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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)

Martin, Myriam LU ; Augustsson, Cecilia ; Lind, Vivian ; Al-Sabti, Riam ; Chi Lam, My ; Gretenkort Andersson, Nadine LU and Strandberg, Karin LU (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)
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author
; ; ; ; ; and
organization
publishing date
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}},
}