Alta variabilidad en la determinación del Factor IX en muestras de plasma adicionadas con nonacog beta pegol entre distintos pares de reactivos/sistemas de detección / High variability in the determination of Factor IX in plasma samples spiked with nonacog beta pegol between different reagents/detection system pairs / Alta variabilidade na determinação do Fator IX em amostras de plasma enriquecidas com nonacog beta pegol entre diferentes pares de reagentes/ sistema de detecção

Resumen La hemofilia B es un trastorno hemorrágico hereditario, ligado al cromosoma X, que se caracteriza por el déficit del factor IX (FIX) de la coagulación. Para mejorar la calidad de vida de los pacientes y la adherencia al tratamiento se han desarrollado concentrados de factores recombinantes modificados para extender su vida media, denominados factores de vida media extendida (EHL: extended half life concentrates). El nonacog beta pegol (N9-GP) es una molécula de FIX humano recombinante glicopegilada que tiene una vida media de 93 h con una sola dosis y ha mostrado un porcentaje de recuperación mayor que otras moléculas. Para diagnosticar y monitorear el tratamiento del paciente hemofílico se determina la actividad del FIX con el ensayo coagulable en una etapa (OSA: one stage assay) y/o en el ensayo cromogénico. El objetivo de este trabajo, realizado en tres centros, fue medir la recuperación de N9-GP con 10 reactivos de APTT diferentes en tres plataformas, en muestras deficientes en FIX adicionadas in vitro con N9-GP, en cuatro niveles de concentración diferentes. Los resultados muestran una heterogeneidad en la actividad de N9-GP medidos por OSA con los diferentes reactivos de APTT cuando se realizaron las calibraciones con el estándar específico de cada coagulómetro. Se obtuvo un porcentaje de recuperación mayor de 92% con Cephascreen, Actin FSL y APTTest elágico en las tres plataformas evaluadas. Estos reactivos serían los únicos apropiados cuando se usa el OSA calibrado con plasma comercial para monitorear el tratamiento de los pacientes que reciben N9-GP.

Abstract Hemophilia B (HB) is an X-linked hereditary bleeding disorder characterised by coagulation factor IX (FIX) deficiency. To improve the quality of life of patients and adherence to treatment, recombinant factor concentrates glicomodified to extend their half-life have been developed. These are called extended half-life factors (EHL: extended half-life concentrates). Nonacog beta pegol (N9-GP) is a glycopegylated recombinant human FIX molecule that has a half-life of 93 h with a single dose and has shown a higher recovery percentage than other molecules. For diagnosis and monitoring the treatment of hemophiliac patients, FIX activity is determined with the One Stage Clotting Assay (OSA) and/or the chromogenic assay. The objective of this work, carried out in three centres, was to measure the recovery of N9-GP with 10 different APTT reagents on three platforms, in FIX deficient samples spiked in vitro with N9-GP, at four different concentration levels. The results show a heterogeneity in the activity of N9-GP measured by OSA with the different APTT reagents when the calibrations were performed with the specific standard of each coagulometer. A recovery percentage greater than 92% was obtained with Cephascreen, Actin FSL and APTTest ellagic in the three platforms evaluated. These reagents would be the only ones appropriate when using the commercial plasma-calibrated OSA to monitor the treatment of patients treated with N9-GP.

Resumo A hemofilia B é uma doença hemorrágica hereditária ligada ao cromossomo X caracterizada pela deficiência do fator de coagulação IX (FIX). Para melhorar a qualidade de vida dos pacientes e a adesão ao tratamento, foram desenvolvidos concentrados de fatores recombinantes modificados para prolongar sua meia-vida, chamados de fatores de meia-vida estendida (EHL: extended half life concentrates). Nonacog beta pegol (N9-GP) é uma molécula de FIX humano recombinante glicopeguilada que tem meia-vida de 93 h com uma dose única e mostrou uma porcentagem de recuperação maior do que outras moléculas. Para diagnosticar e monitorar o tratamento de pacientes hemofílicos, a atividade do FIX é determinada com o ensaio coagulável em um estágio (OSA: One Stage Assay) e/ou o ensaio cromogênico. O objetivo deste trabalho, realizado em três centros, foi medir a recuperação de N9-GP com 10 reagentes de APTT diferentes em três plataformas, em amostras deficiente de fator IX adicionadas in vitro com N9-GP, em quatro níveis de concentração diferentes. Os resultados mostram uma heterogeneidade na atividade de N9-GP medidos por OSA com os diferentes reagentes de APTT quando as calibragens foram realizadas com o padrão específico de cada coagulômetro. Uma porcentagem de recuperação superior a 92% foi obtida com Cephascreen, Actin FSL e APTTest elágico nas três plataformas avaliadas. Esses reagentes seriam os únicos apropriados ao usar o OSA calibrado com plasma comercial para monitorar o tratamento de pacientes tratados com N9-GP.

