Heat treatment of samples improve the performance of the Nijmegen-Bethesda assay in hemophilia A patients undergoing immune tolerance induction.
Nijmegen-Bethesda assay is the gold standard to assess inhibitory antibodies against factor (F) VIII. This method has some limitations, including high coefficient of variation and possible interference of residual endogenous or exogenous factor VIII. Heat-treatment of samples at 56 °C for 30 min could be a strategy to improve the sensitivity of this test. The aim of this study was to compare inhibitor quantification in hemophilia patients with and without inhibitor performed in previously heated and non-heated samples. A total of 109 analyses from 46 patients with severe hemophilia A were performed. Patients were divided into three groups: 20 patients with no history of inhibitor, recently and not recently exposed to FVIII (group I), 21 patients with history of inhibitor not exposed to FVIII (group II), and 5 patients (68 samples) undergoing an immune tolerance induction (ITI) protocol (group III). For patients with no history of inhibitor, heat-treatment did not modify the results (p=0.24). However, differences in inhibitor levels between heated and non-heated samples were observed in patients with history of inhibitor (group II, p<0.05) and in patients in ITI (group III, p<0.001). In 11 samples, inhibitor quantification shifted from negative to positive. Additionally, a longitudinal evaluation of each ITI patient showed similar trend line for the results of heated and non-heated samples. In this study, we demonstrated that heating samples increase sensitivity of Nijmegen-Bethesda assay, with no shift from negative to positive results in patients with no history of inhibitor. Furthermore, this procedure has an important role to patients undergoing an ITI protocol.
IHTC Hemophilia Unit "Cláudio L. P. Corrêa", Instituto Nacional de Ciência e Tecnologia do Sangue, Hemocentro Unicamp, University of Campinas, São Paulo, Brazil. Electronic address: silmara@unicamp.br.
Silmara Aparecida
SA
de Lima Montalvão
IHTC Hemophilia Unit "Cláudio L. P. Corrêa", Instituto Nacional de Ciência e Tecnologia do Sangue, Hemocentro Unicamp, University of Campinas, São Paulo, Brazil.
Alini Camargo
AC
Tucunduva
IHTC Hemophilia Unit "Cláudio L. P. Corrêa", Instituto Nacional de Ciência e Tecnologia do Sangue, Hemocentro Unicamp, University of Campinas, São Paulo, Brazil.
Andrea Luísa
AL
de Almeida Sambo
IHTC Hemophilia Unit "Cláudio L. P. Corrêa", Instituto Nacional de Ciência e Tecnologia do Sangue, Hemocentro Unicamp, University of Campinas, São Paulo, Brazil.
Erich Vinicius
EV
De Paula
IHTC Hemophilia Unit "Cláudio L. P. Corrêa", Instituto Nacional de Ciência e Tecnologia do Sangue, Hemocentro Unicamp, University of Campinas, São Paulo, Brazil.
Samuel
S
de Souza Medina
IHTC Hemophilia Unit "Cláudio L. P. Corrêa", Instituto Nacional de Ciência e Tecnologia do Sangue, Hemocentro Unicamp, University of Campinas, São Paulo, Brazil.
Margareth Castro
MC
Ozelo
D001323:Autoantibodies / Q000276:immunology; D001938:Brazil; D005169:Factor VIII / Q000276:immunology; D005176:Factor XIII / Q000737:chemistry*; D005189:False Positive Reactions; D006403:Hematologic Tests / Q000379:methods*; D006467:Hemophilia A / Q000097:blood / Q000276:immunology*; D006358:Hot Temperature; D006801:Humans; D007107:Immune System; D007108:Immune Tolerance
D001323:Autoantibodies; D005169:Factor VIII; D005176:Factor XIII
Hemophilia; Immune tolerance induction; Inhibitor; Nijmegen–Bethesda assay