Genotipificación del gen RHD en ADN fetal libre en plasma de embarazadas RhD negativo / Genotyping of the RHD gene in free fetal DNA in plasma of RhD negative pregnant women

Objetivo: Determinar el grupo RhD fetal a través del estudio del gen RHD en ADN fetal que se encuentra libre en plasma de embarazadas RhD negativo. Método: Se analizó la presencia de los genes RHD, SRY y BGLO en ADNfl obtenido de plasma de 51 embarazadas RhD negativo no sensibilizadas, utilizando una qPCR. Los resultados del estudio genético del gen RHD se compararon con el estudio del grupo sanguíneo RhD realizado por método serológico en muestras de sangre de cordón, y los resultados del estudio del gen SRY fueron cotejados con el sexo fetal determinado por ecografía. Se calcularon la sensibilidad, la especificidad, los valores predictivos y la capacidad discriminativa del método estandarizado. Resultados: El gen RHD estaba presente en el 72,5% de las muestras y el gen SRY en el 55,5%, coincidiendo en un 100% con los resultados del grupo RhD detectado en sangre de cordón y con el sexo fetal confirmado por ecografía, respectivamente. Conclusiones: Fue posible deducir el grupo sanguíneo RhD del feto mediante el estudio del ADN fetal que se encuentra libre en el plasma de embarazadas con un método molecular no invasivo desarrollado y validado para este fin. Este test no invasivo puede ser utilizado para tomar la decisión de administrar inmunoglobulina anti-D solo a embarazadas RhD negativo que portan un feto RhD positivo.

Objective: To determine the fetal RhD group through the study of the RHD gene in fetal DNA found free in plasma of RhD negative pregnant women. Method: The presence of the RHD, SRY and BGLO genes in fetal DNA obtained from plasma of 51 non-sensitized RhD negative pregnant women was analyzed using qPCR. The results of the genetic study of the RHD gene were compared with the RhD blood group study performed by serological method in cord blood samples, and the results of the SRY gene study were compared with the fetal sex determined by ultrasound. Sensitivity, specificity, predictive values and discriminative capacity of the standardized method were calculated. Results: The RHD gene was present in 72.5% of the samples and the SRY gene in 55.5%, coinciding 100% with the results of the RhD group detected in cord blood, and with the fetal sex confirmed by ultrasound, respectively. Conclusions: It was possible to deduce the RhD blood group of the fetus through the study of fetal DNA found free in the plasma of pregnant women with a non-invasive molecular method developed and validated for this purpose. This non-invasive test can be used to make the decision to administer anti-D immunoglobulin only to RhD-negative pregnant women carrying an RhD-positive fetus.

Uso posnatal de altas dosis de gamma globulina EV en el tratamiento de la enfermedad hemolítica RH / Postnatal use of high doses of EV gammaglobulin in the treatment of hemolytic disease RH

El uso posnatal de altas dosis de inmunoglobulina endovenosa (IgGEV) aparece promisorio, en los escasos trabajos publicados, en reducir la necesidad de exsanguinotransfusiones (EXT) en el recién nacido (RN) con enfermedad hemolítica RH (EHRH). Objetivo: Evaluar la eficacia de la IgGEV para moderar la severidad de la hemólisis: necesidad de EXT, tiempo de luminoterapia (LMT) y número de transfusiones (T) en el RN con EHRH. Diseño: Ensayo clínico controlado y randomizado. Lugar de estudio: Hospital Materno Infantil “Ramón Sardá”. Población y métodos: Todos los RN con EHRH [Coombs directa (+)], fueron estratificados en 3 grados (leve, moderado y severo) según la severidad de la hemólisis evaluada intraútero. Solicitado el consentimiento informado a los padres, los RN de cada grado se randomizaron en: Grupo tratado: recibió IgGEV (500mg/kg/día por tres días, la primera dosis antes de las 2 horas posnatales) más LMT, y grupo control: solo LMT. Las variables estudiadas fueron: mínimo hematocrito (Hto), bilirrubina (Bi) máxima, n° de EXT y/o T, LMT (hs), internación (días) y tiempo hasta la EXT. Resultados: Entre febrero/99 y mayo/00 fueron enrolados 46 RN (grado leve: 24 y grado moderado-severo (gM-S): 22). 23 RN pertenecieron al grupo tratado y 23 al control. Ambos grupos fueron comparables en sexo, peso, EG y Hto, Bi, Hb y prot. totales de cordón. No se observaron diferencias significativas excepto en n° de EXT. De los 23 RN tratados, 5 requirieron EXT (21.74%) cuando su bilirrubina alcanzó el nivel establecido en las guías del hospital; 18 de los 23 RN controles requirieron EXT (78,26%), Chi2 0.0011. El número necesario a tratar (NNT) fue 1,8 para prevenir una EXT. La probabilidad de sufrir EXT es significativamente menor en un paciente tratado que en un control, en cualquier momento del período de estudio (Log Rank test p= 0,0024). Conclusiones: Estos resultados apoyan el beneficio clínico de tratar con IgGEV a los RN con incompatibilidad RH.