Influence of different levels of heparin and antithrombin Ⅲ on human coagulation factor Ⅸ activity / 中国输血杂志

【Objective】 To study the effect of different concentrations of heparin, ATⅢ or a mixture of heparin and antithrombin Ⅲ (ATⅢ) (1∶1)on the activity of human coagulation factor Ⅸ (FⅨ). 【Methods】 The heparin or heparin/ATⅢ with different concentrations were added into human coagulation Ⅸ products or human prothrombin complex (PCC) to prepare heparin or heparin/ATⅢ samples, containing 0, 0.1, 0.3, 0.5, 0.8, 1, 2 and 4 IU per unit. ATⅢ with different concentrations were added into FⅨ or PCC to prepare ATⅢ samples containing ATⅢ 0, 0.1, 0.5 and 1 IU per unit. The FⅨ activity of the samples prepared was tested by one-stage coagulation method. Then corresponding amount of protamine sulfate were added to neutralize heparin or heparin/ATⅢ to detect the FⅨ activity again. Their influence of heparin, ATⅢ and heparin/ATⅢ with different concentrations on the activity of FⅨ were analyzed. 【Results】 When the content of heparin or heparin/ATⅢ was 0, 0.1, 0.3 and 0.5 IU per unit of FⅨ, the detection results of FⅨ titer in samples were consistent. When the content of heparin or heparin/ATⅢ per unit of FⅨ was 0.8, 1, 2 and 4 IU, the detection results of FⅨ titer were all lower than those of samples without heparin. When the ATⅢ content was 0, 0.1, 0.5 and 1 IU, the FⅨ titer of the samples was consistent. 【Conclution】 When the content of heparin or heparin/ATⅢ in the product is less than or equal to 0.5 IU per IU of FⅨ, the step of protamine sulfate adding could be omitted as it has little effect on FⅨ activity. When >0.5 IU per IU of FⅨ, however, protamine sulfate adding, to neutralize heparin, is necessary before FⅨ activity testing.

Hemofilia B o enfermedad de Christmas / Hemophilia B or Christmas disease

La hemofilia B o enfermedad de Christmas se diferenció por primera vez de la hemofilia A en 1947. Su forma clásica consiste en un trastorno hereditario de la coagulación causado por mutaciones en el gen F9, que codifica para el factor IX de la coagulación. Su herencia está ligada al cromosoma X; las mujeres son portadoras, pero se manifiesta clínicamente en hombres, aunque se han descrito casos de mujeres portadoras sintomáticas. El factor IX activado es una proteína dependiente de vitamina K, sintetizada en el hígado, que forma parte del complejo tenasa, cuya función es formar la mayor cantidad de trombina en el nuevo modelo de la coagulación basado en células. De acuerdo a la actividad del factor IX, su deficiencia se puede clasificar en leve (5% a 40%), moderada (1% a 5%), o severa (<1%). Su diagnóstico se realiza con la presencia de un TPT alargado que corrige con plasma normal y con la determinación del nivel funcional del factor IX, y se confirma con el estudio molecular que demuestra la mutación en el gen F9. Su diagnóstico diferencial incluye otras patologías como la hemofilia A. El tratamiento con factor IX recombinante es el más utilizado en la actualidad, pero se vienen desarrollando nuevas terapias con virus adeno-asociados recombinantes que prometen mejorar la calidad de vida para algunos pacientes afectados. La profilaxis juega un papel fundamental, en particular en los casos de enfermedad moderada y severa.

