IgG subclasses of FVIII inhibitors in an argentine cohort of severe hemophilia A patients: Analysis by flow cytometry.

INTRODUCTION: FVIII inhibitors consist of a polyclonal population of antibodies. Previous studies have demonstrated different distribution of IgG subclasses. IgG4 was associated to high level of FVIII inhibitors and failure of immune tolerance induction (ITI) treatment. This study monitored the relative distribution of IgG subclasses of anti-FVIII in patients with severe hemophilia A (SHA). METHODS: Anti-FVIII antibodies were measured employing an immunomethod, developed in our laboratory, that combines flow cytometry (FC) with microspheres coupled (FVIII-m) or not (Control-m) to FVIII. Seventy-five patients with SHA were studied, 17 without inhibitors (Group I); 58 with inhibitor history, 13 low responders: (LR: Group II), and 45 high responders (HR: Group III). Eight patients undergoing ITI were also included. RESULTS: We found anti-FVIII antibodies in 11 of 27 patients (40%) without inhibitors and in 45 of 48 with inhibitors at the moment of the study. IgG4 was predominant only in the Group III: P=0.02 in patients with low level of inhibitors and P=0.0001 with high titer of inhibitors. Longitudinal analysis performed on patients undergoing ITI showed a gradual decrease of IgG4 values that was associated to improvement of clinical parameters during treatment. CONCLUSION: We suggest the use of the FC method to supplement functional traditional assays and to help to improve the management of patients with SHA.

Factor VIII genotype characterization of haemophilia A affected patients with transient and permanent inhibitors: a comprehensive Argentine study of inhibitor risks.

Inhibitor development against exogenous factor VIII is a severe impairment of replacement therapy affecting 18% of Argentine patients with severe haemophilia A (HA). To study the molecular predisposition for inhibitor development, we genotyped 260 HA patients with and without inhibitors, countrywide. The inhibitor-positive population (19 transients, 15 low responders, LR and 70 high responders, HR) of 104 severe-HA patients showed 59 Inv22 (intron 22 inversions), 18 small ins/del-frameshifts, 12 gross deletions, 12 nonsense, one splicing defect and two missense, p.Arg531Pro and p.Leu575Pro, both LR and thought to impair FVIII A2 domain secondary structure. In addition, a patient with mild HA and HR showed the missense p.Glu1704Lys associated with two neutral intronic substitutions potentially affecting the A3 domain. A case/control study (84/143) permitted estimation of F8 genotype-specific inhibitor risks [OR; prevalence (CI)] in severe-HA patients classifying a high-risk group including multi-exon deletions [3.66; 55% (19-100)], Inv22 [1.8; 24% (19-100)] and nonsense in FVIII-LCh [1.2; 21% (7-59)]; an average risk group including single-exon deletions, indel frameshifts and nonsense-HCh; and a low-risk group represented by missense defects [0.14; 3% (0.6-11)]. Analysis of inhibitor concordance/discordance in related patients indicated additional genetic factors other than F8 genotype for inhibitor formation. No significant inhibitor-predisposing factors related to FVIII product exposure were found in age- and F8 genotype-stratified populations of severe-HA patients. In conclusion, the Argentine HA patient series presents similar global and mutation-specific inhibitor risks than the HA database and other published series. This case-specific information will help in designing fitted therapies and follow-up protocols in Argentina.

Severe haemophilia A patients have reduced numbers of peripheral memory B cells.

The development of inhibitors is a complication of replacement treatment in Haemophilia. Loss of factor VIII-specific memory B cells in the spleen is associated with down regulation of antibodies in mice treated with high doses of FVIII, but changes in B cell memory have not been described in haemophilic patients. The aim of this study was to evaluate the phenotype of circulating lymphocytes in severe haemophilia A. Twenty patients with inhibitors (PI), 22 without inhibitors (P), nine patients during immune tolerance induction (ITI) treatment and 20 healthy donors (HD) were included. Peripheral blood lymphocytes were examined using flow cytometry. Anti-FVIII antibodies were measured using Bethesda and flow cytometry. Percentages of T subsets and B lymphocytes were similar in all groups. In contrast, memory B cells (CD27+) were decreased in PI and P compared with HD, but the level of significance was higher in PI (P = 0.001) than P (P = 0.01). PI with high level of anti-FVIII antibodies presented the lowest B memory values. CD70 expression was also lowest in PI. Non-switched CD27+ subpopulation (IgD+) was prevalent in PI, but did not show statistical significance. When ITI failed, the percentages of CD27+ B cells after 12 months of ITI were lowest. In a longitudinal study performed in four patients, an increased percentage of CD27+ and CD70+ B cells during ITI was found. This work suggests that different peripheral lymphocyte markers, such as CD27 and CD70 on B cells, may be helpful to evaluate anti-FVIII response and to monitor the success of ITI.

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

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.