A flow cytometry evaluation of anti-FVIII antibodies: correlation with ELISA and Bethesda assay

Abstract

In this study, we describe a flow cytometry (FC) system for detecting antibodies to factor VIII (FVIII) and compare its results with those of enzyme-linked immunosorbent assay (ELISA) that detects both inhibitory (I-Ab) and non-inhibitory (NI-Ab) antibodies and the Nijmegen modification of the Bethesda method, detecting I-Ab. FC was set up in our laboratory. Recombinant FVIII (rFVIII) was coupled to microspheres (FVIII-m) and reacted with different plasma dilutions. Microspheres without rFVIII were used as control (control-m). Captured anti-FVIII antibodies were detected using anti-human IgG. Plasma samples from the following patients with severe haemophilia A (SHA) patients were evaluated: 17 P (patients without I-Ab, <0.5 BU mL(-1)); 13 PI (patients with I-Ab, 1.1-8200 BU mL(-1)). Of these 13, two PI were referred during immune tolerance induction (ITI), and plasmas from 12 healthy donors (HD) were evaluated. Semiquantitative results were given as an index (the highest mean fluorescence intensity ratio between FVIII-m and control-m multiplied by the inverse of the corresponding plasma dilution). Both plasma and serum were suitable for the test. FC agreed with the Bethesda method (r = 0.8; P = 0.0001). FC and ELISA had 80% of coincidence. Four of 17 patients (23.5%) had NI-Ab by FC, and two of them developed high levels of I-Ab later on. This test provides a useful alternative for measuring FVIII antibodies supplementing Bethesda assay. FC is fast and easy to perform. No more than 200 μL of plasma or serum is required especially making it useful for paediatric patients.

Phenotypes of Allo- and Autoimmune Antibody Responses to FVIII Characterized by Surface Plasmon Resonance

“…Although inhibitory antibodies are the primary concern when attempting to restore hemostatic function, both inhibitory and non-inhibitory antibodies provide information about the immunological state of a patient. A number of sensitive immunoassays have been developed to allow the screening of clinical samples for total (inhibitory+non-inhibitory) anti-FVIII antibodies and to provide complementary information to the Bethesda assay [4]–[9].…”

Differential Expression of Immunogenic Proteins on VirulentMycobacterium tuberculosisClinical Isolates

“…To better describe the circulating populations of anti-Rv2241, Rv0009, Rv0407, and Rv2624c Abs, we performed an immunoisotype characterization based on an Ag-specific FACS analysis [41]. To do this, sera reactive against all the recombinant proteins ( n = 10) were incubated with recombinant protein-coated latex beads (Figure 5(a)).…”

Product‐dependent anti‐factor VIII antibodies

“…[37] It is also possible that non-inhibitory antibodies could be predictors for the development of inhibitors. [38] Additional consideration for the quantitation of non-inhibitory antibodies is related to different mechanisms of FVIII activation in the Bethesda assay and in tissue factor-triggered processes. The Bethesda assay is virtually a modification of the physiologically-irrelevant APTT assay with the majority of FVIII being activated by FXIa.…”

“…It is hard to compare existing immunoassays with the assay used in this study, because the former are non-quantitative with respect to the molar concentration of anti-FVIII antibodies. [38, 45–47]…”