Hemophilia B or Christmas disease was first differentiated from hemophilia A in 1947. Its classic form consists of an inherited bleeding disorder caused by mutations in the F9 gene, which codes for coagulation factor IX. Its inheritance is linked to the X chromosome; women are carriers, but it manifests clinically in men, although cases of symptomatic women carriers have been described. Factor IX activates a vitamin K-dependent protein, synthesized in the liver, which is part of the tenase complex whose function is to form the largest amount of thrombin (factor IIa) in the new model of cell-based coagulation. According to factor IX activity, its deficiency can be classified as mild (5% to 40%), moderate (1% to 5%), and severe (<1%). The diagnosis is made when there is a prolonged TPT that corrects with normal plasma, and by assessing the functional level of factor IX. The diagnosis is confirmed by molecular analysis that demonstrates the F9 gene mutation. Its differential diagnosis includes disorders such as hemophilia A. Treatment with recombinant factor IX is widely used, but also new therapies are being developed with recombinant adeno-associated viruses that promise to improve the quality of life for some of these patients. Prophylaxis plays an important role in cases of moderate and severe disease

A Case of Hemophilia B Diagnosed after Rhinosinus Surgery in 37 Year-Old Patient / 대한이비인후과학회지

Hemophilia is an X-linked recessive disorder, which is classified into hemophilia A, defined by factor VIII deficiency and hemophilia B, defined by factor IX deficiency. The usual clinical presentation is spontaneous bleeding and prolonged activated partial thromboplastin time in a person without history of a coagulation disorder. The severity of hemophilia describes how serious a problem is and has been defined by a traditional classification into three forms: severe, moderate, mild. Hemophilia has never been reported after a rhinosinus surgery in otorhinolaryngology in Korea, but we encountered a 37-year-old man with hemophilia B who had undergone a rhinosinus surgery. He had no bleeding tendency in the past nor a family history for bleeding. But the patient presented with continuous nasal bleeding for a few days after surgery. We report this case of hemophilia B diagnosed after rhinosinus surgery that was cured with Factor IX replacement therapy with a review of the relevant literature.

Características clínicas y epidemiológicas de pacientes con hemofilia del eje cafetero, Colombia / Clinical and epidemiological characteristics of patients with Haemophilia from coffee region, Colombia

INTRODUCCIÓN: La hemofilia es un trastorno hemorrágico hereditario, ligado al cromosoma X, con una prevalencia estimada de 8,4 casos de hemofilia A y 2,7 casos de hemofilia B por cada 100 000 hombres. OBJETIVO: Describir características sociodemográficas, clínicas y percepción del efecto de la patología en el paciente. MATERIALES Y MÉTODOS: Se realizó un estudio transversal, en pacientes con diagnóstico de hemofilia. La fuente de información fueron las historias clínicas, además de una encuesta aplicada para actualizar la caracterización clínica y epidemiológica, el análisis estadístico fue realizado con el software Stata versión 14®. RESULTADOS: 48 pacientes fueron incluidos en el estudio, el 97,9% presenta hemofilia tipo A, de los cuales el 12,5%, 43,8% y 41,7% son leve, moderada y severa respectivamente. El 35% de los pacientes con hemofilia severa desarrollaron anticuerpos contra el factor VIII. El 66% de los pacientes ha presentado alguna manifestación de la patología en los últimos seis meses y el 61,7% refieren que ha tenido un impacto negativo en algún aspecto de su vida. CONCLUSIONES: Los factores asociados a un requerimiento mayor de dosis del factor para el control de la patología fueron antecedente de sangrado, mayor edad y el desarrollo de inhibidores. Los pacientes con hemofilia severa presentaron desarrollo de inhibidores en un 30%, frente a la moderada y leve con un 3 y 0,3% respectivamente.

INTRODUCTION: Hemophilia is an inherited bleeding disorder, linked to the X chromosome, with an estimated prevalence of 8,4 cases of hemophilia A and 2.7 cases of hemophilia B per 100 000 men. OBJETIVE: To describe sociodemographic, clinical characteristics and perception of the effect of the pathology in the patient. MATERIAL AND METHODS: A descriptive cross-sectional study was conducted with patients diagnosed with hemophilia. The required information was extracted from each patient's medical history, additionally, each patient completed a questionnaire to update the clinical and epidemiological information. The analysis was realized in Stata version 14®. RESULTS: From the 48 patients included in the study, 97,9% have type A hemophilia, of whom 12,5%, 43,8% and 41,7% are mild, moderate and severe respectively. 35% of patients with severe hemophilia have developed antibodies against factor VIII. 66% of patients presented some manifestation of the disease in the last six months and 61,7% had a negative impact on some aspect of their life. CONCLUSION: Factors associated with a higher dose requirement of factor for pathology control were antecedent bleeding, increased age and inhibitor development. Patients with severe hemophilia had development inhibitors in 30%, compared to moderate and mild with 3 and 0.3%, respectively.

