Geoffrey Kershaw

Laboratory Identification of Factor Inhibitors: The Perspective of a Large Tertiary Hemophilia Center

Coagulation factor inhibitors are antibodies that bind and neutralize specific procoagulant plasma proteins. The identification of coagulation factor inhibitors by the hemostasis laboratory requires a careful and systematic approach that excludes other possible causes of prolonged screening tests such as the activated partial thromboplastin time and prothrombin time. Once the laboratory is confident that a specific coagulation factor inhibitor is present in a sample, its strength or titer must be measured. The clinician will use this information as a treatment guide. The most frequently occurring factor inhibitors encountered in the hemostasis laboratory are those directed against factor VIII (FVIII), which can arise in individuals with inherited hemophilia A as an immune response to factor replacement therapy or as an autoantibody leading to the condition of acquired hemophilia A. The Bethesda assay is the most widely used test for measuring the FVIII inhibitor titer. The Bethesda assay has several components that must be carefully controlled to achieve consistent results. This overview examines the behavior of various coagulation inhibitors and laboratory tests with an emphasis on the Bethesda assay for factor inhibitors.

Laboratory identification of lupus anticoagulants.

The main laboratory characteristic of lupus anticoagulants (LA) is their ability to prolong phospholipid-dependent clotting time in vitro. The laboratory demonstration of LA requires a systematic approach combined with an awareness of the many variables that can affect test results. The ideal testing procedures are those sensitive enough to detect weak LA and specific enough so as not to produce incorrect conclusions. International guidelines have been published to assist laboratories in applying correct testing processes. The most recently published guidelines from the International Society on Thrombosis and Haemostasis update the criteria for detecting the presence of LA that were presented in the 1995 guidelines. Some of the specific recommendations relate to the key areas of setting cut-off levels for screening, mixing, and confirmatory procedures. The more challenging aspects of testing for LA include maintaining sensitivity and specificity of the assays, especially in the presence of anticoagulant therapy.

Laboratory investigation of lupus anticoagulants: mixing studies are sometimes required

(0.19 ± 0.04 lm, n = 2 experiments) (mean diameter ± standard error of themean) (P = 0.23). Because fiber diameter is dependent upon thrombin concentration, we did not compare these diameters with those of control samples as they were prepared by addition of exogenous thrombin. In conclusion, we have shown that in a parallel plate flow chamber, newly formed fibrin fibers orient in the direction of flow, with increasing alignment as the flow rate increased. Our results are consistent with previous studies [5–7], but were carried out under more physiological conditions, clotting was initiated by platelets on a collagen surface, and the observations were quantified. Because we found similar fiber orientation in our purified in vitro system to the alignment observed in thrombi within coronary arteries, we conclude that the flow of blood serves to orient the fibers that form inside a vessel. The striking degree of orientation even at low shear rates demonstrates the profound effect of flow on clot structure, whichmust be considered in understanding in vivo clotting and thrombosis.

Validation of whole blood impedance aggregometry as a new diagnostic tool for HIT: results of a large Australian study

Heparin-induced thrombocytopenia (HIT) remains a challenge, with diagnosis confirmed only by functional assays. The gold standard 14C-serotonin release assay (SRA) is highly sensitive but technically challenging and unsuitable for routine use. We conducted a large study to validate whole blood impedance aggregometry (WBIA) as a suitable diagnostic tool for HIT. WBIA and SRA were used to test 181 samples positive for H-PF4 antibodies by PaGIA or ELISA. Using the same high responder donor, 77 samples were positive by WBIA (aggregation with low-dose but not high-dose heparin). Using the strict definition for SRA positivity, 72 samples were true HIT. In nine samples, serotonin release with high-dose heparin dropped by > 50% but was still >20%; these were retested after a one-half dilution and 8/9 became positive. Ten other samples were discrepant between the two assays: one strongly positive (89% release) and six weakly positive samples by SRA (average release 56%) were WBIA negative. When these samples were retested using a random donor, only two remained SRA positive. Three samples were strongly WBIA positive but SRA negative; two were retested by SRA with 0.5IU/ml heparin and one became positive. Under controlled conditions, using the same selected high-responder donor, WBIA and SRA performed similarly with slightly increased sensitivity of the WBIA when using the strict definition of SRA positivity. WBIA is easy to perform with rapid turn-around time and warrants a multi-laboratory trial to complete its validation as a confirmatory assay for platelet-activating HIT antibodies.

Laboratory identification of factor inhibitors: an update.

