Factor VIII and IX assays for post-infusion monitoring in hemophilia patients: Guidelines from the French BIMHO group (GFHT).

Replacement therapy with plasma-derived or recombinant FVIII and FIX (pdFVIII/pdFIX or rFVIII/rFIX) concentrates is the standard of treatment in patients with haemophilia A and B, respectively. Measurement of factor VIII (FVIII:C) or factor IX (FIX:C) levels can be done by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). The French study group on the Biology of Hemorrhagic Diseases (a collaborative group of the GFHT and MHEMO network) presents a literature review and proposals for the monitoring of FVIII:C and FIX:C levels in treated haemophilia A and B patients, respectively. The use of CSA is recommended for the monitoring of patients treated with pdFVIII or rFVIII including extended half-life (EHL) rFVIII. Except for rFVIII-Fc, great caution is required when measuring FVIII:C levels by OSA in patients substituted by EHL-rFVIII. The OSA is recommended for the monitoring of patients treated with pdFIX or rFIX. Large discordances in the FIX:C levels measured for extended half-life rFIX (EHL-rFIX), depending on the method and reagents used, must lead to great attention when OSA is used for measuring FIX:C levels in patients substituted by EHL-rFIX. Data of most of recent studies, obtained with spiked plasmas, deserve to be confirmed in plasma samples of treated patients.

APTT therapeutic range for monitoring unfractionated heparin therapy. Significant impact of the anti-Xa reagent used for correlation.

INTRODUCTION: Unfractionated heparin (UFH) therapy is monitored by using the anti-activated factor X (anti-Xa) activity, or the activated partial thromboplastin time (APTT), which remains the most widely used assay. One of the main advantages of anti-Xa relies on its hypothesized standardization, with a unique therapeutic range (0.30-0.70 IU/ml) for all reagents, whereas APTT is influenced by numerous preanalytical and analytical parameters not related to the anticoagulant activity of UFH. METHODS: The aim of this study was to compare the anti-Xa-correlated APTT therapeutic ranges calculated using different combinations of APTT (n = 4) and anti-Xa reagents (n = 4) in frozen citrated plasmas from 87 inpatients on UFH. RESULTS: The median APTT ratio ranged from 2.19 for the less sensitive to 3.23 for the most sensitive reagent, whereas the median anti-Xa activity was between 0.37 IU/ml and 0.57 IU/ml. The APTT therapeutic ranges calculated to correlate with anti-Xa activities between 0.30 and 0.70 IU/ml were found to be highly different from one combination of APTT reagent and analyzer to another. The same applied to the therapeutic range of a single APTT reagent calculated using different anti-Xa assays performed on the same analyzer, leading to a lack of agreement as to whether a sample was classified as subtherapeutic, therapeutic or supratherapeutic in 8.0% to 23.0% of the patients, with kappa coefficients between 0.908 and 0.753. CONCLUSIONS: These results suggest that the APTT therapeutic range calculated to correlate with anti-Xa activities between 0.30 and 0.70 IU/ml is influenced not only by the APTT reagent, but also by the anti-Xa reagent used for calculation.

Age-adjusted D-dimer cut-off levels to rule out venous thromboembolism in patients with non-high pre-test probability: Clinical performance and cost-effectiveness analysis.

BACKGROUND: As aging was found to be associated with increased D-dimer levels, the question arose whether D-dimer measurement was useful in the diagnostic strategy of venous thromboembolism (VTE) in elderly patients. AIM OF THE STUDY: To compare retrospectively the performance of six diagnostic strategies based on the three-level Wells scores and various cut-off levels for D-dimer, evaluated using the HemosIL D-Dimer HS 500 assay, in a derivation cohort of 644 outpatients with non-high pretest probability (PTP) of VTE. The clinical usefulness of the best-performing strategy was then confirmed in a multicenter validation study involving 1255 consecutive outpatients with non-high PTP. RESULTS: The diagnostic strategy based on the age-adjusted cut-off level calculated by multiplying the patient's age by 10 above 50 years was found to perform the best in the derivation study with a better sensitivity-to-specificity ratio than the conventional strategy based on the fixed cut-off level (500 ng/ml), a higher specificity and a negative predictive value (NPV) above 99%. Such an increase in test specificity was confirmed in the validation cohort, with the NPV remaining above 99%. Taking into account the local reimbursement rates of diagnostic tests, using this strategy led to a 6.9% reduction of diagnostic costs for pulmonary embolism and a 5.1% reduction for deep vein thrombosis, as imaging tests would be avoided in a higher percentage of patients. CONCLUSION: The diagnostic strategy of VTE based on the age-adjusted cut-off level for D-dimer in patients over 50 years was found to be safe, with NPV above 99%, and cost-effective.