Human coagulation factor VIII domain-specific recombinant polypeptide expression

BACKGROUND: Hemophilia A is caused by heterogeneous mutations in F8. Coagulation factor VIII (FVIII), the product of F8, is composed of multiple domains designated A1-A2-B-A3-C1-C2. FVIII is known to interact with diverse proteins, and this characteristic may be important for hemostasis. However, little is known about domain-specific functions or their specific binding partners. METHODS: To determine F8 domain-specific functions during blood coagulation, the FVIII domains A1, A2, A3, and C were cloned from Hep3B hepatocytes. Domain-specific recombinant polypeptides were glutathione S-transferase (GST)- or polyhistidine (His)-tagged, over-expressed in bacteria, and purified by specific affinity chromatography. RESULTS: Recombinant polypeptides of predicted sizes were obtained. The GST-tagged A2 polypeptide interacted with coagulation factor IX, which is known to bind the A2 domain of activated FVIII. CONCLUSION: Recombinant, domain-specific polypeptides are useful tools to study the domain-specific functions of FVIII during the coagulation process, and they may be used for production of domain-specific antibodies.

Recent trends and advances in hemophilia – its management and new therapeutic outcomes.

Hemophilia unfortunately a less attracted disease for researchers compared to other life threatening diseases. The prevalence of hemophilia is estimated to be about 1:10,000 birth and that of the severe form of the disease to be about 6% per 1,00,000 population. The most pathetic part of this disease is that even medical personnel are sometimes not familiar with its diagnosis and management. There is obviously a need to establish facilities and treatment options that will help the patient with hemophilia to manage their life with ease. As this is a genetic disorder no complete cure is possible as of now. The only available treatment option is the infusion of factors and some adjuvant therapies depending upon the bleeding conditions .The initiative for the development of new dosage forms, new delivery systems of the existing therapies or new treatment options has to be driven by pharmacy professionals. This article present an overview of hemophilia, in order to drag the attention of medical as well as pharmacy professionals for the benefit of millions of hemophilic patients.

Importancia del diagnóstico de portadoras en familias con antecedentes de hemofilia / Importance of the diagnosis of carriers in families with history of hemophilia

La hemofilia es una enfermedad hemorrágica hereditaria ligada al cromosoma X que se presenta debido a mutaciones en los genes del factor VIII (hemofilia A) y el factor IX (hemofilia B), que ocasiona una disminución o deficiencia funcional de estas proteínas en plasma. Sus frecuencias son de 1 en 5 000 y 1 en 30 000 varones recién nacidos vivos, respectivamente. Afecta casi exclusivamente a los varones y las mujeres portadoras presentan un riesgo del 50 por ciento de transmitir la enfermedad a sus hijos, por lo que es importante en las familias con antecedentes de hemofilia identificar las portadoras a través de las consultas de asesoramiento genético, en el cual se brinda información acerca de la enfermedad, la confección del árbol genealógico, el cálculo del riesgo de recurrencia, el diagnóstico molecular y la posibilidad de diagnóstico prenatal en gestantes portadoras. Es imprescindible que el asesoramiento genético constituya un proceso educativo e informativo, nunca impositivo

Hemophilia is a hereditary bleeding disorderX-linked that a rises dueto mutations in the genes offactor VIII(hemophiliaA)andfactor IX(hemophiliaB), which causes a decrease orfunctional deficiency of these proteins inplasma.Their frequencies are 1 in5 000 males and1 in30 000 live births, respectively. It affects males almost exclusively, and female carriers have a50 percent risk of transmitting the disease to their children. So, it is important in families with history of hemophilia that carriers are identified through genetic counseling, which provides information about the disease, making pedigree,calculation of the risk of recurrence, molecular diagnosis and possibility of prenatal diagnosis in pregnant carriers.It is imperative that genetic counselingconstitutes an educational and informative process,never as an imposition

Prevalence, Incidence, and Factor Concentrate Usage Trends of Hemophiliacs in Taiwan

