6, was added and the increase in absorbance with time was measured. The different CLA-PMN assay versions correlated with each other with r = 0.543-0.782. Cellular fibrinolysis (34 +/- 30% lysis; normal: 25 +/- 10%) did not correlate with the FIPA (72 +/- 27%; normal: 100 +/- 15%), prothrombin time, activated partial thromboplastin time, fibrinogen, C-reactive protein, or the blood counts of thrombocytes, leukocytes, or polymorphonuclear neutrophils. Chloramine (1.5 mmol/l) oxidation of the microclots favours their fibrinolytic breakdown, especially if lysis-resistant microclots are oxidized. The FIPA and CLA-PMN are new economical tests for the fibrinolytic state in patient blood.

Singlet Oxygen Potentiates Thrombolysis

Activated polymorphonuclear neutrophils (PMN) participate in physiologic thrombolysis. PMN produce large amounts of urokinase (u-PA) and oxidants of the hypochlorite/chloramine-type that generate nonradical excited singlet oxygen ((1)O(2)). The u-PA/(1)O(2)-mediated thrombolysis was imitated in vitro. One hundred microliters microclots of normal human plasma were oxidized with 25 microL 0 to 5.0 micromoles of chloramine-T in physiol. NaCl in the absence or presence of 100 microL 6% bovine serum albumin or 100 microL normal plasma. Twenty-five microliters 0 to 167 IU/mL (related to 150 microL added supernatant) u-PA or 0 to 2.08 microg/mL t-PA were added. The absorbance at 405 nm was determined after 0 to 27 hours (37 degrees C). The specific clot turbidity was calculated, subtracting the 100% lysis absorbance from the respective measured absorbance. The chloramine-effective dose 50% (ED(50)) after 27 hours was determined in the presence of 2.6 IU/mL u-PA. The plasminogen activator-ED(25) was determined after 2 hours (37 degrees C), and the ET(25); i.e., the time needed to lyse a microclot by 25%, was determined for each respective clot-oxidation. The ED(25) of u-PA depends on the oxidation of the microclots: 1.25 micromoles chloramine/100 microL clot enhances thrombolysis approximately 20-fold; here, 25% of clot lysis is achieved within 50 minutes (using approximately 20 IU/mL u-PA), whereas approximately 5 hours are needed to lyse an unoxidized microclot by 25%. The present global assay technique imitates the u-PA/(1)O(2) aspects of physiologic thrombolysis by PMN.

68 Evaluation of plasmatic global hypercoagulable assays during normal and LMWH treated pregnancies

68 Evaluation of plasmatic global hypercoagulable assays during normal and LMWH treated pregnancies

A global assay of haemostasis which uses recombinant tissue factor and tissue-type plasminogen activator to measure the rate of fibrin formation and fibrin degradation in plasma

The global assay of Overall Haemostasis Potential we previously described has been refined. The coagulation cascade in platelet-poor plasma is triggered by adding a minimal dose of recombinant tissue factor together with purified phospholipids and calcium; fibrinolysis is initiated by adding recombinant tissue type-plasminogen activator in a concentration similar to what can be obtained during thrombolysis. Numerical differentials of optical densities reflecting rates of fibrin formation and degradation are calculated by a new software, and the Coagulation Profile (Cp) and the Fibrinolysis Profile (Fp) are determined. The combined effect of these counteractive systems is expressed as a ratio of Cp to Fp, called the Overall Haemostasis Index. Commercially available coagulant-deficient patient plasma samples and plasma with various amounts of added PAI-1 are examined; changes of fibrin turbidity demonstrate that this assay can determine Cp and Fp in a physiologically relevant way. Increased Cp and decreased Fp in prothrombotic patients, as well as expected effects of heparin or a thrombin inhibitor on Cp and Fp, suggest that our method can detect hypercoagulability and assist in monitoring antithrombotic treatment. Ongoing studies will show whether this simple assay can be of value in clinical routine.

An abnormal ProC Global test result is associated with an increased risk of venous thromboembolism independent of test sensitivity for protein C pathway abnormalities

The ProC Global assay is a clotting assay primarily developed to globally evaluate the functionality of the protein C (PC) pathway. It was shown to lack both sensitivity and specificity for PC pathway abnormalities, i.e. factor V Leiden mutation, PC and PS deficiency. The hypothesis that an abnormal test result could be associated with venous thromboembolism (VTE) was evaluated in a case-control study. The proportion of reduced response was significantly higher in cases than in controls [n = 71/139 (51.1%) vs. n = 28/147 (19.0%); p < 0.0001] and the same applied after exclusion of those subjects with any PC pathway abnormality [n = 53/119 (44.5%) vs. n = 25/143 (17.5%); p < 0.0001]. An abnormal ProC Global assay result was significantly associated with thrombosis both in the whole population (odds ratio [OR]=4.44, 95% confidence interval [CI]=2.61-7.53) and in those subjects without any PC pathway abnormality (OR = 3.70, 95%CI = 2.16-6.66). The ProC Global assay result was significantly lower in cases with idiopathic VTE than in those with secondary VTE (p < 0.0001). No significant difference was observed when cases were classified according to the presence or absence of recurrent episodes. Moreover, a reduced response was found to be associated with VTE both in subjects with normal or elevated factor VIII (FVIII) level. In vitro, FVIII was found to play a critical role in the ProC Global assay result as suggested by the significant trend toward decreasing response with increasing FVIII levels. In conclusion, our results suggest that an abnormal ProC Global assay result is associated with an increased risk of VTE independently of its sensitivity for PC pathway abnormalities.