Coagulation factor inhibitors comprise antibodies that bind to and then neutralise specific pro-coagulant plasma proteins. Coagulation factor inhibitors can develop against any coagulation factor, although the most common are against factor VIII (FVIII). These can develop in individuals with inherited haemophilia A (HA) as an immune response to factor replacement therapy, or as auto-antibodies leading to the condition of acquired HA. Clinical suspicion for inhibitors may arise when individuals present with bleeding symptoms without any prior bleeding diathesis, or when a patient with known mild haemophilia presents with a bleeding diathesis more extreme to their usual presentation, or when there is failure of factor replacement therapy to arrest bleeding in a known haemophiliac. The laboratory identification of factor inhibitors requires a careful and systematic approach that excludes other possible causes of prolonged screening tests, most commonly the activated partial thromboplastin time (APTT), and sometimes prothrombin time (PT). Coagulation factor inhibitor studies, including the Bethesda assay, are then undertaken to measure inhibitor titre, which guides treatment. This paper overviews the laboratory investigation of factor inhibitors, and also briefly reviews recent cross-laboratory inhibitor studies and the most recent evidence related to differential inhibitor formation according to type of therapy.Summary Coagulation factor inhibitors comprise antibodies that bind to and then neutralise specific pro-coagulant plasma proteins. Coagulation factor inhibitors can develop against any coagulation factor, although the most common are against factor VIII (FVIII). These can develop in individuals with inherited haemophilia A (HA) as an immune response to factor replacement therapy, or as auto-antibodies leading to the condition of acquired HA. Clinical suspicion for inhibitors may arise when individuals present with bleeding symptoms without any prior bleeding diathesis, or when a patient with known mild haemophilia presents with a bleeding diathesis more extreme to their usual presentation, or when there is failure of factor replacement therapy to arrest bleeding in a known haemophiliac. The laboratory identification of factor inhibitors requires a careful and systematic approach that excludes other possible causes of prolonged screening tests, most commonly the activated partial thromboplastin time (APTT), and sometimes prothrombin time (PT). Coagulation factor inhibitor studies, including the Bethesda assay, are then undertaken to measure inhibitor titre, which guides treatment. This paper overviews the laboratory investigation of factor inhibitors, and also briefly reviews recent cross-laboratory inhibitor studies and the most recent evidence related to differential inhibitor formation according to type of therapy.

Investigations from External Quality Assurance Programs Reveal a High Degree of Variation in the Laboratory Identification of Coagulation Factor Inhibitors

The laboratory has a key role in the initial detection of factor inhibitors and an ongoing role in the measurement of inhibitor titers during the course of inhibitor eradication therapy. The most commonly seen factor inhibitors are those directed against factor VIII (FVIII), usually detected either with the original or the Nijmegen-modified Bethesda assay. In addition, several circumstances can arise in which the laboratory may test samples that potentially reflect false identification of factor inhibitors. These include lupus anticoagulants and other events generally related to preanalytical variables, including incorrect sample presentations. This article reviews each of these elements, largely from the perspective of cross-laboratory studies undertaken within the framework of external quality assurance (EQA), a peer-laboratory process that aims to assess the ongoing performance of groups of similar laboratories. This review details the experience of the Royal College of Pathologists of Australasia Haematology Quality Assurance Program, and it also reflects on the experience of other EQA organizations. Our analysis reveals a wide variety of test practice among inhibitor testing laboratories, a wide variation in detected inhibitor levels in cross-tested samples, and substantial evidence of false-positive and false-negative detection of factor inhibitors. These findings hold some significance for the clinical management of patients affected by these inhibitors. There is still much need for standardization and improvement in factor inhibitor detection, and we hope that this report provides a basis for future improvements in this area.

Comparison of intermittent and continuous extracorporeal treatments for the enhanced elimination of dabigatran

Abstract Context. Severe bleeding associated with dabigatran frequently requires intensive care management. An antidote is currently unavailable and data reporting the effect of dialysis on elimination of dabigatran are encouraging, but limited. Objective. To report the effect of intermittent hemodialysis (IHD) and continuous renal replacement therapy (CRRT) at enhancing elimination of dabigatran. Materials and methods. Patients were identified by existing collaborative networks. Pre-filter dabigatran plasma concentrations were measured in all patients, and in dialysate of three patients. Results. Seven patients received dialysis, five with active bleeding and two requiring emergent surgery. Five received IHD and two received CRRT. The plasma elimination half-life of dabigatran was 1.5–4.9 h during IHD, and 14.0–27.5 h during CRRT. Mean dabigatran plasma clearance during IHD was 85–169 mL/min in three patients. Time to obtain a subtherapeutic dabigatran concentration depended on the initial concentration, being 8–18 h for IHD in three patients while 4 h was insufficient in a supratherapeutic case. A 38% rebound in dabigatran levels occurred after one case during IHD, and thrombin time increased after IHD in another, but not after 144 h CRRT or 17 h IHD in two others; data were incomplete in three cases. The amount removed during IHD was proportional to the pre-IHD concentration and clearance, but was consistently low at 3.3–17.4 mg in three patients where this was determined. Moderate bleeding occurred while obtaining vascular access in one patient. Two patients died from intracerebral bleeding, and the influence of treatments could not be determined in these cases. Discussion and conclusions. IHD enhanced elimination of dabigatran more efficiently than CRRT, but their net effect remains poorly defined. Dialysis decisions, including modality and duration, must be individualized based on a risk–benefit assessment.

