Pre-analytical variables in haemostasis: Findings from the United Kingdom National External Quality Assessment scheme for Blood Coagulation (UK NEQAS BC) haemolysis exercise.

INTRODUCTION: Haemolysis is considered one of the major contributors of nonconformities and sample rejection in coagulation testing. MATERIALS AND METHODS: Two lyophilized plasmas were distributed to 800 centres registered for prothrombin time (PT), activated partial thromboplastin time (APTT) and either Clauss fibrinogen or thrombin time (TT) in the UK NEQAS BC programme. The same pool of normal plasma was used to prepare both samples, to one of which red blood cell haemolysate was added to mimic haemolysis at 3 g/L haemoglobin concentration. Participants were asked to complete a questionnaire about their laboratory approach to dealing with haemolysed samples, including strategies used to deal with different levels of haemolysis. RESULTS: Results for tests performed did not show great differences between the two samples. It should be noted that artificially constructed haemolysed samples may not behave in the same way as patient samples (ie, may not be commutable). However, the possibility of carrying out a large multicentre study for detection of haemolysis was demonstrated. Inconsistency in practice was observed with 226/551 (41%) of centres indicated they reject haemolysed samples solely on visual checks, and 163 (30%) using initial visual checks with further sample rejection evaluation by analyser flags. Furthermore, 333 (72%) of centres indicated that the level of haemolysis affects sample rejection decisions, while 132 (28%) stated it did not. CONCLUSION: Variability of responses for dealing with haemolysed samples reflects a lack of clear consistency in the pre-analytical area of sample processing.

Effects of Emicizumab on APTT, FVIII assays and FVIII Inhibitor assays using different reagents: Results of a UK NEQAS proficiency testing exercise.

INTRODUCTION: Emicizumab (Hemlibra: Roche Switzerland) is a, humanized, bi-specific monoclonal modified immunoglobulin G4 (IgG4) which binds human FX, FIX and activated FIX (FIXa) to mimic activated FVIII activity. AIM: Evaluate the effects of emicizumab on the APTT, surrogate FVIII activity and FVIII inhibitor results. METHODS: Two samples were provided, one obtained from an emicizumab treated severe haemophilia A patient with FVIII inhibitors and one constructed by in vitro addition of emicizumab using plasma from a severe haemophilia A patient without FVIII inhibitors. An APTT screen, surrogate FVIII and FVIII inhibitor tests were performed on both samples by participating centres. RESULTS: APTT results were below the lower limit of normal range. Chromogenic FVIII assay results with the Hyphen/Biophen human component-based assay gave higher than expected coefficient of variation (CV) results, 38%-40%. The modified one-stage FVIII assay with emicizumab calibrators showed similar results regardless of the APTT reagent. Inhibitor assay median results for sample S18:23 = 1.43 BU (range 0.9-3.0 BU/ml, CV 38%). S18:24 was classified as below the lower limit of detection. CONCLUSION: APTT screens showed a consistent shortening. Unmodified one-stage Factor VIII assay results were remarkably high. APTT-based assays are unsuitable for measurement of coagulation factors or inhibitors in patients treated with emicizumab. Bovine origin chromogenic assays are insensitive to emicizumab and should be used to monitor FVIII levels/FVIII inhibitors in emicizumab treated patients. Product-specific calibrators should be implemented to reduce result variability.

Discovery of the First α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Antagonist Dependent upon Transmembrane AMPA Receptor Regulatory Protein (TARP) γ-8.

Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients.

Interlaboratory variation in factor VIII:C inhibitor assay results is sufficient to influence patient management: data from the UK national quality external assessment scheme for blood coagulation.

