Search

Non-parallelism in factor assays can lead to incorrect factor activities. Parallelism can be assessed by calculating the coefficient of variation (CV) of results obtained on 3 dilutions of the same sample. Some authors have proposed that if there is15% then the average activity is reportable. Some analysers use a slope ratio (SR) to calculate parallelism, with an acceptance range of approximately 0.9-1.1.We evaluated CV and SR in one stage FII-FXII assays on Sysmex CS5100i using Innovin or Actin FS. Frozen normal and pathological plasmas, plasmas containing Direct Oral Anticoagulants, Direct Thrombin Inhibitors or Lupus Anticoagulant were analysed to assess possible non-parallelism.In plasmas with factor levels25 IU/dl (plus no interfering substances) all CVs were 15%. One sample (low factor activities 10-15 IU/dl), had CVs 15% in FII, FVII and FXII assays only. SR outside of 0.9-1.1 were seen in FII and FXII assays at different levels of clotting factor including some within the normal range. Non-parallelism was detected more frequently with SR than CV for those with interfering substances.SR outside of 0.9-1.1 were seen in different levels of clotting factors, including samples which did not contain interfering substances. The target of 15% CV was a better discriminator than a SR for acceptance. When factor levels were reduced to around 10-15 IU/dl, a target 20 %CV was more appropriate than 15%. It might be appropriate for laboratories to assess locally whether their acceptance criteria need to be wider at low levels of clotting factors.

Introduction The Summary of Product Characteristics for the direct thrombin inhibitor argatroban states monitoring should be by activated partial thromboplastin time (APTT), with a target range of 1.5-3.0 times the patients' baseline APTT. APTT may be influenced by coagulopathies, lupus anticoagulant and raised FVIII levels. Previous studies have shown sensitivity differences of APTT reagents to argatroban. Some recent publications have favoured the use of anti-IIa methods to determine the plasma drug concentration of argatroban. This study aims to compare the anti-IIa assays: Hemoclot thrombin inhibitor assay (HTI) and Ecarin chromogenic assay (ECA) alongside the APTT. Methods Residual plasma of 25 samples from 8 patients (3 with COVID-19 and HIT: n = 18, 5 with HIT: n = 7) was tested at two sites: site A: Sysmex CS5100 by HTI and APTT (Actin FS and SynthASil), and also on Stago STA Compact Max: ECA and APTT (CK Prest); and site B: Stago STA R Max 2 by ECA and APTT (Cephascreen). Results Mean APTT ratio was 1.96 (Actin FS), 1.84 (SynthASil), 1.59 (CK Prest) and 2.48 (Cephascreen). Mean argatroban concentration by HTI was 0.60 µg/mL and by ECA was 0.65 µg/mL (site A) and 0.70 µg/mL (site B). There was a poor correlation to HTI in APTT ratios (range r2 = .0235-0.4181) with stronger correlations between ECA methods to HTI (r2 = .8998 site A, r2 = .8734 site B). Conclusion This study confirms previous publications on the unsuitability of APTT and expands this by being multisited and included APTT reagents that use mechanical clot detection. Both anti-IIa methods are more suitable.

Introduction The measurements of clotting factor activities are usually performed using a one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). Advances in automated coagulation analysers have led to the utilization of stored calibration curves. There are sometimes substantial intervals between test calibration and analysis of samples. Variability in results can be influenced by calibrant and methodology. Several guidelines recommend calibration and patient samples be performed together in parallel; this incurs costs, but reliance on a stored calibration curve may lead to inaccuracy of results over time. Methods We evaluated inclusion of a live truncated (3 point) calibration curve using calibrator plasma alongside test samples and compared results calculated against the stored calibration curves and live truncated calibration to assess the impact on precision and accuracy. The feasibility of this was tested on two hospital sites in the UK; OSA and CSA were performed on Sysmex CS5100 using Actin FS, SynthASil, Innovin, Biophen chromogenic VIII and Rossix chromogenic IX. Results Results of two batches of IQC were compared for FII, FV, FVII, FX, FIX:C, FXI, FXII and OSA FVIII (FVIII:C1) and CSA FVIII:C (FVIII:CR) and FXI:C. By utilizing a live truncated calibration, precision improved with the most striking examples: FXII %CV 23.1% to 3.1% (site A) FXI 7.3% to 2.4% (site B). The improvement in other clotting factors was more modest. Conclusion To the best of our knowledge, this is the first study that demonstrates that the use of a live truncated calibration curve will improve precision and accuracy.

