False‐positive results in ELISA‐based anti FVIII antibody assay may occur with lupus anticoagulant and phospholipid antibodies

Abstract

Summary. The evaluation of a prolonged aPTT often includes Lupus Anticoagulant, Antiphospholipid Antibodies, and Factor VIII (FVIII) inhibitors. We have noticed that patient samples positive for lupus antibody (LA) are frequently also positive for FVIII IgG antibodies in an enzyme‐linked immunosorbent assay (ELISA), indicating the need for follow‐up testing with a more labour‐intensive functional assay for FVIII inhibition. This study evaluates the potential for a FVIII IgG ELISA to yield false‐positive results in patient samples positive for LA or other antiphospholipid antibodies. A total of 289 residual de‐identified patient samples positive for LA (n = 143), anti‐cardiolipin IgG (n = 84), or beta2‐glycoprotein antibody (n = 62) were tested for FVIII IgG using a commercial ELISA. Samples with positive FVIII IgG ELISA results were further tested for FVIII activity using a clot‐based FVIII inhibitor assay. The FVIII IgG ELISA yielded positive results in 39 (13%) of the samples tested, including 13/143 (13%) LA‐positive, 15/85 (18%) aCL IgG‐positive and 6/62 (10%) β2‐glycoprotein IgG‐positive samples. The clot‐based FVIII inhibitor assay yielded negative results in all 39 FVIII IgG‐positive specimens tested, indicating discrepancy with the FVIII IgG ELISA results. Patient specimens positive for LA, aCL IgG, or β2‐glycoprotein IgG may yield false‐positive results for FVIII antibodies. Caution is warranted in interpreting FVIII antibody results in these cases.

Distinguishing lupus anticoagulants from factor VIII inhibitors in haemophilic and non‐haemophilic patients

“…These results are similar to those observed in healthy subjects by Whelan et al using an ELISA and by Boylan et al using the FLI. The negative results in the FLI strongly support our hypothesis that this assay is unaffected by the presence of Las, whereas the commercial anti‐FVIII ELISA, which is reported to measure IgG but is not specific for IgG4, has been reported to give positive results in some LA patients . Our results show IgG subclasses other than IgG4 may be present in LA patients and could influence results of this ELISA test.…”

Optimization of pre‐analytical heat treatment for inhibitor detection in haemophilia A

“…A commercially available, solid phase, indirect anti‐FVIII IgG ELISA (Immucor, Norcross, Georgia, USA), precoated with H1 haplotype rFL‐FVIII (Kogenate®, Bayer Corp., Pittsburg, PA, USA) was performed as per the manufacturer’s instruction. Samples and controls (diluted 1:3 in diluent buffer) were added to the microwells and incubated at 37°C for 30 minutes.…”

Laboratory testing for factor VIII and IX inhibitors in haemophilia: A review

“…5,21,64,72 IgG 1 antibodies are present in both inhibitor-negative and inhibitor-positive patients; preliminary evidence suggests that they appear earlier and can be predictive of subsequent inhibitor development in HA, 26 as well as remaining present when high-titre inhibitors decline. 72 The commercial anti-FVIII ELISA has also detected antibodies in some non-haemophilic patients with LA, 73 although the IgG subclass was not reported. FIX inhibitors have similarly been linked to the presence of anti-FIX antibodies of IgG 4 subclass with ELISA 28,49 and FLI.…”