Role of chromogenic assays in haemophilia A and B diagnosis.

The laboratory diagnosis and monitoring of factors VIII and IX have been primarily by one-stage clotting assay (OSA) for many years. Chromogenic assays (CSA) have been available only in specialist laboratories and not for routine use. Significant differences, of more than 1.5-fold in results between the 2 assay methods, have been described in Europe and Australia in approximately one-third of patients with mild haemophilia A. In certain discrepant groups with restricted F8 gene mutations, the OSA results are more than 1.5-fold higher than CSA and risk a missed or misleading diagnostic result. More recently, an assay discrepancy in haemophilia B has been reported. With the introduction of extended half-life (EHL) FVIII and FIX products, it is likely most coagulation laboratories will need to evaluate at least one CSA and gain experience with this technique. The validation of CSA involves a careful appraisal of calibration curve linearity, limit of detection, precision, reference range, quality control material, sample analysis, method comparison and cost. This review will discuss the current status of FVIII and FIX CSA for the diagnosis of haemophilia A and B and describe approaches to implement CSA into the laboratory repertoire.

Is there thrombin-activatable fibrinolysis inhibitor in saliva?

The purpose of this study was to identify thrombin-activatable fibrinolysis inhibitor (TAFI) in saliva and to investigate the correlation between TAFI levels in saliva and plasma. Subjects included were healthy adults without diseases or medication that could affect coagulation. Samples of stimulated saliva and blood samples were obtained from 33 subjects. Levels of TAFI in saliva and plasma were analysed. The association between levels of TAFI in saliva and plasma was calculated using linear regression. Low levels of TAFIa/TAFIai were found in most saliva samples but only one sample had levels that were above the lower limit of detection of the assay used. TAFI (proenzyme) was not found in saliva, so no correlations could be calculated. In this study there was no indication that there is TAFI present in secreted saliva. Either TAFIa/TAFIai in saliva were much lower than in plasma and under the detection limit of the assay used, or there was no TAFIa/TAFIai in the saliva tested.

Age-adjusted D-dimer cut-off leads to more efficient diagnosis of venous thromboembolism in the emergency department: a comparison of four assays.

Essentials Age-adjusted D-dimer cut-offs decrease the false positives in the elderly. Four D-dimer assays were compared in venous thromboembolism outpatients in an emergency ward. Age-adjusted cut-off resulted in improved specificity with maintained sensitivity for all assays. There was a substantial decrease in false positive results, especially in the older population. SUMMARY: Background The study compares different D-dimer assays and age-adjusted cut-offs in outpatients with suspected venous thromboembolism (VTE). The plasma concentration of this sensitive biomarker is increased by activated coagulation, but also by several conditions that are linked to an increased risk of VTE. One such condition is old age, which poses a common clinical problem where many prefer not to analyze D-dimer in elderly patients. Age-adjusted cut-offs have been validated for both deep venous thrombosis (DVT) and pulmonary embolism, aiming to increase specificity without notably decreasing sensitivity. Objectives We evaluated four common D-dimer assays in parallel, with and without applying age-adjusted cut offs for VTE. Patients/methods The prospective single-center study was conducted in 940 outpatients attending the emergency department with clinically suspected pulmonary embolism or DVT. Four automated D-dimer assays were compared (Siemens INNOVANCE® , Roche Tina-quant, Medirox MRX and STA® -Liatest® D-Di PLUS). Results All assays performed with areas under the ROC curve (AUC) > 0.9 and maintained their sensitivities after implementation of age-adjusted cut-offs. Specificities increased by 6-7% and number needed to test decreased by < 0.3. The rate of false positive results decreased by 6% overall and by 10-20% for patients ≥ 70. Conclusions Age-adjusted cut-offs resulted in maintained high sensitivity and a modest improvement in specificity and number needed to test for all evaluated D-dimer assays. There was a significant reduction in false positive results, which reflects avoidable unnecessary imaging without any compromise of clinical safety. This suggests a potential to benefit the management of VTE in elderly patients, both clinically and economically.

Evaluation of small-volume tubes for venous and capillary PT (INR) samples.