Un caso de grupo sanguíneo raro: fenotipo p / A rare blood group: p phenotype

Un individuo con un fenotipo eritrocitario raro carece de uno o varios antígenos presentes en la mayor parte de la población de pertenencia. Cuando presenta el anticuerpo correspondiente, se pueden producir complicaciones perinatales, transfusionales y/o transplantológicas. Se presenta el caso de una embarazada aloinmunizada derivada a nuestro servicio en la semana 12 de su tercera gesta para su evaluación y seguimiento. El diagnóstico inmunohematológico le asignó el excepcional fenotipo "p" (aproximadamente 1/200 000 individuos), asociado con una mayor tasa de abortos espontáneos y a reacciones transfusionales graves cuando se transfunden unidades incompatibles. El estudio del gen A4GALT demostró la presencia de la mutación c.752C > T en doble dosis. Esta mutación lleva a un cambio de una prolina por una leucina en el residuo 251 de la 4-α-galactosiltransferasa. Por parto inducido por sufrimiento fetal, nace a las 36 semanas una bebé con prueba de antiglobulina (Coombs) directa negativa, eluido reactivo, con ictericia que requirió luminoterapia. Una semana después el neonato fue externado sin secuelas aparentes. Posteriormente, a raíz de una cirugía inminente y la improbabilidad de encontrar sangre compatible, se elaboró un plan para cubrir las posibles demandas. Este caso pone en evidencia la necesidad de contar a nivel nacional con un laboratorio de referencia de inmunohematología y un banco de sangre de grupos raros, que permita resolver con celeridad situaciones que requieran transfundir a estos individuos.

A rare blood group is usually defined as the absence of a high prevalence antigen or the absence of several antigens within a single blood group system. These individuals may develop clinically significant red cell antibodies to the high incidence red cell antigens they lack. A 33-year-old alloimmunized woman was referred to our center at the 12th week of her third pregnancy for evaluation and follow up. The laboratory work-up grouped her as belonging to "p" phenotype, associated with difficulties to find compatible blood for transfusion and a high incidence of recurrent miscarriage. At 36 weeks, a baby girl was born by induced labor due to fetal suffering. With a negative direct antiglobulin test but a positive elution test, she was in the neonatology ward for one week receiving luminotherapy. Homozygosity for a missense mutation at position 752 (c.752C > T) in the A4GALT gene was found to be responsible for the p phenotype. This mutation changes a proline to a leucine at codon 251 of the 4-α-galactosyltransferase. Recently, due to an imminent chirurgical intervention and the impossibility to have compatible blood available for transfusion, an autologous donation plan was designed to satisfy probable demand. This case showed the need for blood bank facilities capable to respond satisfactorily to these situations in Argentina. This would facilitate the storage of cryopreserved blood from individuals with rare blood groups for homologous use or to develop rare blood donors programs.

Enfermedad hemolítica del recién nacido por incompatibilidad Duffy: reporte de un caso / Hemolytic disease of the newborn due to Duffy incompatibility: a case report

En el feto los antígenos Duffy pueden ser detectados a las 6 o 7 semanas de gestación y están bien desarrollados al nacimiento. A pesar de su temprana expresión, la enfermedad hemolítica por incompatibilidad de grupo sanguíneo Duffy no es usual. Se presenta el caso un recién nacido con enfermedad hemolítica por incompatibilidad Duffy. Para su tratamiento se empleó fototerapia unida a un procedimiento hemoterapéutico: la exanguinotransfusión. Aunque la incompatibilidad por este sistema de grupo sanguíneo suele ser moderada, se debe estar alerta ante la ocurrencia de un conflicto con curso inusual, para brindar un tratamiento óptimo en el momento adecuado y disminuir la morbilidad de esta enfermedad.

