Anticuerpos anti-FVIII: su evaluación por citometría de flujo / Anti FVIII antibodies: evaluation by flow cytometry

En este trabajo se describe un sistema para evaluar y caracterizar los anticuerpos anti-FVIII en pacientes con Hemofilia A Severa (HAS) que reciben el Factor como tratamiento de sustitución. Consiste en el empleo combinado de microesferas y Citometria de Flujo (CF). El rFVIII fue acoplado a microesferas de 2 µm de diámetro (m-FVIII) las cuales se incubaron con diluciones de plasma o suero de pacientes con (n=13) o sin (n=17) inhibidor, pacientes en Tratamiento Inmunotolerante (TIT)(n=5) y dadores normales (N) (n=12). Los anticuerpos se revelaron con anti-lgG humana, anti-lgG1, anti-lgG2, anti-IgG3 o anti-lgG4 biotiniladas, seguido por streptavidina-ficoeritrina. Se registraron los valores de Intensidad de Fluorescencia Media (IFM). Microesferas sin FVIII (m-Control) se utilizaron como control. El resultado se expresó como índice: (IFM de m-FVIII/IFM de m-Control) multiplicado por la inversa de la dilución de máxima respuesta. Se determinó el porcentaje de contribución de cada subclase de IgG. Los resultados presentaron un 86 por ciento de concordancia con la prueba de Bethesda y un 80 por ciento con ELISA. El método fue útil para el seguimiento de los pacientes durante el TIT. La IgG4 prevaleció en pacientes con alto título y al comienzo del TIT. La CF es fácil y rápida y requiere sólo 200 µl de muestra.

In this study, a Flow Cytometry (FC) system is described for detecting and characterizing antibodies (inhibitors) to Factor VIII (FVIII) in Severe Haemophilia A (SHA) patients following FVIII infusion. A combination of microspheres and Flow Cytometry (FC) was employed. First, rFVIII was coupled to microspheres of 2 µm of diameter (m-FVIII). Then, they were reacted with dilutions of plasma or serum of patients with (n=13) or without (n=17) inhibitors. Five patients receiving Immunotolerant Treatment (ITI) and 12 normal donors were included. Microspheres without rFVIII were used as control (m-Control). Captured anti-FVIII antibodies were detected using biotinylated anti-Human IgG, IgG1, IgG2, IgG3 or IgG4 followed by streptavidin-phycoerythrin. FC analysis was performed recording Mean Fluorescence Intensity (MFI). Results were given as an Index: the highest MFI ratio between m-FVIII and m-Control multiplied by the inverse of the corresponding plasma dilution. The contribution of each IgG subclass was expressed as percentage. FC results had 86 per cent and 80 per cent of coincidence with the Bethesda method and ELISA respectively. The test was useful to measure anti-FVIII antibodies during the ITI. IgG4 was the prevalent IgG subclass in patients with high level of inhibitors and previously to ITI. FC was easy, fast and requires only 200 µl of sample.

Transient Acquired Hemophilia Associated with Mycoplasma Pneumoniae Pneumonia

Acquired hemophilia is a rare disorder caused by autoantibodies to factor VIII (FVIII) (also referred to as factor VIII inhibitors or anti-FVIII) and may be associated with pregnancy, underlying malignancy, or autoimmune disorders. A 33-month-old girl who presented with hematochezia and ecchymotic skin lesions was diagnosed with Mycoplasma pneumoniae pneumonia by serology and polymerase chain reaction. Hematologic studies showed a prolonged activated partial thromboplastin time (aPTT), partially corrected mixing test for aPTT, reduced levels of FVIII, and the presence of antibodies against FVIII. She was treated conservatively with prednisone and intravenous immunoglobulin (IVIG) without FVIII transfusion and recovered without sequelae. This report provides the first description of acquired hemophilia due to anti-FVIII in association with M. pneumoniae in Korea. We discuss this case in the context of the current literature on acquired hemophilia in children.