Introduction Acquired haemophilia A (AHA) is a rare bleeding disorder caused by the development of autoantibodies to endogenous human factor VIII (hFVIII). If treatment of bleeding is required, one option is recombinant porcine FVIII (rpFVIII). Cross-reactivity between factor VIII inhibitors and rpFVIII has previously been described. Aim The aim of this study was to retrospectively assess the incidence of cross-reacting anti-porcine inhibitors in patients diagnosed with AHA in two UK centres. Methods The plasma of fifty-one patients diagnosed with AHA via reduced FVIII:C and positive FVIII inhibitor titre as detected with a Nijmegen-Bethesda assay (NBA) was also tested by a porcine Bethesda assay (PBA). The NBA was modified by replacement of human FVIII with rpFVIII in the PBA, with determination of residual FVIII by one-stage clotting assay. Results The median FVIII inhibitor titre by NBA was 22.8 BU/mL (range: 0.8-1000 BU/mL). 37% of samples exhibited linear, type 1 kinetics in the NBA. Negative PBA was observed in 26 patients, and 25 were positive (median PBA: 3.5 BU/mL; range: 0.8-120 BU/mL). Type 1 kinetics were observed in 40% of PBA-positive patients. At NBA tires of greater than 100 BU/mL, the positive predictive value for the presence of porcine cross-reactivity was 100%. At NBA below 5 BU/mL, the negative predictive value for the presence of porcine cross-reactivity was 71%. Conclusion Cross-reactivity between FVIII inhibitors and rpFVIII was observed in 49% of patients. The presence of inhibitors to rpFVIII may influence the treatment choice for patients with acquired haemophilia A.

Introduction Argatroban is a direct thrombin inhibitor used as an anticoagulant for patients who have Heparin induced thrombocytopenia. Quantification can be performed using a dilute thrombin time or anti-iia assay. Our preferred method is Hemoclot Thrombin Inhibitor Assay (HTI).To the best of our knowledge, no one has published on the stability of plasma argatroban in whole citrated blood at room temperature. Methods Forty matched samples obtained from 4 patients receiving argatroban. 1 mL of the whole blood was removed from the sample into a labelled plastic tube and left on the laboratory bench at room temperature for 24 hours prior to centrifugation. The remaining sample was spun at 2000 g for 10 minutes and the plasma aliquoted off and labelled 0 hour and stored at -80°C prior to testing. At 24 hours the plastic tube containing whole blood was centrifuged at 2000 g for 10 minutes and the plasma aliquoted off and labelled 24 hours and stored at -80°C prior to testing. HTI assay was used for plasma argatroban determination. Results The lowest argatroban level obtained was 0.16 μg/mL at 0 hour; 0.18 μg/mL at 24 hours from the same pair, the highest argatroban level obtained was 1.72 μg/mL at 0 hour;1.76 μg/mL for the same pair at 24 hours. The mean result for 0 hour was 0.57 μg/mL and 0.60 μg/mL at 24 hours. Conclusion This study proposes that patients receiving treatment in hospitals who cannot provide a dedicated argatroban plasma concentration method could have their samples sent on whole blood within 24 hours of venepuncture to a laboratory who could provide the test.

SummaryBackground Argatroban is licensed for patients with heparin-induced thrombocytopenia and monitoring is conventionally by activated partial thromboplastin time (APTT) ratio with a target of 1.5–3.0 and not exceeding 100 s. The APTT may be influenced by coagulopathies, lupus anticoagulant and raised FVIII levels. Variable but not clinically significant sensitivity of APTT reagents to argatroban has been highlighted in other studies. Methods Residual plasma of 15 patients (n = 124 samples) was tested on Sysmex™ CS series using the Hemoclot® thrombin inhibitor assay (HTI) and APTT ratio (Actin FS and SynthASil). A subgroup from four patients (n = 31) were tested on ACL TOP™ to compare the different platforms with the HTI. Spiked normal pooled plasma was tested with Actin FS, Actin FSL, SynthASil and APTT-SP on their respective platforms (CS5100™ and ACL TOP™) to assess reagent sensitivity. Results Mean concentration of argatroban by HTI assay for patient plasma was 0.47 μg/mL; the mean APTT ratio using Actin FS was 1.89 and for SynthASil 1.56. There was a poor correlation between APTT and the HTI. In the spiked normal pooled plasma, Actin FS gave a significantly higher APTT ratio than the other three reagents for the various argatroban concentrations. Conclusions Hemoclot ® thrombin inhibitor assay should be considered in patients on argatroban, particularly if there is concern the APTT may not be reflective of the degree of anticoagulation with argatroban due to other factors including coagulopathies in critically ill patients, the presence of a lupus anticoagulant or very high FVIII levels.

Lepirudin (r-hirudin) is one of the two alternative anticoagulants licensed to treat patients with heparin-induced thrombocytopenia (HIT). Manufacturer's guidelines state that lepirudin should be monitored using the activated partial thromboplastin time (APTT) ratio. However, several studies have demonstrated a plateau effect of higher concentrations of lepirudin on APTT ratios and variable results when comparing different APTT reagents. This study compares APTT ratios (using two different APTT reagents) with two other commercially available methods for directly quantifying plasma lepirudin levels: ecarin chromogenic assay and prothrombinase-induced clotting time in 95 samples from five patients receiving lepirudin anticoagulation for HIT.