PURPOSE: Hemophilia A and B (HA, HB) are the most common X-linked inherited bleeding disorders. The introduction of factor concentrates has allowed for control of the lifelong chronic disease. However, no studies have been published regarding the epidemiology of hemophilia in Taiwan. Our aim was to determine the prevalence, incidence, and mortality rate, as well as trends in the use of factor concentrates, in individuals with hemophilia in Taiwan. MATERIALS AND METHODS: A retrospective study was conducted using the National Health Insurance Research Database between 1997 and 2007. RESULTS: We identified 988 males with hemophilia (HA : HB ratio=5.4 : 1). The mean prevalence per 100000 males was 6.7+/-0.1 for HA and 1.2+/-0.1 for HB. The estimated mean annual incidence per live male birth was 1 in 10752 for HA and 1 in 47619 for HB. Standardized mortality ratios for males with hemophilia (all severities) or severe hemophilia were 1.3- and 2.1-fold higher than that of the general male population, respectively. Mean factor VIII (FVIII) and factor IX (FIX) usage was 1.5003+/-0.4029 and 0.3126+/-0.0904 international units (IUs) per capita, respectively. Mean FVIII and FIX usage per patient with hemophilia (all severities) or severe hemophilia was 44027+/-11532 and 72341+/-17298, respectively, and 49407+/-13015 and 74369+/-18411 IUs per person with HA or HB, respectively. CONCLUSION: Our data revealed epidemiologic and factor concentrate usage trends in males with hemophilia in Taiwan, highlighting a need for improvements in the mandatory National Health Insurance registry. A better-designed, patient-centered registry system would enable more detailed patient information collection and analysis, improving subsequent care.

Hemofilia II. Aspectos moleculares y de genética poblacional / Hemophilia II. Molecular and population genetics

La prevalencia de la hemofilia esporádica fue estimada hace más de 40 años y se demostró que aproximadamente un tercio de los casos son de novo. La mayoría de las mutaciones que ocurren en la hemofilia se producen durante la espermatogénesis masculina; en otros casos, los cambios ocurren en los estadios tempranos de desarrollo del embrión o una mutación germinal en la madre. El proceso de inactivación del cromosoma X es al azar. Extensos estudios han evidenciado que son más frecuentes las mutaciones en las meiosis masculinas que en las femeninas, con una proporción global de 3,5/1, especialmente las inversiones de los intrones 22 y 1. Se revisaron aspectos moleculares y bioquímicos de los factores VIII y IX. Destacamos la importancia del dominio B del factor VIII que contribuye a múltiples funciones esenciales, como el control de la calidad de la síntesis, la secreción, la unión con los fosfolípidos plaquetarios, la inactivación y el aclaramiento de la molécula completa

The sporadic hemophilia prevalence was estimated more than 40 years ago and it was shown that approximately a third of the cases are novo. Most of the mutations that occur in hemophilia are produced during the male spermatogenesis; in other cases, they occur in early stages of the embrión development or in the mother a germinal mutation. The X-cromosoma- inactivation process is at random. Extended studies have shown that male meiosis are more frequent than female ones, with a global proportion of 3,5/1, specially introns inversions 22 and 1. There were revised molecular and biochemical aspects of factors VIII and IX. We ruled out the importance of B domain in factor VIII, which contributes to multiple essential functions, as the quality control of synthesis, secretion, union with platelet phospholipids, inactivation and complete clearance of the molecule

Factor IX mutations in Haemophilia B patients in Malaysia: apreliminary study

Haemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. Identifi cation of mutations contributing to defective factor IX may be advantageous for precise carrier and prenatal diagnosis. We studied 16 patients from 11 families, consisting of 8 patients of the Malay ethnic group, of which 6 were siblings. Factor IX mutations have not been previously reported in the Malay ethnic group. The functional region of the factor IX gene was sequenced and mutations were identifi ed in either the exon or intronic regions in 15 of the patients. One novel mutation, 6660_6664delTTCTT was identifi ed in siblings with moderate form of haemophilia B. Mutations identifi ed in our patients when linked with disease severity were similar to fi ndings in other populations. In summary, this preliminary data will be used to build a Malaysian mutation database which would facilitate genetic counseling.

A Case of Heel Reconstruction with a Reverse Sural Artery Flap in a Hemophilia B Patient

Hemophilia B is a rare blood coagulation disorder. Complications such as bleeding and hematoma can cause necrosis of flaps, wound disruption, and the disturbance of wound healing. In particular, guidelines for flap operations in hemophilia B patients have still not been defined, and case reports are rare. We reconstructed the heel of a 41-year-old male hemophilia B patient using a reverse sural artery flap operation. The patient presented with mild hemophilia, having 27% of the normal value of coagulation factor IX. Coagulation and the changing value of the coagulation factor were regularly measured, and 70% of the normal value of coagulation factor IX was maintained through the injection of recombinant coagulation factors and antihemorrhagics. Hematoma developed twice (postoperative day [POD] 5 and POD 7) and in each case the hematoma was removed. Injections of recombinant coagulation factors and antihemorrhagics were continuously administered until postoperative week 2. When the coagulation factors were within normal ranges. In this article, a hemophilia B patient underwent reverse sural artery flap surgery and the healing progress was analyzed. We conclude that higher than baseline levels of coagulation factors are needed for successful healing in reverse sural artery flap surgery.

