Base de datos sistematizada de historias clínicas pertenecientes al I. I. HEMA: informe preliminar / Systematized database of medical records belonging to I. I. HEMA: preliminary report

Se procedió a confeccionar una base de datos de los casos atendidos en Clínica Hematológica del Instituto de Investigaciones Hematológicas. Se registró información sobre: a) Datos demográficos; b) Enfermedad actual; c) Métodos diagnósticos y d) Clasificación de las enfermedades según CIE 10. Sobre un total de 3573 casos registrados entre junio de 2002 y noviembre 2015 se analizaron 1300 (42%) casos. Los principales resultados muestran un predominio de las anemias, y entre ellas las ferropénicas. El mielograma y el frotis de sangre periférica predominaron entre los procedimientos diagnósticos. El tiempo entre primera consulta y diagnóstico muestra que en la mayoría (79,24%) de los casos este fue menor a 3 meses. En un 55,9 % de los casos se inició tratamiento antes del mes. (AU)

A data base was made from cases treated in the Hematological Clinic service. The following information was recorded: a) Demographics; b) Current disease; c) Diagnostic methods and d) Disease classification according to CIE 10. There were analyzed 1300 (42%) out of 3573 cases between June 2002 and November 2015. The main results show predominance of anemia and among them iron deficiency. The myelogram and peripheral blood smear predominated among the diagnostic procedures. Time between first consultation and diagnosis shows that in the majority (79.24%) of cases was less than 3 months. In 55.9% of cases it started treatment before the month. (AU)

Assessment of the F9 genotype-specific FIX inhibitor risks and characterisation of 10 novel severe F9 defects in the first molecular series of Argentinian patients with haemophilia B.

In haemophilia B (HB) (factor IX [FIX] deficiency), F9 genotype largely determines clinical phenotype. Aimed to characterise Argentinian families with HB, this study presents F9 genotype frequencies and their specific FIX inhibitor risk and 10 novel F9 mutations. Ninety-one DNA samples from HB patients and relatives were subjected to a new scheme: a primary screen for large deletions, a secondary screen for point mutations using conformation sensitive gel electrophoresis, DNA-sequencing and bioinformatic analysis. Our unbiased HB population (N=52) (77% with severe, 11.5% moderate and 11.5% mild HB) showed 32 missense (61.5%), including three novel mutations predicting specific structural/functional defects in silico , seven nonsense (13.5%) (one novel), five large deletions, four splice including three novel mutations affecting predicted splicing scores, three indels (two novel) and one Leiden mutation. Our comprehensive HB population included five patients with long-lasting FIX inhibitors: three nonsense (p.E35* (novel), p.R75*, p.W240*) and two entire- F9 deletions. Another patient with an indel (p.A26Rfs*14) developed transient inhibitors. A case-control analysis, based on our global prevalence of 3.05% for developing inhibitors in HB revealed that missense mutations were associated with a low risk odds ratio (OR) of 0.05 and a prevalence of 0.39%, whereas nonsense and entire- F9 deletions had significantly higher risks (OR 11.0 and 32.7) and prevalence (14.3% and 44.5%, respectively). Our cost-effective practical approach enabled identification of the causative mutation in all 55 Argentine families with HB, analysis of the molecular pathology of novel F9 defects and determination of mutation-associated FIX inhibitor risks.

Secondary prophylaxis with rFVIIa in hemophilia and inhibitors: recommendations from an experts committee from Argentina.

Secondary prophylaxis with rFVIIa has been the subject of several publications in the past few years. However, there is no general consensus on how this treatment should be put into practice, as publications have been very heterogeneous in the dosing schedule they report. Furthermore, the mechanism of action of rFVIIa and its short half life have been used as arguments against its role in prophylaxis. There have been a series of recent publications that show that rFVIIa can traffic through the intact endothelium and be stored in the subendothelium of several organs for a prolonged period of time. In order to consensuate the role of rFVIIa in prophylaxis, a group of experts from Argentina, resumed available information regarding pharmacology and clinical experience with this treatment, and developed a series of recommendations to use this drug in the prophylaxis setting.

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.