Duffy antigens may be detected in the fetus at 6 or 7 weeks of gestation and be well developed at birth. Despite its early expression, the hemolytic disease due to Duffy blood group incompatibility is rare. The case of a newborn with hemolytic disease caused by Duffy incompatibility is presented. For its treatment, it was used phototherapy combined with a hemotherapeutic procedure: exanguinotransfusion. Although the incompatibility produced by this blood group system is usually moderate, one should be alert to the occurrence of a conflict with unusual course in order to apply an optimum treatment at the right time and to reduce the morbidity of this disease.

A Fatal Case of Severe Hemolytic Disease of Newborn Associated with Anti-Jk(b)

The Kidd blood group is clinically significant since the Jk antibodies can cause acute and delayed transfusion reactions as well as hemolytic disease of newborn (HDN). In general, HDN due to anti-Jk(b) incompatibility is rare and it usually displays mild clinical symptoms with a favorable prognosis. Yet, we apparently experienced the second case of HDN due to anti-Jk(b) with severe clinical symptoms and a fatal outcome. A female patient having the AB, Rh(D)-positive boodtype was admitted for jaundice on the fourth day after birth. At the time of admission, the patient was lethargic and exhibited high pitched crying. The laboratory data indicated a hemoglobin value of 11.4 mg/dL, a reticulocyte count of 14.9% and a total bilirubin of 46.1 mg/dL, a direct bilirubin of 1.1 mg/dL and a strong positive result (+++) on the direct Coomb's test. As a result of the identification of irregular antibody from the maternal serum, anti-Jk(b) was detected, which was also found in the eluate made from infant's blood. Despite the aggressive treatment with exchange transfusion and intensive phototherapy, the patient died of intractable seizure and acute renal failure on the fourth day of admission. Therefore, pediatricians should be aware of the clinical courses of hemolytic jaundice due to anti-Jk(b), and they should be ready to treat this disease with active therapeutic interventions.

Hiperbilirrubinemia neonatal prolongada devido à associação entre síndrome de Gilbert e doença hemolítica por incompatibilidade RhD / Persistent neonatal hyperbilirubinemia resulting from Gilbert's syndrome in association with RhD hemolytic disease

OBJETIVO: Relatar associação infreqüente de patologia que cause aumento considerável de produção de bilirrubina e outra diminuição importante na sua excreção. DESCRIÇÃO: Mãe tercigesta, Rh negativo. Na primeira gestação, gerou recém-nascido normal, de termo, não tendo recebido imunoglobulina humana anti-RhD. A segunda gestação complicou-se por isoimunização Rh, dando à luz neonato de termo, o qual necessitou três exsanguinotransfusões e faleceu com 8 dias de vida. Na gestação atual, conseguiu dar à luz a termo recém-nascido tipo ORh positivo, Coombs direto positivo, bilirrubina de cordão 6,5 mg/dl e hematócrito 44 por cento. Com 5 horas de vida, estava ictérico, tendo sido iniciados fenobarbital (por 3 dias) e fototerapia intensiva. A hiperbilirrubinemia foi logo controlada, porém ascendia rapidamente sempre que a fototerapia era suspensa. No 10° dia de vida, a criança foi transfundida por anemia importante. Em vista da persistência da icterícia, no 13° dia de vida pensou-se em associação com síndrome de Gilbert, e o seqüenciamento de DNA foi solicitado. O resultado mostrou genótipo mutante homozigoto UDPT1A1[TA]7TAA. Permaneceu em fototerapia até o 17° dia de vida. Recebeu alta no dia seguinte, após controle de bilirrubinemia. Voltou para acompanhamento ambulatorial e apresentou desenvolvimentos pondo-estatural e neurológico normais. COMENTARIOS: O caso ressalta a importância da associação do aumento de produção/diminuição de excreção de bilirrubina na gênese de hiperbilirrubinemias prolongadas, intensas e passíveis de causar kernicterus, se não tratadas vigorosamente. Demonstra, ainda, a eficácia da fototerapia intensiva, reduzindo os riscos de tratamentos mais agressivos. Ressalta, também, a importância do acompanhamento das icterícias neonatais até a completa remissão dos sintomas.