Síndrome de anticuerpos antifosfolípidos asociado a la presencia de inhibidor adquirido del factor IX en una paciente sin hemofilia / Antiphospholipid antibody syndrome associated with acquired factor IX inhibitor in a non-hemophilic patient

Se presenta el caso de una paciente de 23 años de edad, con diagnóstico de síndrome de anticuerpos antifosfolípidos, con un cuadro típico de trombosis venosa profunda recurrente y pérdidas fetales, en presencia de anticoagulante lúpico y anticuerpos anticardiolipina positivos. En el transcurso de su enfermedad presenta manifestaciones hemorrágicas, no explicadas por el uso de anticoagulantes orales. Los estudios de hematología permitieron documentar una disminución de los factores IX y VIII, con evidencia de inhibidor específico del factor IX. La deficiencia de factores de la coagulación mejoró gracias al uso de esteroides y ciclofosfamida. (Acta Med Colomb 2011; 36: 196-199).

We report the case of a 23-year-old female patient with an antiphospholipid syndrome characterized by recurrent deep venous thromboses and fetal losses, with positive lupus anticoagulant and anticardiolipin antibodies. During the course of the disease she had hemorrhagic manifestations not attributable to oral anticoagulants. Hematological analyses documented reduced levels of factors IX and VIII, with evidence of a specific inhibitor of factor IX. The deficiency of clotting factors improved with steroids and cyclophosphamide. (Acta Med Colomb 2011; 36: 196-199).

Estudio familiar de las hemofilias A y B: 5 años de experiencia en la detección de portadoras / Family study of hemophilia A and B: 5 years of experience in carrier detection

La hemofilia se caracteriza por ser una enfermedad congénita del trastorno de la coagulación y constituye un desorden recesivo ligado al cromosoma X. El estudio molecular se realiza por estudios indirectos por ser causada por mutaciones heterogéneas en los genes del FVIII y FIX. Se realizó el estudio de 40 familias afectadas con hemofilia A (HA) y 10 hemofilia B (HB). La extracción de ADN se realizó por el método de precipitación salina a 293 muestras de sangre y 19 de líquido amniótico, y se hizo el análisis de los polimorfismos St14, Bcl I y Hind III para la HA y Taq I, Xmn I y Dde I para la HB. Se usó la técnica de PCR. En el caso de la HA se obtuvo el 35 por ciento de informatividad para St14 y Hind III y 32,5 para Bcl 1. El polimorfismo Dde I fue el más informativo para la HB con el 33 por ciento; mientras que Taq I representó el 10 por ciento de informatividad y XmnI el 0 por ciento. Se comprobó que de las 40 familias analizadas con HA, 23 fueron informativas. Por otra parte, fueron informativas 4 familias de las afectadas con HB. Se realizaron 19 diagnósticos prenatales con previa determinación del sexo fetal, incluidos 3 varones enfermos

Hemophilia is a congenital disease of coagulation disorder and it is a recessive disorder linked to X-chromosome. The molecular study is conducted by indirect studies due to it is caused by heterogeneous mutations in gen of FVIII and FIX in 40 families with hemophilia A (HA) and 10 with hemophilia B (HB). DNA extraction was carried out by saline precipitation method in 293 blood samples and 19 samples of amniotic fluid, as well as the analysis of St14, Bcl I and Hind III polymorphism for the AH and Taq I, Xmn I and Dde I for BH. The PCR technique was used. In the caser of AH it was possible to achieve a 35 percent of information for St14 and Hind III and a 32.5 percent for Bcl. Dde polymorphism supplied more information for BH for a 33 percent; whereas the Taq I represented the 10 percent of information and Xmn I the 0 percent. We verified that from the families analyzed with HA, in 23 of them we there was information. Besides, in 4 families affected by HB there was information. A total of 19 prenatal diagnoses were made with a previous determination of fetus sex, including 3 males ill