Profilaxis en hemofilia / Prophylaxis in hemophilia

Se desarrolla el tema de profilaxis sustitutiva de factores de coagulación en hemofilia severa cuyo objetivo es cambiar el fenotipo a moderada, reducir las hemorragias y prevenir la artropatía hemofílica discapacitante. Se describen las características de profilaxis primaria y secundaria; se discuten las controversias del momento de inicio, dosis y esquemas óptimos, y cuándo suspenderla. Por último, se muestran los datos de la experiencia en profilaxis en Argentina en 113 niños, 20 en primaria y 93 en secundaria. Se destaca que la profilaxis primaria es la más óptima, pues reduce casi a 0 las hemartrosis, previene la artropatía hemofílica y mejora la calidad de vida comparado contra profilaxis secundaria y a demanda.

It develops the theme of prophylactic replacement of clotting factors in severe hemophilia whose goal is to change the phenotype to moderate, reduce bleeding and prevent disabling haemophiliac arthropathy. Describes the characteristics of primary and secondary prophylaxis, we discuss the controversies of the moment of initiation, optimal doses and schedules, and when suspended it. Finally, we show the data of expeience in Argentina: prophylaxis in 113 children, 20 primary and 93 secondary. It also emphasized that the primary prophylaxis is the most optimal, since reduced to almost 0 hemarthrosis, prevents haemphiliac arthropathy and improves quality of life compared to secondary and treatment on demand.

Sixteen novel hemophilia A causative mutations in the first Argentinian series of severe molecular defects.

Hemophilia A (HA) is caused by heterogeneous mutations in the factor VIII gene (F8). This paper reports 16 novel small F8-mutations and rearrangements in a series of 80 Argentinian families with severe-HA. Using an updated scheme for F8-analysis, we found 37 F8-inversions (46%), 10 large deletions (13%), 13 small ins/del (16%), 7 nonsense (9%) and 8 missense mutations (10%), including 4 new ones (p.T233K, p.W1942R, p.L2297P and p.L2301S). The potential changes leading to severe-HA of these latter mutations were suggested by bioinformatics. The F8-mutation was characterised in 76 families (95%). They received genetic counselling and precise information about treatment design.

Prevalencia de anticuerpos neutralizantes y no neutralizantes en personas con hemofilia en Argentina / Prevalenceof neutralizing antibodies and not in people with henophilia in Argentina

El desarrollo de inhibidores (1nh) o anticuerpos neu-ralizantes (Ac Ntr) es una de las complicaciones del tratamiento de personas con hemofilia (PCH). Los Ac Ntr actúan inhibiendo la actividad coagulante del FVIII/IX. Se conoce la existencia de Ac anti FVIII no neutralizantes (Ac no Ntr), con posible influencia en la vida media del FV111 transfundido. Los Ac Ntr se detectan en ellaboratorio en ensayos coagulométricos y se cuantifican por método Bethesda o Nijmegen. Los Ac no Ntr sólo pueden detectase en ensayos inmunológicos. Con el objeto de determinar la prevalencia de Ac Ntr y no Ntr estudiamos 801 muestras de PCH. La detección de Ac anti FV111 se realizó mediante una técnica de ELISA y los resultados positivos fueron confirmados por método Bethesda modificado o Nijmegen para obtener el título de Ac Ntr. En PCH B se realizó la titulación del inh por método Bethesda. La prevalencia de Ac Ntr en la población estudiada fue de 14.7% en hemofilia A y 5.0% en hemofilia B. Encontramos una baja prevalencia de Ac no Ntr (3.3%) en hemofilia A. Estos pacientes serán evaluados con mayor frecuencia dado el alto riesgo de desarrollar Ac Ntr en el futuroEl desarrollo de inhibidores (inh) o anticuerpos neutralizantes (Ac Ntr) es una de las complicaciones del tratamiento de personas con hemofilia (PCH). La Ac Ntr actúan inhibiendo la actividad coagulante.

Genética de la hemofilia humana. Diagnóstico molecular de defectos en los genes de los factores VIII y IX de coagulación / Genetics of human haemophilia. Molecular diagnosis of defects affecting coagulation factors VIII and IX

La hemofilia A (HA) y B (HB) son enfermedades hemorrágicas hereditarias ligadas al sexo causadas por defectos de los factores VIII y IX, respectivamente. Excepto grandes inversiones recurrentes involucradas en la mitad de las HA severas, el resto de las hemofilias son causadas por distintos tipos de mutaciones grandes y pequeñas. Fueron estudiadas 70 familias con HA severa (se), 6 con seHB, 1 con HA moderada-leve (m) y 2 con mHB. Primero, en seHA, se estudio la inversión del intrón 22 (Inv22) usando un nuevo abordaje basado en PCR inversa. En los casos negativos para las inversiones se estudiaron primariamente las grandes deleciones y secundariamente las mutaciones pequeñas. En familias con HA, encontramos la Inv22 en 43 por ciento de las seHAs, una única inversión del intrón 1, 10 grandes deleciones (catorce por ciento)y 23 mutaciones pequeñas (incluyendo 10 deleciones, 3 inserciones, 4 cambios nonsense, 5 missense y 1 de splicing); y en HB, 1 deleción afectando un sitio de splicing, 4 missense y 3 nonsense. Este esquema de caracterización de mutaciones permite un estudio y análisis molecular preciso de HA y HB y beneficiará tanto al asesoramiento genético como a la provisión de información clave para el diseño del tratamiento.

