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.

Frequency and characterization of RHD variants in serologically D- Surinamese pregnant women and D- newborns.

BACKGROUND: Numerous RHD variant genes affect the expression of D on the red blood cell surface. In Suriname, 4.3% of pregnant women were D-, ranging from virtually zero to 7% among ethnic groups. Characterization of RHD variants, which are associated with a variable potential to induce anti-D, is of practical clinical importance especially in case of limited access to preventive measures. Here we report on the occurrence of RHD variant genes in Surinamese serologically D- pregnant women and their D- newborns from different ethnic groups. STUDY DESIGN AND METHODS: The RheSuN study is a cross-sectional cohort study in D- pregnant women and their newborns, who visited hospitals in Paramaribo, Suriname, during routine pregnancy care. The presence of RHD variants was investigated using quantitative polymerase chain reaction targeting RHD Exons 5 and 7 and RH-multiplex ligation-dependent probe amplification. RESULTS: Seven RHD variant genes were detected in 35 of 84 women and four RHD variant genes in 15 of 36 newborns. The RHD*03 N.01 and RHD*08 N.01 variants represented 87% of a total of 62 variant genes. Variants were comparably frequent among ethnicities. In four cases genotyping would have changed anti-D prophylaxis policy: one woman with a RHD*01EL.01 variant, not associated with anti-D formation and three D- newborns with RHD*09.01 and RHD*09.03.01 variants, potentially capable of inducing anti-D. CONCLUSION: RHD variants at risk for anti-D are common among serologic D- individuals from African descent in Suriname. While genotyping D- women has limited added value, it may be considered in newborns from D- women.

Noninvasive approach for the management of hemolytic disease of the fetus.

Hemolytic disease of the fetus and newborn (HDFN) is due to maternal alloantibodies directed against paternally inherited antigens on fetal red cells, and it is still a problem in affected pregnancies despite the routine use of anti-D immunoglobulin during pregnancy and shortly after delivery. The current noninvasive management of HDFN starts with the determination of fetal RhD genotype by use of cell-free fetal DNA in maternal plasma. When the fetus is antigen positive, the follow-up is performed by Doppler ultrasonography for the detection of moderate or severe anemia on the basis of an increase peak velocity of systolic blood in the middle cerebral artery. Finally, if anemia is suspected, an invasive approach is required in order to perform an intrauterine blood transfusion, which should only be attempted when the fetus needs transfusion. This approach reduces the iatrogenic conversion of mild-to-severe disease, which occurred as a result of the previous invasive management, and prevents unnecessary administration of human-derived blood products. These changes represent one of the genuine successes of fetal therapy.

Early detection of RhD status in pregnancies at risk of hemolytic disease of the newborn.

The aim of this work was to investigate the presence of the RHD gene in fetal cells obtained from amniotic fluid. We studied 65 samples of amniotic fluid, 11 from RhD-negative mothers sensitized with anti-D alloantibodies. The fetal origin of the DNA was confirmed with the analysis of 1 VNTR locus and 3 STR loci in DNA samples from amniotic fluid and maternal blood. The RHD genotyping was performed in non-contaminated samples (n=62) using a multiplex polymerase chain reaction strategy that yields three amplification products from RhD-positive phenotypes (intron 4 of both RHCE and RHD genes and exon 10 of the RHD gene) and I DNA fragment from RhD-negative phenotypes (intron 4 of the RHCE gene). We genotyped 54 RhD-positive fetuses (8 from RhD-negative sensitized mothers) and 8 RhD-negative fetuses (3 from RhD-negative sensitized mothers). The fetal DNA genotyping allows the diagnosis, from a single amniocentesis, of fetuses at real risk of hemolytic disease of the newborn. When the fetus is determined to be RhD-negative invasive procedures can be avoided.

Enfermedad hemolítica fetoneonatal: estudio prenatal del genotipo RHD / Feto-neonatal hemolytic disease: prenatal study of RHD genotype

El objetivo de este trabajo fue determinar la presencia del gen RHD en células fetales obtenidas de líquido amniótico por PCR. Se estudiaron 65 muestras de líquido amniótico, 11 de madres RhD negativas sensibilizadas con anti-D. Se confirmó el origen fetal del ADN analizando un locus VNTR y 3 loci STR en las muestras de ADN de líquido amniótico y sangre materna. En las muestras no contaminadas (n = 62) se realizó la genotipificación RHD utilizando una estrategia de PCR multiplex que permite la obtención de tres productos de amplificación en los fenotipos RhD positivos y sólo un fragmento de ADN en los fenotipos RhD negativos. Se genotipificaron 54 fetos RhD positivos (8 de madres RhD negativas sensibilizadas) y 8 fetos RhD negativos (3 de madres RhD negativas sensibilizadas). La genotipificación del ADN fetal permite diagnosticar con una única amniocentesis fetos en riesgo real de enfermedad hemolítica fetoneonatal y evitar la utilización de métodos invasivos en casos de fetos RhD negativos. (AU)

[Rh system genotyping in amniotic fluid]. / Genotipificación del sistema RH en líquido amniótico.

