Brian Boylan

Bleeding episodes in patients who have haemophilia A (HA), a hereditary bleeding disorder caused by a deficiency in factor VIII (FVIII), are treated or prophylactically prevented with infusions of exogenous FVIII. Neutralizing antibodies, referred to as inhibitors, against infusion products are a major complication experienced by up to 30% of patients who have severe HA. Bypassing agents (BPA), a class of therapeutics given to patients who have inhibitors, bypass the need for FVIII in the coagulation cascade, and long-term inhibitor eradication is accomplished using immune tolerance induction therapy (ITI). Data examining the antibody levels in patients receiving BPA and ITI are limited. Measure anti-FVIII antibody levels in specimens from patients receiving ITI or BPA in order to evaluate the anti-FVIII antibody response in those patients. Specimens were tested using the CDC-modified Nijmegen-Bethesda assay (NBA) and the CDC fluorescence immunoassay (FLI) for anti-FVIII IgG1 and IgG4 . NBA-negative specimens from patients undergoing ITI or receiving BPAs have a higher frequency of anti-FVIII IgG4 positivity compared with the previously published level for NBA-negative HA patients. Analysis of anti-FVIII antibody levels in serial samples from patients undergoing ITI reveals that antibodies can persist even after the patient's NBA result falls into the negative range. Measurement of anti-FVIII antibodies may be a useful means to better contextualize NBA results in specimens from patients receiving BPA or ITI. In addition, assessment of anti-FVIII antibody levels has the potential to improve inhibitor surveillance and clinical decision-making related to the progress of ITI.

Introduction: Accurate diagnosis of an inhibitor, a neutralizing antibody to infused factor VIII (FVIII), is essential for appropriate management of haemophilia A (HA). Low‐titre inhibitors may be difficult to diagnose due to high rates of false‐positive inhibitor results in that range. Transient low‐titre inhibitors and false‐positive inhibitors may be due to the presence of a lupus anticoagulant (LA) or other non‐specific antibodies. Fluorescence immunoassay (FLI) to detect antibodies to FVIII is a sensitive method to identify inhibitors in HA. Evaluations of antibody profiles by various groups have demonstrated that haemophilic inhibitors detected by Nijmegen‐Bethesda (NBA) and chromogenic Bethesda (CBA) assays correlate with positivity for anti‐FVIII immunoglobulin (Ig) G1 and G4.Aim: This study sought to determine whether FLI could distinguish false‐positive FVIII inhibitor results related to LAs from clinically relevant FVIII inhibitors in HA patients.Methods: Samples from haemophilic and non‐haemophilic subjects were tested for LA, specific FVIII inhibitors by NBA and CBA, and anti‐FVIII immunoglobulin profiles by FLI.Results: No samples from LA‐positive non‐haemophilic subjects were positive by FLI for anti‐FVIII IgG4. Conversely, 91% of NBA‐positive samples from haemophilia subjects were positive for anti‐FVIII IgG4. Two of 11 haemophilia subjects had samples negative for anti‐FVIII IgG4 and CBA, which likely represented LA rather than FVIII inhibitor presence.Conclusions: Assessment of anti‐FVIII profiles along with the CBA may be useful to distinguish a clinically relevant low‐titre FVIII inhibitor from a transient LA in HA patients.

Introduction: The use of pre‐analytical heat treatment (PHT) with the Nijmegen‐Bethesda assay (NBA) for inhibitors to factor VIII (FVIII) can remove/destroy infused or endogenous FVIII from patient plasma samples, allowing testing of recently infused patients with haemophilia. Two PHT methods have been described as follows: heating to 56°C for 30 minutes and heating to 58°C for 90 minutes. Data examining the effects of PHT on anti‐FVIII IgG₄, the antibodies known to correlate most closely with the presence of FVIII inhibitors, are limited.Aim: To assess the effect of PHT on the levels of detectable anti‐FVIII IgG₄.Methods: Nijmegen‐Bethesda assay‐positive specimens were incubated at 56, 58 or 60°C for 90 minutes, and anti‐FVIII IgG₄ was measured by fluorescence immunoassay (FLI) at 30‐minute intervals. The effects of PHT on the ability of recombinant FVIII (rFVIII) to inhibit detection of patient antibodies by FLI was also examined to assess the stability of rFVIII under the various PHT conditions tested.Results: Levels of anti‐FVIII IgG₄ showed little change following incubations at 56°C (mean 101% of original value at 30 minutes and 100% at 60 minutes) but decreased upon exposure to 58°C (mean 85% at 30 minutes and 66% at 60 minutes). In addition, heating to 56°C effectively decreased the ability of rFVIII to block antibody binding compared to unheated rFVIII.Conclusion: The optimal temperature for PHT in the FVIII NBA is 56°C. Higher temperatures may lead to loss of inhibitory antibodies.

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