Analysis of factor VIII gene intron 1 inversion in Argentinian families with severe haemophilia A and a review of the literature.

Besides intron 22 factor VIII gene inversion (Inv22), intron 1 inversion (Inv1) has recently been reported as a further recurrent mutation that causes approximately 5% of severe haemophilia A (HA) cases. We analysed the presence of the Inv1 in a group of 64 severe HA-affected families from Argentina, and found only one positive case. This Inv1 patient has not developed a factor VIII inhibitor, and the screening for small mutations in the coding sequences of the factor VIII gene did not detect any additional defect in this case. The Inv1 genotyping was further applied to analyse the haemophilia carrier status of the proband's sister. In addition, we studied the accuracy of the current polymerase chain reaction-based method to investigate the Inv1, and confirmed the absence of amplimer length polymorphisms associated to the Inv1-specific polymerase chain reaction amplifications in 101 X-chromosome haplotypes from unrelated Argentinian healthy males. In order to discuss Inv1 mutation frequency in severe HA and the risk of inhibitor formation, a review of the literature was included. Our data highlight the importance of analysis of the Inv1 in Inv22-negative severe HA cases. This will benefit both genetic counselling and the study of the relationship between genotype and inhibitor development.

[Avascular osteonecrosis of femoral head and neck in an AIDS patient]. / Necrosis avascular de cabeza y cuello de fémur en un paciente con sida.

Avascular osteonecrosis (AON) has increased in the last few years in patients infected with the human immunodeficiency virus type-1 (HIV-1). The most commonly affected bone is the femoral head and neck. Frequently these bilateral and clinical findings include moderate to severe pain and functional impotence of the affected joints. The etiology is multifactorial and highly active antiretroviral therapy (HAART) with protease inhibitors (PI) is probably related to its development. In the evolution, a total hip replacement may be needed. We present an hemophilic patient with AIDS, who developed a bilateral AON of the femoral head and neck during HAART.

Necrosis avascular de cabeza y cuello de fémur en un paciente con sida. / [Avascular osteonecrosis of femoral head and neck in an AIDS patient]

Avascular osteonecrosis (AON) has increased in the last few years in patients infected with the human immunodeficiency virus type-1 (HIV-1). The most commonly affected bone is the femoral head and neck. Frequently these bilateral and clinical findings include moderate to severe pain and functional impotence of the affected joints. The etiology is multifactorial and highly active antiretroviral therapy (HAART) with protease inhibitors (PI) is probably related to its development. In the evolution, a total hip replacement may be needed. We present an hemophilic patient with AIDS, who developed a bilateral AON of the femoral head and neck during HAART.

[Splenic artery embolization for the treatment of hypersplenism in hemophilic, HIV-1 and HCV seropositive patients]. / Embolización de la arteria esplénica como tratamiento del hiperesplenismo en pacientes hemofílicos, HIV-1 y HCV seropositivos.

Thrombocytopenia is an important and common hematological abnormality in patients with HIV-1/HCV coinfection. Splenomegaly is a frequent finding in these patients and usually causes hypersplenism and thrombocytopenia. We analyzed the clinical results of a minimal invasive treatment (splenic artery embolization) for thrombocytopenia secondary to hypersplenism and refractory to other therapies in two hemophiliac patients, HIV seropositive and with cirrhosis due to chronic HCV infection. The results suggest that splenic artery embolization is a safe, relatively atraumatic and effective method for the treatment of splenomegaly and hypersplenism in selected patients with HIV-1/HCV coinfection.

