Methods for anti-factor VIII antibody levels in haemophilia A patients-validation of a multiplec immunoassay and comparability with assays measuring non-neutralising and neutralising antibodies (inhibitors)
(2023) In Haemophilia 29(1). p.336-347- Abstract
- Introduction
The development of neutralising (inhibitors) and non-neutralising antibodies (NNAs) is a complication to factor replacement therapy in haemophilia. The diagnostic methods available lack standardisation, have high inter-laboratory variation, and false-negative as well as false-positive results may affect treatment. Both functional inhibitors and NNAs may be detected with higher reproducibility, sensitivity and specificity using the immunological Luminex xMAP-based fluorescence-immunoassay (xFLI).
Aim
Validation of our xFLI and comparability with enzyme-linked immunosorbent assay (ELISA) and chromogenic Nijmegen-Bethesda assay (CBA) for anti-FVIII antibodies in haemophilia A (HA)... (More) - Introduction
The development of neutralising (inhibitors) and non-neutralising antibodies (NNAs) is a complication to factor replacement therapy in haemophilia. The diagnostic methods available lack standardisation, have high inter-laboratory variation, and false-negative as well as false-positive results may affect treatment. Both functional inhibitors and NNAs may be detected with higher reproducibility, sensitivity and specificity using the immunological Luminex xMAP-based fluorescence-immunoassay (xFLI).
Aim
Validation of our xFLI and comparability with enzyme-linked immunosorbent assay (ELISA) and chromogenic Nijmegen-Bethesda assay (CBA) for anti-FVIII antibodies in haemophilia A (HA) patients.
Methods
The xFLI method was developed with full-length and B-domain deleted factor coupled to magnetic beads, optimised and validated for performance characteristics. Comparability with ELISA and CBA was evaluated in HA patient samples (n = 112), serial samples in six inhibitor patients and reference interval and decision-limits in healthy donors (n = 44).
Results
The intra- and inter-assay precision (CV%) for the xFLI method was below 6% and detection limit (LLOQ) .084 ng/mL (NovoEight). All ELISA-positive samples were positive with either Advate or NovoEight. Additionally, 10.7%–14.3% were xFLI-positive and ELISA-negative. All but one CBA-positive sample was above 3SD with xFLI; one was between 2 and 3SD. 29.1% were xFLI-positive and CBA negative. The overall concordance between xFLI and ELISA was 82.1% and xFLI and CBA 77.9%.
Conclusion
The anti-FVIII antibody xFLI method is adaptable to clinical practice and more sensitive and reproducible than ELISA and CBA. Actual NNA titers are determined to both full-length and B-domain deleted FVIII. The xFLI is thus valuable for confirmation of all anti-FVIII antibodies. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/c41f2193-dd8f-48d4-bfee-4ef3ee33ae93
- author
- Martin, Myriam LU ; Augustsson, Cecilia ; Lind, Vivian ; Al-Sabti, Riam ; Chi Lam, My ; Gretenkort Andersson, Nadine LU and Strandberg, Karin LU
- organization
- publishing date
- 2023
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Haemophilia
- volume
- 29
- issue
- 1
- pages
- 336 - 347
- publisher
- Wiley-Blackwell
- external identifiers
-
- scopus:85139237440
- pmid:36193002
- ISSN
- 1351-8216
- DOI
- 10.1111/hae.14669
- language
- English
- LU publication?
- yes
- id
- c41f2193-dd8f-48d4-bfee-4ef3ee33ae93
- date added to LUP
- 2023-02-09 11:50:30
- date last changed
- 2023-05-12 03:00:02
@article{c41f2193-dd8f-48d4-bfee-4ef3ee33ae93, abstract = {{Introduction<br/>The development of neutralising (inhibitors) and non-neutralising antibodies (NNAs) is a complication to factor replacement therapy in haemophilia. The diagnostic methods available lack standardisation, have high inter-laboratory variation, and false-negative as well as false-positive results may affect treatment. Both functional inhibitors and NNAs may be detected with higher reproducibility, sensitivity and specificity using the immunological Luminex xMAP-based fluorescence-immunoassay (xFLI).<br/><br/>Aim<br/>Validation of our xFLI and comparability with enzyme-linked immunosorbent assay (ELISA) and chromogenic Nijmegen-Bethesda assay (CBA) for anti-FVIII antibodies in haemophilia A (HA) patients.<br/><br/>Methods<br/>The xFLI method was developed with full-length and B-domain deleted factor coupled to magnetic beads, optimised and validated for performance characteristics. Comparability with ELISA and CBA was evaluated in HA patient samples (n = 112), serial samples in six inhibitor patients and reference interval and decision-limits in healthy donors (n = 44).<br/><br/>Results<br/>The intra- and inter-assay precision (CV%) for the xFLI method was below 6% and detection limit (LLOQ) .084 ng/mL (NovoEight). All ELISA-positive samples were positive with either Advate or NovoEight. Additionally, 10.7%–14.3% were xFLI-positive and ELISA-negative. All but one CBA-positive sample was above 3SD with xFLI; one was between 2 and 3SD. 29.1% were xFLI-positive and CBA negative. The overall concordance between xFLI and ELISA was 82.1% and xFLI and CBA 77.9%.<br/><br/>Conclusion<br/>The anti-FVIII antibody xFLI method is adaptable to clinical practice and more sensitive and reproducible than ELISA and CBA. Actual NNA titers are determined to both full-length and B-domain deleted FVIII. The xFLI is thus valuable for confirmation of all anti-FVIII antibodies.}}, author = {{Martin, Myriam and Augustsson, Cecilia and Lind, Vivian and Al-Sabti, Riam and Chi Lam, My and Gretenkort Andersson, Nadine and Strandberg, Karin}}, issn = {{1351-8216}}, language = {{eng}}, number = {{1}}, pages = {{336--347}}, publisher = {{Wiley-Blackwell}}, series = {{Haemophilia}}, title = {{Methods for anti-factor VIII antibody levels in haemophilia A patients-validation of a multiplec immunoassay and comparability with assays measuring non-neutralising and neutralising antibodies (inhibitors)}}, url = {{http://dx.doi.org/10.1111/hae.14669}}, doi = {{10.1111/hae.14669}}, volume = {{29}}, year = {{2023}}, }