INTRODUCTION: Frequent PT (INR) testing may represent a problem for patients on warfarin treatment, and capillary or small-volume tubes may be more appropriate for such patients. A demand for small-volume tubes also comes from pediatric wards. Yet, while various small-volume tubes are available, they have not been properly evaluated. METHODS: Three small-volume tubes were tested (MiniCollect 3.8% citrate, MiniCollect 3.2% citrate and Microvette EDTA) and compared with a standard 4.5-mL 3.2% citrated tube. Samples were taken by venipuncture from the back of the hand and by capillary sampling from the tip of the finger. The measures were compared with those after standard venipuncture of the arm fold. A total of 180 samples, using different combinations of tubes and sampling sites, were collected from 30 volunteers. RESULTS: There were no differences in the results obtained using citrate tubes for venous samples in comparison with those obtained by standard sampling, while the results when using EDTA tubes were not comparable to those obtained by standard sampling (P < 0.001), expressing systematically lower values (by about 10%). The results observed after capillary sampling were significantly different to those obtained after standard sampling. CONCLUSIONS: The MiniCollect 3.2% tube may be used for PT (INR) venipuncture samples when withdrawal of a small amount of blood is preferable, while EDTA tubes should not be used for PT (INR) testing.

Does thrombin activatable fibrinolysis inhibitor (TAFI) contribute to impairment of fibrinolysis in patients with preeclampsia and/or intrauterine fetal growth retardation?

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a relatively recently described glycoprotein (MM 55 KDa) that can be converted into its active form by the thrombin/thrombomodulin complex and potentially inhibits fibrinolysis. Since it represents a link between coagulation and fibrinolysis, TAFI can be expected to play a part in various clinical conditions associated with a thrombotic tendency. Preeclampsia (PE) and intrauterine fetal growth retardation (IUFGR) are fairly common complications of pregnancy that are characterized by hemostatic abnormalities. TAFI antigen and its influence on hemostasis was investigated in 46 women with PE and/or IUFGR and in 16 normal pregnancies. We found a significant decrease of TAFI antigen in the patient group. Using the recently described method Overall Hemostatic Potential (OHP) in plasma we measured clot lysis time (CLT) and overall fibrinolytic potential (OFP). We found that CLT is prolonged and OFP decreased in patients with PE and/or IUFGR. Since OFP did not increase after addition of the specific inhibitor of TAFI (potato tuber carboxypeptidase inhibitor), it seems that TAFI does not contribute to the impairment of fibrinolysis in these patients. Since serum albumin was decreased together with presence of proteinuria and aminotransferases were increased in the patients, it seems that one explanation for the decrease in TAFI could be reduced hepatic synthesis and an increased loss in urine. It an be speculated that this mechanism can prevent more serious thrombotic complications in patients with PE and/or IUFGR.

Is a decrease of microparticles related to improvement of hemostasis after FVIII injection in hemophilia A patients treated on demand?

BACKGROUND: Microparticles (MPs) are small membrane vesicles (0.1-1 µm) released from various cells after activation and/or apoptosis. There are limited data about their role in hemophilia A. PATIENTS AND METHODS: Blood samples were taken before and 30 min after FVIII injection in 18 patients with severe hemophilia A treated on demand. Flow-cytometric determination of total MPs (TMPs) using lactadherin, platelet MPs (PMPs) (CD42a), endothelial MPs (EMPs) (CD144) and leukocyte MPs (LMPs) (CD45) was performed. The results were compared with data on endogenous thrombin potential (ETP), overall hemostatic potential (OHP), fibrin gel permeability and thrombin-activatable fibrinolysis inhibitor (TAFI). RESULTS AND CONCLUSIONS: TMPs and PMPs decreased after treatment (to 1015 ± 221 [SEM] and 602 ± 134 × 10(6)  L(-1) ) in comparison with values before treatment (2373 ± 618 and 1316 ± 331; P < 0.01). EMPs also decreased after treatment (78 ± 12 vs. 107 ± 13; P < 0.05) while LMPs were not influenced. Both TMP and PMP counts were inversely correlated, moderately but statistically significantly, with data on OHP, ETP, fibrin network permeability and TAFI/TAFIi (P < 0.05 for all). EMP counts were correlated only with ETP (P < 0.05), while LMP counts did not show any correlation. TMP and PMP counts were also inversely correlated with FVIII levels (P < 0.05). TMP, PMP and EMP counts decreased after on-demand treatment with FVIII concentrate in hemophilia A patients. The decrease in circulating MPs, which were inversely correlated with hemostatic activation, may imply that MPs are incorporated in the hemostatic plug formed after FVIII substitution at the site of injury.

