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Laboratory Scientist, AMPATH, Moi Teaching and Referral Hospital


To characterize the antibody profiles of patients with hemophillia A inhibitors

A major complication associated with factor eight infusion therapy for the treatment of hemophillia A is the development of inhibitors against the factor eight. Some researchers have shown that a subset of patients with hemophillia A have anti-factor eight antibodies that do not promote the development of inhibitors. Therefore the current study seeks to assess the anti-factor eight antibody profiles of patients with hemophillia A inhibitors as a potential predictor of clinical outcomes. This is a cross-sectional study involving 90 participants with hemophillia A. The presence of inhibitors was determined using Nijmegen- Bethesda assay, and those with inhibitor negative status recruited as controls. The antibody profiles will be determined using flouresence immunoassay method. Findings from this study is expected to provide a rationale for future studies aiming to utilize the anti-factor eight antibody profiles in hemophillia A patients as a potential predictor of inhibitor development. Additionally, it will assess the value of flouresence immunoassay as a supplement to the traditional bethesda assay.


Characterization of the anti-factor VIII immunoglobulin profile in patients with hemophilia A by use of a fluorescence-based immunoassay.

Abstract: The development of neutralizing antibodies, referred to as inhibitors, against factor VIII is a major complication associated with FVIII infusion therapy for the treatment of hemophilia A (HA). Previous studies have shown that a subset of HA patients and a low percentage of healthy individuals harbor non-neutralizing anti-FVIII antibodies that do not elicit the clinical manifestations associated with inhibitor development.To assess HA patients' anti-FVIII antibody profiles as potential predictors of clinical outcomes.A fluorescence immunoassay (FLI) was used to detect anti-FVIII antibodies in 491 samples from 371 HA patients.Assessments of antibody profiles showed that the presence of anti-FVIII IgG1 , IgG2 or IgG4 correlated qualitatively and quantitatively with the presence of an FVIII inhibitor as determined with the Nijmegen-Bethesda assay (NBA). Forty-eight patients with a negative inhibitor history contributed serial samples to the study, including seven patients who had negative NBA titers initially and later converted to being NBA-positive. The FLI detected anti-FVIII IgG1 in five of those seven patients prior to their conversion to NBA-positive. Five of 15 serial-sample patients who had a negative inhibitor history and had anti-FVIII IgG1 later developed an inhibitor, as compared with two of 33 patients with a negative inhibitor history without anti-FVIII IgG1 .These data provide a rationale for future studies designed both to monitor the dynamics of anti-FVIII antibody profiles in HA patients as a potential predictor of future inhibitor development and to assess the value of the anti-FVIII FLI as a supplement to traditional inhibitor testing.

Pub.: 30 Oct '14, Pinned: 29 Apr '18

Effects of pre-analytical heat treatment in factor VIII (FVIII) inhibitor assays on FVIII antibody levels

Abstract: 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.To assess the effect of PHT on the levels of detectable anti-FVIII IgG4.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.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.The optimal temperature for PHT in the FVIII NBA is 56°C. Higher temperatures may lead to loss of inhibitory antibodies.

Pub.: 20 Feb '18, Pinned: 29 Apr '18

Limit of detection and threshold for positivity of the Centers for Disease Control and Prevention assay for factor VIII inhibitors.

Abstract: Essentials Immunologic methods detect factor VIII (FVIII) antibodies in some inhibitor-negative specimens. Specimens were tested by modified Nijmegen-Bethesda assay (NBA) and fluorescence immunoassay. The NBA with preanalytical heat inactivation detects FVIII inhibitors down to 0.2 NBU. IgG4 frequency validates the established threshold for positivity of ≥ 0.5 NBU for this NBA.Background The Bethesda assay for measurement of factor VIII inhibitors called for quantification of positive inhibitors by using dilutions producing 25-75% residual activity (RA), corresponding to 0.4-2.0 Bethesda units, with the use of 'more sensitive methods' for samples with RA closer to 100% being recommended. The Nijmegen modification (Nijmegen-Bethesda assay [NBA]) changed the reagents used but not these calculations. Some specimens negative by the NBA have been shown to have FVIII antibodies detectable with sensitive immunologic methods. Objective To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)-modified NBA (CDC-NBA), which includes preanalytic heat inactivation to liberate bound anti-FVIII antibodies. Methods Specimens with known inhibitors were tested with the CDC-NBA. IgG4 anti-FVIII antibodies were measured by fluorescence immunoassay (FLI). Results Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). With four statistical methods, the limit of detection of the CDC-NBA was determined to be 0.2 NBU. IgG4 anti-FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥ 0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU. Conclusions The CDC-NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti-FVIII IgG4 in some patients with negative (< 0.5) NBU. The sharp increase in IgG4 frequency between 0.4 and 0.5 NBU validates the established threshold for positivity of ≥ 0.5 NBU for the CDC-NBA, supporting the need for method-specific thresholds.

Pub.: 11 Aug '17, Pinned: 29 Apr '18