The aim of this work was to determine the presence of the RHD gene in fetal cells obtained from amniotic fluid (AF). We studied 65 samples of AF, 11 from RhD- mothers sensitized with anti-D. The fetal origin of the DNA was confirmed with the analysis of 1 VNTR locus and 3 STR loci in DNA samples from AF and maternal blood. The RHD genotyping was performed in non contaminated samples (n = 62) using a multiplex PCR strategy that yields 3 amplification products from RhD+ phenotypes and 1 DNA fragment from RhD- phenotypes. We genotyped 54 RhD+ fetuses (8 from RhD- sensitized mothers) and 8 RhD- fetuses (3 from RhD- sensitized mothers). Fetal DNA genotyping allows the diagnosis, from a single amniocentesis, of fetuses at real risk of hemolytic disease of the newborn. When the fetus is determined to be RhD- all invasive procedures can be avoided.

Genotipificación del sistema RH en líquido amniótico / Rh system genotyping in amniotic fluid

El objetivo de este trabajo fue determinar la presencia del gen RHD en células fetales obtenidas de líquido amniótico (LA) por PCR. Se estudiaron 65 muestras de LA, 11 de madres RhD-sensibilizadas con anti-D. Se confirmó el origen fetal del AND analizando un locus VNTR y 3 loci STR en las muestras de AND de LA y sangre materna. En las muestras no contaminadas (n=62) se realizó la genotipicación RHD utilizando una estrategia de PCR multiplex que permite la obtención de 3 productos de amplificación en los fenotipos RhD+ y sólo un fragmento de AND en los fenotipos RhD-. Se genotipificaron 54 fetos RhD+ ( de 8 madres RhD- sensibilizadas) y 8 feto RhD- ( 3 de madres RhD- sensibilizadas). La genotipificación del AND fetal permite diagnosticar con una única amniocentesis fetos en riesgo real de enfermedad hemolítica fetoneonatal (EHFN) y evitar la utilización de métodos invasivos en casos de fetos RhD-.

Genotipificación del sistema RH en líquido amniótico / Rh system genotyping in amniotic fluid

El objetivo de este trabajo fue determinar la presencia del gen RHD en células fetales obtenidas de líquido amniótico (LA) por PCR. Se estudiaron 65 muestras de LA, 11 de madres RhD-sensibilizadas con anti-D. Se confirmó el origen fetal del AND analizando un locus VNTR y 3 loci STR en las muestras de AND de LA y sangre materna. En las muestras no contaminadas (n=62) se realizó la genotipicación RHD utilizando una estrategia de PCR multiplex que permite la obtención de 3 productos de amplificación en los fenotipos RhD+ y sólo un fragmento de AND en los fenotipos RhD-. Se genotipificaron 54 fetos RhD+ ( de 8 madres RhD- sensibilizadas) y 8 feto RhD- ( 3 de madres RhD- sensibilizadas). La genotipificación del AND fetal permite diagnosticar con una única amniocentesis fetos en riesgo real de enfermedad hemolítica fetoneonatal (EHFN) y evitar la utilización de métodos invasivos en casos de fetos RhD-. (AU)

Mothers at risk of alloimmunization to the Rh(D) antigen and availability of gamma-globulin at the Mexican Institute of Social Security

Hemolytic disease of the newborn develops mainly when an Rh negative (D-) mother becomes sensitized and produces anti-Rh possitve (anti-D) antibodies capable of hemolysing D+ fetal erythrocytes. Maternal alloimmunization can be prevented by the administration of anti-D gamma-globulin immediately after the birth of each Rh positive child. In order to identify the frequency of prevention of alloimmunization at the Instituto Mexicano del Seguro Social(IMSS), the amount of mothers at risk of sensitization from 1985 to 1995 was estimated from Rh and ABO blood group frequencies and with the number of deliveries and abortions at the Medical Institutions. Also, information in regard to the dose of gamma-globulin units purchased by the Institute of Social Security from 1985 to 1993 was obtained. The number of mothers at risk stedily increased from 16,616 in 1985 to 21,071 in 1995, amounting to a total of 203,203 in the 10-year period, while only 120,800 gamma-globulin units were purchased in that same period. The findings in this study suggest the need to define reasonable policies for the acquisition of gamma-globulin lots to prevent alloisoimmunization of mothers at risk

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

Early diagnosis of ABO haemolytic disease of the newborn.

To assess the usefulness of cord blood tests in diagnosing ABO-haemolytic disease of the newborn (ABO-HDN), 132 term, adequate for gestational age (AGA) neonates were evaluated. The tests studied and their significant results were: quantitative elution test (greater than or equal to 1/16), direct Coombs test (positive), bilirubin concentration (greater than or equal to 4 mg/dl). In none of the 56 O+ newborn infants delivered by O+ women were the results of any test positive. Of the 76 A+ and B+ newborn infants delivered by O+ women, 17 (22%) developed ABO-HDN. When the combined result of any two tests was positive, the sensitivity, the specificity and the positive predictive accuracy for the diagnosis of ABO-HDN was higher than for any one of the isolated tests. The probability that ABO-HDN was present when the results of at least two cord blood tests were positive was 70%, and the probability that ABO-HDN was not present when less than two cord blood tests gave positive results was 93%. It is suggested that the combination of quantitative elution test, bilirubin concentration and direct Coombs test in the cord blood is useful for an early diagnosis of ABO-HDN.