Determination of Fetal RHD Genotype Including the RHD Pseudogene in Maternal Plasma.

OBJECTIVE: To examine the accuracy of fetal RHD genotype and RHD pseudogene determination in a multiethnical population. METHODS: Prospective study involving D-negative pregnant women. Cell-free DNA was extracted from 1 ml of maternal plasma by an automated system (MagNA Pure Compact, Roche) and real-time PCR was performed in triplicate targeting the RHD gene exons 5 and 7. Inconclusive samples underwent RHD pseudogene testing by real-time PCR analysis employing novel primers and probe. RESULTS: A positive result was observed in 128/185 (69.2%) samples and negative in 50 (27.0%). Umbilical cord blood phenotype confirmed all cases with a positive or negative PCR result. Seven (3.8%) cases were found inconclusive (exon 7 amplification only) and RHD pseudogene testing with both conventional and real-time PCR demonstrated a positive result in five of them, while two samples were also RHD pseudogene negative. CONCLUSION: Real-time PCR targeting RHD exons 5 and 7 simultaneously in maternal plasma is an accurate method for the diagnosis of fetal D genotype in our population. The RHD pseudogene real-time PCR assay is feasible and is particularly useful in populations with a high prevalence of this allele.

Frequency of human platelet antigens in oncohematological patients with thrombocytopenia and the probability of incompatibility to platelet transfusions.

OBJECTIVE: The objective of this study was to evaluate the frequencies of human platelet antigens in oncohematological patients with thrombocytopenia and to analyze the probability of their incompatibility with platelet transfusions. METHODS: Platelet antigen genotyping was performed by sequence-specific primer polymerase chain reaction (SSP-PCR) for the HPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-4a, HPA-4b, HPA-5a, HPA-5b; HPA-15a, HPA-15b alleles in 150 patients of the Hematology Service of the Hospital das Clínicas (FMUSP). RESULTS: THE ALLELE FREQUENCIES FOUND WERE: HPA-1a: 0.837; HPA-1b: 0.163; HPA-2a: 0.830; HPA-2b: 0.170; HPA-3a: 0.700; HPA-3b: 0.300; HPA-4a: 1; HPA-4b: 0; HPA-5a: 0.887; HPA-5b: 0.113; HPA-15a: 0.457 and HPA-15b: 0.543. CONCLUSIONS: Data from the present study showed that the A allele is more common in the population than the B allele, except for HPA-15. This suggests that patients homozygous for the B allele are more predisposed to present alloimmunization and refractoriness to platelet transfusions by immune causes. Platelet genotyping could be of great value in the diagnosis of alloimmune thrombocytopenia and to provide compatible platelet concentrates for these patients.

Frequency of human platelet antigens in oncohematological patients with thrombocytopenia and the probability of incompatibility to platelet transfusions

OBJECTIVE: The objective of this study was to evaluate the frequencies of human platelet antigens in oncohematological patients with thrombocytopenia and to analyze the probability of their incompatibility with platelet transfusions. METHODS: Platelet antigen genotyping was performed by sequence-specific primer polymerase chain reaction (SSP-PCR) for the HPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-4a, HPA-4b, HPA-5a, HPA-5b; HPA-15a, HPA-15b alleles in 150 patients of the Hematology Service of the Hospital das Clínicas (FMUSP). RESULTS: The allele frequencies found were: HPA-1a: 0.837; HPA-1b: 0.163; HPA-2a: 0.830; HPA-2b: 0.170; HPA-3a: 0.700; HPA-3b: 0.300; HPA-4a: 1; HPA-4b: 0; HPA-5a: 0.887; HPA-5b: 0.113; HPA-15a: 0.457 and HPA-15b: 0.543. CONCLUSIONS: Data from the present study showed that the A allele is more common in the population than the B allele, except for HPA-15. This suggests that patients homozygous for the B allele are more predisposed to present alloimmunization and refractoriness to platelet transfusions by immune causes. Platelet genotyping could be of great value in the diagnosis of alloimmune thrombocytopenia and to provide compatible platelet concentrates for these patients.

Sistema de grupo sangüíneo Duffy: biologia e prática transfusional / Duffy blood group system: biology and transfusion practice

Após a introducão da técnica de antiglobulina indireta por Coombs em meados da década de 40, vários anticorpos antieritrocitários foram descobertos. O grupo sanguíneo Duffy foi descoberto quando Cutbush e Ikin detectaram, no início da década de 50, os primeiros anticorpos desse sistema. Os anticorpos Duffy são clinicamente significantes na prática transfusional, pois mostraram ser causadores de reacão hemolítica transfusional e de doenca hemolítica do recém-nascido, sendo de ocorrência mundial. O gene FY é constituído por dois exons e seu lócus foi mapeado no cromossomo 1q22-q23. Os antígenos Fyª e Fy b são codificados pelos alelos FYA e FYB e são responsáveis pelos fenótipos Fy(a+b-), Fy(a-b+) e Fy(a+b+). São carreados por uma glicoproteína de 336 aminoácidos também chamada DARC (Duffy Antigen/Receptor for Chemokines), que tem alta afinidade a quimiocinas, sendo também os receptores para Plasmodium vivax. Os polimorfismos relacionados aos seus alelos permitiram o desenvolvimento da técnica de genotipagem por PCR, que é de grande utilidade para a seguranca transfusional e incompatibilidade feto-materna. Na última década, inúmeras pesquisas têm sido feitas quanto ao papel biológico dos antígenos de grupos sangüíneos. Nesse artigo iremos revisar o sistema de grupo sangüíneo Duffy, em especial quanto à prática transfusional e suas funcões biológicas.

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%). 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.

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