Hemolytic disease of the newborn due to anti-U

Anti-U is a rare red blood cell alloantibody that has been found exclusively in blacks. It can cause hemolytic disease of the newborn and hemolytic transfusion reactions. We describe the case of a female newborn presenting a strongly positive direct antiglobulin test due to an IgG antibody in cord blood. Anti-U was recovered from cord blood using acid eluate technique. Her mother presented positive screening of antibodies with anti-U identified at delivery. It was of IgG1 and IgG3 subclasses and showed a titer of 32. Monocyte monolayer assay showed moderate interaction of Fc receptors with maternal serum with a positive result (3.1 percent). The newborn was treated only with 48 hours of phototherapy for mild hemolytic disease. She recovered well and was discharged on the 4th day of life. We conclude that whenever an antibody against a high frequency erythrocyte antigen is identified in brown and black pregnant women, anti-U must be investigated

Haemolytic disease of newborn.

Hemolytic disease of the newborn (HDN) occurs due to maternal IgG antibodies crossing the placenta thereby producing hemolysis mainly due to Rh, ABO and Kell groups. A systematic approach to the Rh HDN involves an obstetric history of previous isoimmunized baby, timing and regular monitoring of maternal Rh antibodies and pigment assay of amniotic fluid. Timely decision regarding in utero transfusion and early termination of pregnancy based on the maternal monitoring has radically improved the outcome of these babies. Antenatal prophylaxis with anti D has resulted in great reduction in the magnitude of Rh problem. The fetal blood sampling and in-utero intravenous transfusions has made it possible for almost 100% survival of isoimmunized pregnancies without hydrops. Alternative methods--IVIG and plasma exchange are still of limited application. ABO HDN though common is not a serious form of disease and dose not warrants invasive antenatal monitoring. Anti-Kell is found in patients having received multiple transfusions and the rapid progress of hemolysis in them may not allow such systematic follow up as in Rh HDN.

An unusual case of ABO-haemolytic disease of the newborn.

Haemolytic disease of the newborn (HDN) is a clinical condition in which foetal red blood cells are destroyed by maternal alloantibodies directed against red cells antigens acquired from the father. These antibodies usually belong to the Rhesus (Rh) or ABO blood group systems. ABO-HDN is usually a sub-clinical condition and less severe than Rh-HDN. The placenta is relatively impermeable to naturally occurring IgM anti-A/anti-B antibodies. However, immune anti-A and anti-B of the IgG type will cross the placenta and may thus cause ABO-HDN. ABO-HDN is the commonest in Group O mothers having A infants. Occasionally it is seen in Group O or A (A2) mothers of Group B infants. The most severe disease is seen in immune anti-B rather than anti-A antibodies. There are at present no satisfactory methods to predict ABO-HDN in the antepartum period. We report here a case of ABO-HDN where mother who was B, Rh-positive; delivered a baby girl of A1B, Rh-positive who developed severe haemolytic disease. The baby was the third child. To our knowledge this is the first of this kind of severe ABO-HDN in Bangladesh and one of the rarest ABO-HDN cases in the world.

Detection of high risk pregnancies with relation to ABO haemolytic disease of newborn.

IgM and IgG anti A and anti B antibody status of 100 antenatal O group mothers (who had non O group husbands) were studied. Of these, 3 mothers had an IgM anti A antibody levels ranging from 1:512 to 1:2048 and IgG ranging from 1:1204 to 1:2048, IgM anti B ranging from 1:128 to 1:512 and IgG anti B ranging from 1:256 to 1:512. All these mothers had A + ve children and all these children suffered from ABO haemolytic disease of newborn (HDN). A 4th mother had an IgM anti A titre of 1:64 and IgG anti A titre of 1:16. The titre of IgM anti B in this mother was 1:512 and that of IgG 1:1024. The child was A + ve and did not suffer from ABO haemolytic disease of newborn.

Anti-E as a cause of hemolytic disease of the newborn.

A case of HDN caused by anti-E antibody is reported. A group A, E-positive, hemoglobin E trait female infant was born from a group A, E-negative, beta-thalassemia/hemoglobin E mother. Hyperbilirubinemia was noted at the first day of life. The DAT was positive. Anti-E was detected in the maternal serum. Jaundice and anemia occurring to the baby were severe enough to require phototherapy intervention for 9 days and 50 ml of group A, E-negative packed red blood cells was transfused. The baby's condition improved. She was discharged at 12 days of age. Follow-up of the baby at 1 year old showed that she was alive and in good health.