Recombinant to modified factor VIII and factor IX – chromogenic and one-stage assays issues

The recent development of modified recombinant factor VIII (FVIII) and factor IX (FIX) therapeutic products with extended half‐lives will create challenges for the haemostasis laboratory in obtaining recovery estimates of these products in clinical samples using existing assays. The new long‐acting therapeutic concentrates contain molecular modifications of Fc fusion, site‐specific of polyethylene glycol or albumin fusion. The optimum methods for monitoring each new product will need to be assessed individually and laboratories should select an assay which gives similar results to the assay used to assign potency to the product in question. For some extended half‐life FVIII and FIX products some one stage assays are entirely unsuitable for monitoring purposes. For most products and assay reagents studied so far, and reviewed in this manuscript, chromogenic FVIII or FIX assays can be safely used with conventional plasma standards. If one stage assays are used then they should be performed using carefully selected reagents/methods which have been shown to recover activity close to the labelled potency for the specific product being monitored.

Laboratory identification of factor VIII inhibitors in the real world: the experience from Australasia

Summary.  The laboratory has a key role in the initial detection of factor inhibitors and an ongoing role in the measurement of inhibitor titres during the course of inhibitor eradication therapy. The most commonly seen factor inhibitors are those directed against factor VIII (FVIII), usually detected either using the original or Nijmegen‐modified Bethesda assay. In view of previously demonstrated high variability in laboratory results for inhibitor assays, we have more extensively examined laboratory performance in the identification of FVIII inhibitors. Over the past 3 years, we conducted two questionnaire‐based surveys and two wet‐challenge surveys utilizing eight samples comprising no FVIII inhibitor (n = 1), or low‐titre (n = 2), medium‐titre (n = 3) or high‐titre (n = 2) FVIII inhibitor. Four samples were tested by 42 laboratories in 2007, and four by 52 laboratories in 2009. High inter‐laboratory variation was evident, with CVs around 50% not uncommon, and some 10% of all laboratories (or around 15% of laboratories using Bethesda method) failed to detect low‐level inhibitors of around 1 BU mL−1. Laboratories using the Nijmegen method appeared to perform better than those using a standard Bethesda assay, with lower evident assay variation and no false negatives. There was a wide variety of laboratory practice, with no two laboratories using exactly the same process for testing and interpretation of factor inhibitor findings. In conclusion, our study indicates that there is still much need for standardization and improvement in factor inhibitor detection, and we hope that our findings provide a basis for future improvements in this area.

Validation of 4% albumin as a diluent in the Bethesda Assay for FVIII inhibitors.

INTRODUCTION External quality assurance programs show the Nijmegen Bethesda Assay for FVIII inhibitors improves test specificity compared to the Classic Bethesda Assay but its uptake has been slow possibly due to the cost of using FVIII deficient plasma as diluent. This study was conducted to determine if modifying the Nijmegen Bethesda assay by replacement of FVIII deficient plasma with 4% as a diluent would be suitable for for measuring FVIII inhibitors. MATERIALS AND METHODS The titres of 59 samples from 35 patients with FVIII inhibitors were determined in parallel tests by the Nijmegen Bethesda Assay and and the modified Nijmegen assay. Method reproducibility was assessed on inhibitor-containing samples from seven individuals covering a range of titres from 1-200 Bethesda units/mL. RESULTS The all-sample geometric mean titre was 6.73 Bethesda units/mL for the Nijmegen Bethesda Assay and 7.54 Bethesda units/mL for the modified Nijmegen assay. No sample was found where a difference in measured titre between methods would have altered clinical management. Agreement was very close in samples with titres less than 2BU/mL. Both assays gave inhibitor titres in external quality assurance samples of close to consensus values. The average between-run coefficients of variation were 8.6% for the Nijmegen Bethesda Assay and 7.9% for the modified Nijmegen assay. CONCLUSIONS The modified Nijmegen assay using 4% albumin as the sample diluent showed good overall comparability to our existing Nijmegen Bethesda Assay and is substantially more cost-effective, making it a reasonable alternative for measuring FVIII inhibitors.