We report the results of external quality assessment exercises in which 60 to 120 centers performed factor VIII (FVIII) inhibitor testing on a series of samples over a 13-year period. Samples from seven different subjects were distributed for analysis comprising the following: four different subjects with severe hemophilia A with antibodies following replacement therapy, one subject with acquired hemophilia A and antibodies to FVIII, one subject with normal FVIII and an easily detected lupus anticoagulant, and one subject with mild hemophilia A and a difficult-to-detect lupus anticoagulant but without antibodies to FVIII. In all of the surveys the results obtained in different centers analyzing the same sample varied to an extent that would influence patient management decisions. In the UK National External Quality Assessment Scheme surveys reported here, there was considerable interlaboratory variation in the results of FVIII inhibitor testing that did not improve over the survey period. The coefficient of variation of results in different centers was between 33% and 106% in samples from patients with severe congenital hemophilia A. In some cases, results were affected by assay components. For one plasma, the mean FVIII inhibitor results in centers using one source of normal plasma was 3.9 Bethesda unit (BU)/mL compared with a mean of 5.7 BU/mL in centers using a different normal plasma source ( P = 0.04). Our data indicate that the detection of FVIII inhibitors is not the same in different centers, and the degree of variability noted makes it likely that assay variability has contributed to the lack of international consensus in relation to the real incidence of FVIII inhibitors in different clinical settings. Improvements in assay standardization are urgently needed.

Point-of-care International Normalised Ratios: UK NEQAS experience demonstrates necessity for proficiency testing of three different monitors.

External quality assessment (EQA) or proficiency testing is widely considered to be necessary for International Normalised Ratio (INR) determinations performed in conventional laboratory settings. There is increasing use of near-patient-test (NPT) or point-of-care (POC) INR devices and it is not known whether EQA is also necessary for these monitors. We report here on six years experience of proficiency testing for POC monitors used by health care professionals. Three devices were used by >10 centres who participated in the programme, the CoaguChek (CUC), the CUC-S and the TAS or Rapidpoint Coag. Not all users of the same type of monitor obtained the same INR result when analysing the same plasma sample. For the three monitors the CV of results in different centres was 11-14%. The variation between results in different centres could relate to inappropriately handled proficiency testing material, inaccuracies in the calibration of the system by the manufacturer or deterioration during transport/storage of the test strips. In each survey 10-11% of centres using POC monitors obtained INR results which were >15% different from those in other centres using the same monitors. For hospital laboratories using conventional INR techniques this figure was 12%. The relationship between INR results obtained by users of the Rapidpoint Coag or TAS monitor and results obtained by conventional techniques was not constant over the period of study. During one period INRs with TAS were 13.7% greater than with conventional methods. For the remaining three time periods results were similar. Our data suggest that the variation between INR results determined with three POC monitors show similar variation to that observed in hospital laboratories using conventional methods. Based on our data we recommend that users of these POC monitors participate regularly in an independent external proficiency testing programme.

Laboratory tests for measurement of von Willebrand factor show poor agreement among different centers: results from the United Kingdom National External Quality Assessment Scheme for Blood Coagulation.

In recognition of the importance of von Willebrand factor (vWF) testing in the diagnosis of von Willebrand disease (vWD), the United Kingdom National External Quality Assessment Scheme for Blood Coagulation regularly distributes samples for determination of vWF:antigen (vWF:Ag). Data from 10 separate surveys performed between 2001 and 2005 are reviewed. These include results from ~200 different centers, of which 55% are within the United Kingdom and the remainder are from other countries. During the period of the surveys, the use of immunoelectrophoresis for determination of vWF:Ag practically disappeared and was largely replaced by latex agglutination assays. The coefficient of variation (CV) of results in different centers was approximately 15 to 20% for most vWF:Ag techniques, with CVs of approximately 7% for a fluorescence-based assay. Several different techniques were used for determination of vWF ristocetin cofactor activity (vWF:RCo), all of which were associated with poor agreement among centers as indicated by CVs of 40 to 50%. Several centers calculated the ratio of vWF:Ag/vWF:RCo but with variable success. Ratios compatible with either type 1 or type 2 vWD were obtained on samples from subjects with type 1 vWD, as well as on samples from subjects with genetically confirmed type 2 vWD. Overall, our data show that laboratory testing for vWD remains problematic. It remains to be seen whether newer techniques will offer consistently improved precision.