Gene Therapy in Rats with a Lentiviral Vector Containing the Human Coagulation Factor IX Gene / 대한수혈학회지

BACKGROUND: Hemophilia B is an inheritable X-linked bleeding disorder that occurs as a consequence of genetic alterations within the factor IX (IX) gene. In the present study, pseudotyped HIV-I-derived lentiviral vectors expressing human IX (lentivirus-IX) were assessed for the ability to produce an active human IX in the animals transduced with lentivirus-IX. METHODS: The IX concentrations and activated partial thromboplastin times (aPTT) were measured from the supernatants of HeLa cells that were transduced with lentivirus-IX. In an animal study, we injected 1microgram of lentivirus-IX into the hind limbs of Sparague-Dawley (SD) rats. The IX concentrations were measured from the plasma of the vehicle injected rats and the plasma of the lentivirus-IX injected rats for 8 weeks. RESULTS: The in vitro expression of human IX was detected in a dose-dependent manner following the transduction of lentivirus-IX into the HeLa cells (control: 10+/-3 vs. 100 ng of lentivirus-IX: 1486+/-50 ng/mL, P<0.05). The aPTT also showed the tendency of dose-dependent decrease (control: 83.9+/-0.5 vs. 50 ng of lentivirus-IX: 80.1+/- 0.8 sec), but this was not statistically significant. In the animal experiment, the plasma IX concentration from the lentivirus-IX transduced rats (n=3) was significantly increased compared to the vehicle-injected rats (n=4) (5.9+/-3.9 vs. 46.4+/-20.6 ng/mL) at post-injection 1 week. CONCLUSION: This study demonstrated that in vivo delivery of lentiviral vectors expressing human IX to the muscle cells has the potential to be a therapeutic modality for hemophilia B.

Purificación del factor IX recombinante expresado en leche de cerdas transgénicas / Purification of recombinant factor IX expressed in milk of transgenic sows

Se purificó a partir de la leche de cerdas transgénicas, factor IX recombinante, y se obtuvieron rendimientos entre 1 a 2 g de esta proteína por litro, lo que resulta una nueva vía para la obtención de este producto con una alta eficiencia, ya que su expresión es 1 000 veces superior a la del factor IX plasmático humano. Mediante la combinación de 2 pasos cromatográficos: intercambio iónico en DEAE-Shephadex A-50 y cromatografía de afinidad con heparina, se realizó la purificación del factor IX, con esta leche como material de partida. Se estudiaron diferentes métodos para la eliminación de las caseínas, contaminante principal del proceso, y se escogió finalmente la ultracentrifugación, por las numerosas ventajas que presenta con respecto a la precipitación isoeléctrica y la precipitación por sales. El factor IX puede ser purificado de la leche transgénica con una alta pureza utilizando métodos cromatográficos que no usan inmunoafinidad y son finalmente escalables en la producción industrial, lo cual proporciona nuevas perspectivas para el tratamiento de la hemofilia B mediante la preparación de posibles formulaciones orales.

Recombinant factor IX was purified from milk of transgenic sows, and yieldings between 1 and 2 g of this protein per liter were obtained. This is a new way to get this product with a high efficiency, since its expression is 1 000 times higher than of the human plasmatic factor IX. By combining 2 chromatographic steps (ion exchange in DEAE-Shephadex A-50 and affinity chromatography with heparin), the factor IX was purified, with this milk as a starting material. Different methods were studied to eliminate caseins, the main pollutant of the process, and ultracentrifugation was selected due to its numerous advantages over the isoelectric precipitation and salt precipitation. Factor IX may be purified from transgenic milk with an elevated purity by chromatographic methods that do not use immunoaffinity and are finally scalable in industrial production, which provides new perspectives for treating hemophilia B by preparing new oral formulations.

Molecular diagnosis of hemophilia A and B. Report of five families from Costa Rica

Hemophilia A and B are X-chromosome linked bleeding disorders caused by deficiency of the respective coagulation factor VIII and IX. Affected individuals develop a variable phenotype of hemorrhage caused by a broad range of mutations within the Factor VIII or Factor IX gene. Here, were report the results of the molecular diagnosis in a five Costa Rican families affected with Hemophilia. Methods of indirect and direct molecular diagnosis are applied in three Hemophilia A and two Hemophilia B families from Costa Rica as well as preconditions, practicability and facilities of this diagnosis. In two families with Hemophilia A and both families with Hemophilia B the causative mutation could be detected by Southern blotting, polymerase chain reaction or sequence analysis. One Hemophilia A family could only analyzed by linkage analysis using genomic markers.