Enfermedad hemolítica del recién nacido por isoinmunización por grupos sanguíneos / Hemolytic disease of the newborn by immunization by groups sanguineous

Se estudiaron 251 recién nacidos a término y sus respectivas madres en la Maternidad Concepción Palacios, Caracas-Venezuela, con el fin de establecer la frecuencia de enfermedad hemolítica (EH) y tratar de determinar parámetros útiles para la predicción de la severidad de la EH-ABO. Hubo 23 casos de incompatibilidad ABO con los siguientes hallazgos serológicos: 9 (39) por ciento presentaron la prueba de autoaglutinación positiva, 5(21 por ciento) el Coombs directo positivo, 20 (36 por ciento) el eluido positivo. De cuatro (2 por ciento) casos con EH-ABO, dos (50 por ciento) tuvieron Coombs directo positivo y tres (75 por ciento) la prueba de autoaglutinación y el eluido positivo. El título de anticuerpos maternos varió entre 1:12 y 1:4096. La determinación semicuantitativa de las subclases IgG en 3 casos de EH-ABO demostró en forma constante la presencia de IgG3 e IgG4 y en 2 casos se asoció la IgG2. Se determinó la presencia de sustancia A en el suero de 14 niños con incompatibilidad ABO, no observándose aparentemente el efecto protector del carácter secretor. Se presentaron 2 casos de EH-Rh, los cuales tuvieron un comportamiento diferente, lo cual podría atribuirse a las subclases de IgG presentes en el suero materno

Transfusao intravascular por cordocentese na terapeutica da doenca hemolitica perinatal / Intravascular intrauterine transfusion by cordocentesis in Rh alloimmunization

Os autores relatam a experiencia com 65 transfusoes intravasculares no tratamento da Doenca Hemolitica Perinatal realizada em 25 gestacoes complicadas por aloimunizacao Rh no periodo de janeiro a dezembro de 1991. As transfusoes comecaram entre 20-34 semanas e foram repetidas ate 6 vezes, com intervalos de 3-28 dias...

Concentraçäo de bilirrubinas em cordäo umbilical e sua relaçäo com a doença hemolítica pelo sistema ABO / Bilirubin levels in umbilical cord and yours relation with hemolytic disease the ABO system

Os autores estudaram a concentraçäo de bilirrubinas indireta 9BI) e total (BT) no cordäo umbilical de 64 RN de termo, AIG, distribuídos em três grupos: 35 sem incompatibilidade sanguínea materno-fetal pelo sistema ABO (IH-ABO); 21 IH-ABO e 8 com doença hemolítica por este sistem (DH-ABO). Obtiveram concentraçöes de BI e BT mais elevadas nos RN com DH-ABO, sendo esta diferença estatisticamente significativa em relaçäo aos demais grupos. Também verificaram que níveis de BI superiores a 2,0 mg/dl e BT maiores do que 4,0 mg/dl elevavam o risco de DH-ABO em 3,8 vezes, embora näo houvesse uma associaçäo entre os níveis de cordäo e os máximos atingidos nas primeiras 72 horas de vida. Concluíram que a determinaçäo das concentraçöes de bilirrubinas em cordäo umbilical podem contribuir, de forma bastante satisfatória, para o diagnóstico precoce de DH-ABO

Transfusión fetal intravascular en el manejo de la enfermedad hemolítica perinatal / Intravascular fetal transfusion in the management of perinatal hemolytic disease

Cinco fetos con enfermedad hemolítica severa por isoinmunización al factor RH fueron tratados con transfusiones intravasculares repetidas. De 16 transfusiones intentadas, 14 se completaron exitosamente. Los 5 fetos requirieron más de una transfusión. Uno de ellos falleció antes de nacer. Los otros cuatro nacieron vivos y actualmente están sin secuelas aparentes. Se discuten aspectos técnicos del procedimiento, así como sus indicaciones, ventajas y antecedentes históricas

Enfermedad hemolítica neonatal por incompatibilidad del sistema Diego (Aloinmunización por anticuerpos IgG anti Diegos) / Hemolitic neonatal disease due to Diego system incompatibility (Alloimmunization by IgG antibodies anti Diegos)

Se describe un caso de enfermedad hemolítica por incompatibilidad del sistema Diego. Se revisan las características antigénicas y étnicas de este sistema sanguíneo de distribución restirngida, llamado también sistema "familiar" o "privado", en contraste con los sistemas "comunes" o "firmemente establecidos" como el ABO y Rh. Por último, se analiza la importancia de este factor en el estudio de las migraciones de las poblaciones precolombinas