Abstract Number: LPB0061
Meeting: ISTH 2021 Congress
Background: The development of inhibitors to infused factor VIII (FVIII) in congenital hemophilia A (HA) patients display a serious complication. Moreover, FVIII inhibitors can occur due to an autoimmune disorder (acquired HA). The inhibitors are classified as type I and II according to their different kinetics. Routinely the inhibitor titer is quantified using the modified Nijmegen-Bethesda assay (MNBA). The resulting titers are often difficult to compare due to the different criteria used for calculation.
Aims: To analyze the impact of different FVIII inhibitor kinetics on the titer calculation in the MNBA applying different criteria.
Methods: Two type I FVIII antibodies [4A4 (Green Mountain Antibodies), BO2C11 (Creative Biolabs)] and two type II FVIII antibodies [2-54 (Green Mountain Antibodies), ESH-8 (ImmBioMed)] were analyzed at different concentrations (0.25-4 µg/mL) with the MNBA using the cryocheck™ Factor VIII Inhibitor Kit (Precision BioLogic). FVIII residual activities (RAs) were measured in duplicates with a FVIII One-Stage Clotting Assay using a cobas t 511/711 analyzer in combination with the FVIII assay kit (Roche Diagnostics). The RAs were plotted against inhibitor dilution in a semi-log(x)-plot.
Results: The kinetics of four different FVIII antibodies were compared to the theoretical kinetic model (black lines), where every RA would lead to the same calculated titer result (Figure 1). As a result, only the inhibition kinetic of one analyzed antibody 4A4 (C) was perfectly represented by the theoretical kinetic while the other kinetics showed significant deviations from the used model. These deviating inhibition kinetics (A/B and D) can have a significant impact on titer calculation when using different criteria (Table 1).
|Criteria*||Limitations for inhibitors with deviating kinetics**|
|Number||Reference||Inhibitor titer calculation|
|1||C. Miller et al., Transfusion Medicine and Hemostasis 132 (2019)||first dilution with RA > 25 %||A/B: long plateau phase (> 25 % RA) ↓
selected dilution with RA close to 25 % ↓
D: selected dilution with RA close to 25 % ↑
|2||S. Kitchen et al., WFH 34 (2010)||mean value of all titers with RAs between 25 and 75 %||A/B: long plateau phase (> 25 %) ↓
uneven distribution of values ↓↑
D: uneven distribution of values ↓↑
|3||S. Kitchen et al., WFH 34 (2010)||titer of the dilution closest to 50 % RA||A/B, D: RAs far from 50 % ↓↑|
|4||B. Verbruggen et al., Thrombosis and haemostasis 74 (1995)||RAs (25-75 %) were plotted versus dilution in a semi-log(y)-plot
titer calculation with the concentration that gives 50 % RA by extrapolation
|D: no calculation possible when only one value is between 25-75 %|
|5||sigmoidal regression of all RAs in a semi-log(x)-plot
RA [%] = bottom + (100 – bottom)/(1 + 10^((LogEC50 – x) ∙ HillSlope))
titer calculation with the concentration that gives 50 % RA
|* Criteria 1-3 describe different strategies to select dilutions with RAs which can be used for a reliable inhibitor titer calculation. Criteria 4+5 use a recalculation to determine the concentration that gives 50 % RA in different ways.
** A – D corresponding to the generated kinetics as presented in figure 1. Showing either a kinetic profile with a lower slope (e.g. A +B) or a higher slope (e.g. D) at 50 % RA. ↓↑ indicate a resulting over- or underestimation of the calculated titer depending on the criteria in combination with an outlined deviation of the kinetic profile.
Conclusions: The theoretical kinetic model of the MNBA does not perfectly reflect the inhibition profile of various different FVIII inhibitors. This consequently leads to differences in the calculated titer especially depending on the criteria used for quantification.
COBAS and COBAS T are registered trademarks of Roche.
To cite this abstract in AMA style:Ketteler C, Hoffmann I, Davidson S, Tiede A, Richter N. Impact of Characteristic Inhibition Kinetics of Different FVIII Inhibitors on the Inhibitor Titer Quantification in the Modified Nijmegen-Bethesda Assay [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/impact-of-characteristic-inhibition-kinetics-of-different-fviii-inhibitors-on-the-inhibitor-titer-quantification-in-the-modified-nijmegen-bethesda-assay/. Accessed August 19, 2022.
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