Embolizacion de la arteria esplenica como tratamiento del hiperesplenismo en pacientes hemofilicos, HIV-1 y HCV seropositivos / Splenic artery embolization for the treatment of hypersplenism in hemophylic, HIV-1 and HCV seropositive patients

La trombocitopenia es una anomalía usual e importante en pacientes con coinfección por HIV-1/HCV. La esplenomegalia es un hallazgo frecuente en estos pacientes y, usualmente, causa hiperesplenismo y trombocitopenia. Analizamos los resultados clínicos de un método invasivo mínimo (embolización de la arteria esplénica) para el tratamiento de la trombocitopenia secundaria al hiperesplenismo y refractaria a otras terapias en dos pacientes hemofílicos, infectados por el HIV-1 y con cirrosis causada por la infección crónica por HCV. Estos resultados sugieren que la embolización de la arteria esplénica es un método seguro, poco traumático y efectivo para el tratamiento de la esplenomegalia y el hiperesplenismo en pacientes con coinfección por HIV-1/HCV.(AU)

Embolización de la arteria esplénica como tratamiento del hiperesplenismo en pacientes hemofílicos, HIV-1 y HCV seropositivos. / [Splenic artery embolization for the treatment of hypersplenism in hemophilic, HIV-1 and HCV seropositive patients]

Thrombocytopenia is an important and common hematological abnormality in patients with HIV-1/HCV coinfection. Splenomegaly is a frequent finding in these patients and usually causes hypersplenism and thrombocytopenia. We analyzed the clinical results of a minimal invasive treatment (splenic artery embolization) for thrombocytopenia secondary to hypersplenism and refractory to other therapies in two hemophiliac patients, HIV seropositive and with cirrhosis due to chronic HCV infection. The results suggest that splenic artery embolization is a safe, relatively atraumatic and effective method for the treatment of splenomegaly and hypersplenism in selected patients with HIV-1/HCV coinfection.

Embolizacion de la arteria esplenica como tratamiento del hiperesplenismo en pacientes hemofilicos, HIV-1 y HCV seropositivos / Splenic artery embolization for the treatment of hypersplenism in hemophylic, HIV-1 and HCV seropositive patients

La trombocitopenia es una anomalía usual e importante en pacientes con coinfección por HIV-1/HCV. La esplenomegalia es un hallazgo frecuente en estos pacientes y, usualmente, causa hiperesplenismo y trombocitopenia. Analizamos los resultados clínicos de un método invasivo mínimo (embolización de la arteria esplénica) para el tratamiento de la trombocitopenia secundaria al hiperesplenismo y refractaria a otras terapias en dos pacientes hemofílicos, infectados por el HIV-1 y con cirrosis causada por la infección crónica por HCV. Estos resultados sugieren que la embolización de la arteria esplénica es un método seguro, poco traumático y efectivo para el tratamiento de la esplenomegalia y el hiperesplenismo en pacientes con coinfección por HIV-1/HCV.

Carga viral en liquido cefalorraquideo de pacientes hemofílicos HIV-1 positivos bajo tratamiento con HAART / Cerebrospinal fluid viral load in HIV-1 positive hemophilic patients treated with HAART

As HIV seropositive patients with undetectable CSF viral load have a lower likelihood of developing neurologic disease, the determination of CSF viral load levels may be useful to evaluate the efficacy of HAART. We compared plasma viral load levels with HIV-1 RNA CSF levels in 18 hemophilic patients without neurocognitive involvement under HAART. We detected a significant correlation between plasma viral load levels and CSF viral load levels. Fourteen patients with undetectable plasma viral load had undetectable RNA HIV-1 CSF levels as well. Four patients with detectable plasma viral load had detectable HIV-RNA in CSF, but the latter were significantly lower. Viral load is usually lower in non-blood fluids and HAART decreases the viral load in CSF as well as in blood.

Carga viral en liquido cefalorraquideo de pacientes hemofílicos HIV-1 positivos bajo tratamiento con HAART / Cerebrospinal fluid viral load in HIV-1 positive hemophilic patients treated with HAART

As HIV seropositive patients with undetectable CSF viral load have a lower likelihood of developing neurologic disease, the determination of CSF viral load levels may be useful to evaluate the efficacy of HAART. We compared plasma viral load levels with HIV-1 RNA CSF levels in 18 hemophilic patients without neurocognitive involvement under HAART. We detected a significant correlation between plasma viral load levels and CSF viral load levels. Fourteen patients with undetectable plasma viral load had undetectable RNA HIV-1 CSF levels as well. Four patients with detectable plasma viral load had detectable HIV-RNA in CSF, but the latter were significantly lower. Viral load is usually lower in non-blood fluids and HAART decreases the viral load in CSF as well as in blood. (Au)