Anesthetic Management of a Patient with Hemophilia B / 대한마취과학회지

A 22 year old male with hemophilia B was scheduled for the correction of a right subtrochanteric femur fracture. Plasma concentration of coagulation factor IX in this patient was 50% of the normal level and the partial thromboplastin time was 57 seconds on the first day of hospital administration. We started the intravenous administration of facnyne injection (factor IX) 11 days before the operation when his plasma concentration was 20%. His plasma concentration was 40% on the operation day. The operation and general anesthesia were accomplished safely without severe hemorrhage or major complication. He received facnyne injection continuously until the 5th postoperative day: 25-43% of his plasma concentrations of factor IX was maintained. He had no problem with postoperative care.

Sensibilidad del tiempo parcial de tromboplastina activado a la deficiencia de factores VIII y IX Y a la heparina / Sensitivity of the activated partial thromboplastin time to the deficiency of factors VIII and IX and heparin

Se reevaluó el rango de referencia del tiempo parcial de tromboplastina activada (TPTA). Se obtuvo un rango de referencia de 29 a 40 seg. con una media (X) de 34,01 seg. Con nuestro sistema de reactivos se alcanzaron resultados aceptables en cuanto a la sensibilidad a diferentes niveles de actividad de los factores VIII y IX, se logró una alta correlación en ambos casos (r2 = 0,9842 y 0,9846, respectivamente) cuando se relacionó el TPTa con los diferentes niveles de actividad de los factores VIII y IX. Se obtuvo una respuesta lineal en el intervalo de actividad de heparina utilizado normalmente en la terapia (0,2 a 0,5 UI/mL) y se alcanó una buena respuesta a altas concentraciones de heparina (0,8 UI/mL), expresada por un valor finito del TPTa. Se corroboró la necesidad de alargar el tiempo de incubación con el activador a 10 min para mejorar la sensibilidad del ensayo a la heparina.

The reference range of the activated partial thromboplastin time (APTT) was reevaluated. A reference range from 29 to 40 seconds with a mean (X) of 34.01 sec. was obtained. By using our system of reagents, acceptable results were obtained as regards the sensitivity to different levels of activity of factors VIII and IX. A high correlation was attained in both cases (r2 = 0.9842 and 0.9846, respectively) when the APTT was associated with the different levels of activity of factors VIII and IX. A linear response was obtained in the interval of heparin activity commonly used in therapy (0.2 to 0.5 UI/mL) and a good response was attained at high concentrations of heparin (0.8 UI/mL) expressed by a finite value of APTT. The need to extend the incubation time with the activator to 10 min. to improve the sensitivity of the test to heparin was corroborated.

RFLP Analysis of a Gene for Blood Coagulation Factor IX in Korean / 대한산부인과학회잡지

OBJECTIVE: The purpose of this study was to investigate the methods for analysis of restriction fragment length polymorphisms of hemophilia B (coagulation factorIX) gene in Korean population. METHODS: Genomic DNAs were extracted from 40 Korean females. In order to amplify genomic DNAs at the region of the polymorphic sites, two sets of primers (Hha I and Dde I) were synthesized. The primers were named as FIX1, FIX2 for Hha I, and Dde I 59, Dde I 39 for Dde I, respectively. Hha I primers annealed 3'-flanking region of the FactorIX gene and amplified 230 bp long fragment. The PCR fragment (230 bp) treated with Hha I endonuclease produced two fragments (150 bp and 80 bp), when the polymorphic site existed. Dde I primers annealed the region of the first intron of Factor IX gene and amplified 319 bp long fragments. People cases with Dde I polymorphic site are supposed to produce 369 bp long fragment. Results: It has been found that seven (14 X chromosomes) out of forty individuals showed Hha I polymorphism. However, none of the experimental People cases showed the Dde I polymorphism. CONCLUSIONS: By the analysis of 80 chromosomes, the PICs calculated from allele frequency of Hha I-RFLP (0.175/0.825) and that of Dde I-RFLP (0.0/1.0) were 0.289=[1-(0.1752+0.8252)] and 0=[1-(02+12)], respectively. From these results, it can be postulated that Hha I and Dde I polymorphisms of the Factor IX gene in Korean exhibited different patterns from those of Caucasian.