Monitoring unfractionated heparin therapy: Lack of standardization of anti-Xa activity reagents.

INTRODUCTION: One of the main advantages of using anti-Xa instead of activated partial thromboplastin time in monitoring of unfractionated heparin (UFH) therapy relies on its hypothesized standardization, with a unique therapeutic range defined to be 0.30 to 0.70 IU/mL. The aim of the present study was to compare the inter-reagent agreement of anti-Xa activity. METHODS: Citrate tubes were obtained from 104 inpatients on UFH. Plasma samples were stored frozen in aliquots at -70°C before being shipped to three accredited coagulation laboratories to be evaluated for anti-Xa activity using their routine assay(s). Pooled normal plasmas spiked with dilutions of the 6th International Standard of UFH to achieve anti-Xa activities up to 1.0 IU/mL were evaluated using the same techniques. RESULTS: In the plasmas from patients on UFH, the median anti-Xa activity ranged from 0.37 IU/mL with one reagent to 0.57 IU/mL with another; results were in between (0.45 IU/mL) using two other reagents. Comparisons of results obtained using the different reagents demonstrated unacceptable bias up to 0.24 IU/mL between some reagents (41% difference). The concordance as whether anti-Xa activities measured using different reagents were within or outside the therapeutic range was between 0.411 and 0.939 (kappa). Similar discrepancy was demonstrated for anti-Xa activities when evaluating normal plasma spiked with the International Standard. A discrepancy of the same order of magnitude was demonstrated in the 2017 External Quality Assessment Program provided by the External Quality Control in Assays and Tests exercises. CONCLUSIONS: The reported discrepancy between test results obtained using different anti-Xa assays clearly suggests a lack of standardization of that assay with potentially significant impact on the patients' anticoagulation.

[Factor IX assays in treated hemophilia B patients]. / Dosage de l'activité du facteur IX chez les patients hémophiles B substitués.

Replacement therapy with plasma-derived or recombinant FIX (pdFIX or rFIX) concentrates is the standard of treatment in patients with hemophilia B. The method predominantly used for measuring factor IX (FIX:C) levels is the one-stage clotting assay (OSA) but this method depends on the activated partial thromboplastin time (APTT) reagent and the coagulation analyzer used, and wide variations in the measurements of FIX recovery have been reported with some factor concentrates. The French study group on the biology of hemorrhagic diseases (a collaborative group of the GFHT and MHEMO network), presents a review of the literature and proposals for the monitoring of FIX:C levels in treated hemophilia B patients. The use of OSA calibrated with a plasma reference tested against the current FIX WHO International Standard is recommended for the monitoring of patients treated with pdFIX or rFIX. Chromogenic substrate assays (CSA) are adequate for the monitoring of patients treated with Rixubis®, but data available for Benefix® are currently too limited. For extended half-life rFIX (EHL-rFIX), large discordances in the FIX:C levels measured were evidenced, depending on the method and reagents used. Great attention is therefore required for measuring FIX:C levels by OSA in patients substituted by EHL-rFIX. Commercial kits for CSA are not equivalent, and although potentially useful, they are not validated for all EHL-rFIX. Most of recent studies reported data obtained with spiked plasmas, which deserve to be confirmed on plasma samples collected in treated patients.

[Factor VIII assays in treated hemophilia A patients]. / Dosage de l'activité du facteur VIII chez les patients hémophiles A substitués.