The evaluation of two automated soluble fibrin assays for use in the routine hospital laboratory.

INTRODUCTION: The soluble fibrin monomer (sFM) assay, like the D-dimer (DDi) assay, has the potential to be used both as an aid in the diagnosis of disseminated intravascular coagulation (DIC) and as a thrombotic marker. It differs from DDi in that it is a much earlier produced fragment produced only by thrombin action on fibrinogen, whereas DDi is a much later produced fragment formed by plasmin cleavage of cross-linked fibrin. METHODS: In our study, we compared two commercially available automated sFM assays in the routine hospital setting using samples obtained from the general hospital ward and the emergency room. The results obtained with the two automated assays (Stago LIA sFM assays and the LPIA-Iatro SF assay) were compared with each other and with the results obtained using the routine semiquantitative hemagglutination assay. RESULTS: The study showed that both automated assays were comparable with each other. No patient sample previously classified as positive would be missed, but with the higher sensitivity in the automated tests, more samples are positive. CONCLUSION: In conclusion, we suggest that both automated tests are suitable for routine laboratory use. Both assays had the advantage over the hemagglutination assay in that previously frozen samples could be used, and the assays are easier and quicker to perform. The LIA sFM Stago has slightly better sensitivity but has a tendency to lower specificity than the Iatro SF test.

Comparison of five point-of-care D-dimer assays with the standard laboratory method.

INTRODUCTION: D-dimer (DD) assays are effective for the exclusion of deep-vein thrombosis (DVT), but point-of-care (POC) DD assays have not been fully evaluated. METHODS: We have compared five POC DD assays (Pathfast, Cardiac, Vidas, Stratus and NycoCard) with our routine DD method (Tinaquant), testing 60 samples from patients with suspected DVT. RESULTS: Using 0.5 µg/mL as a cut-off value, four samples tested negative with Tinaquant were positive with Pathfast. There were no Tinaquant-positive samples tested negative with Pathfast, while the overall agreement (k = 0.81) was very good. Four samples were discrepant between Tinaquant and Cardiac (cut-off, 0.4 µg/mL), while k = 0.72. One of two Tinaquant-negative samples was shown to be positive for either Vidas (cut-off, 0.5 µg/mL) or Stratus (cut-off, 0.4 µg/mL), respectively. The agreement with Tinaquant was excellent for both Vidas (k = 0.92) and Stratus (k = 0.94). Total CV was <10% for all four assays. Eight samples (of 27) were negative with NycoCard although they were positive with Tinaquant, while CV was 41%. CONCLUSION: Vidas cannot be considered a POC assay because the sample has to be centrifuged before testing. Our findings have also shown that the use of NycoCard is inappropriate. Stratus and Pathfast have a similar analytical profile in comparison with the Tinaquant method. Cardiac is potentially less sensitive but may still be acceptable for use. It seems that the employment of these three assays for rapid bed-side analysis offers a possibility to adequately rule out DVT in outpatients within minutes after admission.

On-demand but not prophylactic treatment with FVIII concentrate increase thrombin activatable fibrinolysis inhibitor activation in severe haemophilia A patients.

INTRODUCTION: Thrombin activatable fibrinolysis inhibitor (TAFI) down-regulates fibrinolysis after activation by thrombin/thrombomodulin. We investigated the effect of treatment with FVIII concentrate on plasma levels of pro-TAFI and activated TAFI in haemophilia A patients. METHODS: Samples were collected pre and posttreatment from patients treated prophylactically or on-demand. Pro-TAFI, TAFI/TAFIi and FVIII levels were measured in all samples. RESULTS: Treatment had no effect on pro-TAFI levels. Pro-TAFI was similar in both patient groups but higher than in controls. Patients from the prophylactic treatment group had measurable FVIII levels pretreatment while in the treatment-on-demand group FVIII levels were ≤0.01 IU/mL. In the prophylactic treatment group, the levels of TAFI/TAFIi were significantly lower pre- and posttreatment (4.31 ± 3.14 and 3.48 ± 2.65 ng/mL respectively) than in the on-demand group (13.02 ± 3.47 and 14.87 ± 3.47 ng/mL respectively). This difference may be due to release of tissue factor at the injury site in the on-demand group. This could induce thrombin and TAFI activation within the clot counterbalancing fibrinolysis in these patients. In the prophylactic group, no injury existed, thus there was insufficient thrombin generation within the clot to activate TAFI. CONCLUSION: These findings suggest that in patients to whom FVIII is administered on demand the fibrinolysis activity is more down regulated than in patients following a prophylactic treatment regime.