Replacement therapy with plasma-derived or recombinant FVIII (pdFVIII or rFVIII) concentrates is the standard of treatment in patients with hemophilia A. The reference method used for measuring factor VIII (FVIII:C) levels in patients treated by FVIII concentrates is the chromogenic substrate assay (CSA). However, the one-stage clotting assay (OSA) is predominantly used in current clinical practice, but this method depends on the activated partial thromboplastin time (APTT) reagent and the coagulation analyzer used, and wide variations in the measurements of FVIII recovery have been reported with some factor concentrates. The French study group on the biology of hemorrhagic diseases (a collaborative group of the GFHT and MHEMO network) presents a review of the literature and proposals for the monitoring of FVIII:C levels in treated hemophilia A patients. The use of CSA calibrated with a plasma reference tested against the current FVIII WHO (World Health Organization) International Standard is recommended for the monitoring of patients treated with pdFVIII or rFVIII including extended half-life (EHL) rFVIII. OSA are adequate for the monitoring of patients treated with pdFVIII or with most of rFVIII concentrates. However, preliminary comparison with CSA is mandatory before measuring FVIII:C by OSA in patients treated by Refacto AF®. For rFVIII-EHL, OSA are only acceptable for Elocta®. Great caution is therefore required when measuring FVIII:C levels by OSA in patients substituted by other EHL-rFVIII. Indeed, most of recent studies reported data obtained with spiked plasmas, which deserve to be confirmed on plasma samples collected in treated patients.

A new plastic collection tube made of polyethylene terephtalate is suitable for monitoring traditional anticoagulant therapy (oral anticoagulant, unfractionated heparin, and low molecular weight heparin).

To improve the safety of blood collection, plastic tubes have been developed but various interactions with the coagulation system and/or antithrombotic drugs were reported with the first generation of such tubes. The aim of this multicentre study was to compare hemostasis test results measured in evacuated plastic tubes made of polyethylene terephtalate (VenoSafe, Terumo Europe) and in siliconized glass tubes containing the same citrate concentration (0.129 M). In addition, the impact of aging of the plastic tube was investigated by collecting blood samples in tubes at 8 months and at 1 month before expiry. Blood was drawn in 3 centres from untreated patients (n=269), patients on oral anticoagulant treatment (OAT, n=221), and patients treated with either unfractionated heparin (UFH, n=73) or a low molecular weight derivative (LMWH, n=48). Prothrombin time (PT) or INR, activated partial thromboplastin time (APTT) and anti-FXa activity were locally performed, when applicable. In untreated patients and in patients on OAT, PT and APTT values were found statistically shorter (p<0.05) when evaluated in plastic tubes than in glass tubes, except when PT was evaluated using a human thromboplastin. Surprisingly, significantly longer APTT and higher anti-FXa activities were obtained when blood from patients on UFH was drawn in plastic than in glass tubes. However, none of the differences had any clinical relevance (Bland-Altman analysis). In patients on anticoagulant treatment, there was no effect of aging of the plastic tubes. These results suggest that the plastic tube VenoSafe is suitable for coagulation testing both in untreated subjects and more interestingly in patients on traditional anticoagulant therapy during the whole shelf life indicated by the manufacturer.

Monitoring treatments with unfractionated heparin: CTAD must be used instead of citrate as the anticoagulant solution when using partial-draw collection tubes. Results of a multicenter evaluation.

BACKGROUND: Sampling small volumes of blood may be necessary, particularly in pediatric patients, or in case of difficult or recurrent venipunctures. METHODS: Routine hemostasis test results evaluated in partial- and full-draw evacuated polymer tubes obtained in 4 centers were compared. RESULTS: No relevant discrepancy (Bland-Altman) was found between test results measured in partial- and full-draw tubes obtained from untreated patients and from patients on vitamin K-antagonist or low molecular weight heparin. In patients on unfractionated heparin (UFH), significantly lower anti-FXa activity [median=0.29IU/mL (range:0.04-1.15) vs. 0.39 (0.05-1.25), n=89, p<0.0001] and shorter aPTT were measured in partial-draw tubes. This discrepancy was likely to be related to the release of higher amounts of PF4 after increased platelet activation in partial-draw tubes. As CTAD is known to counteract platelet activation, we then collected blood into partial-draw CTAD tube and full-draw citrate tube. Both in patients on UFH and in untreated patients, no relevant difference could be demonstrated for all studied parameters (Bland-Altman), including aPTT and anti-FXa activity, even if analytical comparison showed significantly higher anti-FXa activity in partial-draw CTAD than in full-draw citrated tubes with a mean bias of 0.02 IU/mL, identical throughout the measuring range. CONCLUSIONS: These results suggest that samples collected into partial-draw citrate tubes allow accurate routine coagulation testingin all patients but those requiring UFH assessment,in which their use led to a significant underestimation ofanticoagulation. In such cases, partial-draw tubes containing CTAD could be validly used to monitor heparin therapy as well as to perform routine coagulation testing.