Overall haemostatic potential can be used for estimation of thrombin-activatable fibrinolysis inhibitor-dependent fibrinolysis in vivo and for possible follow-up of recombinant factor VIIa treatment in patients with inhibitors to factor VIII.

Thrombin generation induced by recombinant factor VIIa (rFVIIa) in patients with haemophilia and/or inhibitors to factor VIII/IX could enhance generation of thrombin-activatable fibrinolysis inhibitor (TAFI), a recently described link between coagulation and fibrinolysis. TAFI is unstable and it is not easy to measure its active form in vivo. Overall haemostatic potential (OHP) is a novel method for haemostasis estimation, based on determination of the fibrin aggregation curve in which tiny amounts of thrombin are used for activation of clotting. We measured OHP in six patients with inhibitors to factor VIII before injection of rFVIIa and 10 and 120 min thereafter. Overall fibrinolytic potential (OFP) and clot lysis time (CLT) analysed by this method could be used for indirect estimation of TAFI generation. We found no change in pro-TAFI and total TAFI antigen before and after treatment with rFVIIa. OHP was almost undetectable before treatment but increased into the range of normal pooled plasma 10 and 120 min after rFVIIa treatment, as did CLT. However, after addition of potato tuber carboxypeptidase inhibitor, a specific inhibitor of TAFI, the shortening of CLT was lower than that in NPP. OFP was increased in patient plasma both 10 and 120 min after treatment compared with NPP. There was a strong positive correlation between pro-TAFI concentration and shortening of CLT after PTCI addition and a negative correlation between pro-TAFI concentration and OFP 10 min after rFVIIa injection. Thus, rFVIIa normalizes OHP and CLT 10 min after injection. While this improvement slightly decreases, but still exists after 2 hours, it suggests efficacy in bleeding prevention using a protocol based on rFVIIa administration every 2 hours.

Does an enzyme other than thrombin contribute to unexpected changes in the levels of the different forms of thrombin activatable fibrinolysis inhibitor in patients with hemophilia A, hemophilia B and von Willebrand disease?

Pro-thrombin activatable fibrinolysis inhibitor (pro-TAFI), also called plasma procarboxypeptidase B or U, is one of the modulators of fibrinolysis in blood. Pro-TAFI is activated by thrombin/thrombomodulin complex or by plasmin to a carboxypeptidase B-like enzyme (TAFI) of 35.8 kD molecular weight. TAFI spontaneously becomes inactive as a result of a temperature-dependent conformational change in the protein (TAFIi). In this study, pro-TAFI, total TAFI antigen and TAFI-TAFIi antigen levels were measured in 32 patients with hemophilia A, 4 patients with hemophilia B, 21 patients with von Willebrand disease (VWD) and 13 healthy controls. A statistically significant decrease in pro-TAFI was found in all groups (10.72+/-4.57 mg/L (p<0.001); 8.00+/-2.35 mg/L (p<0.01) and 8.98+/-2.33 mg/L (p <0.001) for hemophilia A, hemophilia B and VWD, respectively) compared to controls (17.85+4.61 mg/L). A statistically significant increase in TAFI-TAFIi antigen was found in hemophilia A (1.05+/-1.01 mg/L) (p<0.05) and in VWD patients (0.96+/-1.01 mg/L) (p<0.05) compared to controls (0.55+/-0.36 mg/L). There was no difference in total TAFI antigen levels between any group of patients and the controls. Neither did pro-TAFI nor TAFI-TAFIi levels differ within the group of hemophilia A patients in relation to severity (mild, moderate and severe) or among the VWD patients in relation to subtype (type 1, type 2A and type 3). These findings indicate an increased conversion of pro-TAFI to TAFI and/or TAFIi in patients with bleeding disorders. As thrombin generation is seriously impaired in these patients and almost absent in hemophilia A and B and in type 3 VWD, it is possible that plasmin mediates pro-TAFI activation in these patients. Enhanced fibrinolysis via generation of plasmin has previously been reported in hemophilia and VWD. Activation of pro-TAFI by plasmin may be a feedback mechanism that counterbalances increased fibrinolysis in patients with bleeding disorders. The relationship between the TAFI activation pathway and bleeding complications associated with hemophilia A, hemophilia B and VWD requires further investigation.