Marcia Regina Dezan

RHD Allelic Identification Among D−Brazilian Blood Donors as a Routine Test Using Pools of DNA

RHD alleles leading to a reduced expression of D antigen of the red blood cell (RBC) surface may be erroneously typed as D− by serology and may cause anti‐D immunizations when transfused to recipients.

Molecular HPA genotyping by microarray in Brazilian blood donors.

Human platelet antigens (HPA) polymorphisms may cause HPA alloimmunization, platelet (PLT) refractoriness, fetomaternal alloimmune thrombocytopenia, and posttransfusion purpura. Characterized by significant racial admixture, the Brazilian population might benefit from the knowledge about HPA frequency to guide decision‐making concerning PLT transfusion.

RHD and RHCE genotyping by next-generation sequencing is an effective strategy to identify molecular variants within sickle cell disease patients

BACKGROUND The complexity of Rh genetic variation among sickle cell disease (SCD) patients is high. Conventional molecular assays cannot identify all genetic variants already described for the RH locus as well as foresee novel alleles. Sequencing RHD and RHCE is indicated to broaden the search for Rh genetic variants. AIMS To standardize the Next Generation Sequencing (NGS) strategy to assertively identify Rh genetic variants among SCD patients with serologic suspicion of Rh variants and evaluate if it can improve the transfusion support. METHODS Thirty-five SCD patients with unexplained Rh antibodies were enrolled. A NGS-based strategy was developed to genotype RHD and RHCE using gene-specific primers. Genotype and serological data were compared. RESULTS Data obtained from the NGS-based assay were gene-specific. Ten and 25 variant RHD and RHCE alleles were identified, respectively. Among all cases of unexplained Rh antibodies, 62% had been inaccurately classified by serological analysis and, of these, 73.1% were considered as relevant, as were associated with increased risk of hemolytic reactions and shortage of units suitable for transfusion. CONCLUSION The NGS assay designed to genotype RH coding regions was effective and accurate in identifying variants. The proposed strategy clarified the Rh phenotype of most patients, improving transfusion support.

-318C/T polymorphism of the CTLA-4 gene is an independent risk factor for RBC alloimmunization among sickle cell disease patients

Cytotoxic T‐lymphocyte‐associated antigen 4 (CTLA‐4) molecule is expressed on T‐lymphocyte membrane and negatively influences the antigen‐presenting process. Reduced expression of CTLA‐4 due to gene polymorphisms is associated with increased risk of autoimmune disorders, whose physiopathology is similar to that of post‐transfusion red blood cell (RBC) alloimmunization. Our goal was to evaluate if polymorphisms of CTLA‐4 gene that affect protein expression are associated with RBC alloimmunization. This was a case–control study in which 134 sickle cell disease (SCD) patients and 253 non‐SCD patients were included. All patients were genotyped for the polymorphisms 49A/G and ‐318C/T of CTLA‐4 gene. The genotype frequency of ‐318C/T differed significantly between alloimmunized and nonalloimmunized SCD patients, irrespective of clinical confounders (p = .016). SCD patients heterozygous for ‐318T allele presented higher risk of alloantibody development (OR: 5.4, CI: 1.15–25.6). In conclusion, the polymorphism ‐318C/T of CTLA‐4 gene is associated with RBC alloimmunization among SCD patients. This highlights the role played by CTLA‐4 on post‐transfusion alloantibody development.

High-throughput strategy for molecular identification of Vel- blood donors employing nucleic acids extracted from plasma pools used for viral nucleic acid test screening.

Serologic methods to determine the Vel– phenotype require the use of rare human antisera and do not allow for many samples to be tested simultaneously, which limits their application as a tool to search for rare donors. This study developed a low‐cost molecular screening strategy using real‐time polymerase chain reaction (PCR) and DNA, extracted from plasma pools for viral nucleic acid test (NAT) screening, to identify Vel– and Vel+W donors.

Evaluation of the applicability and effectiveness of a molecular strategy for identifying weak D and DEL phenotype among D- blood donors of mixed origin exhibiting high frequency of RHD*Ψ

Molecular tests designed to detect the presence of active RHD gene among D– donors have been successfully applied in people of European ancestry, but not in admixed populations with a considerable frequency of RHD*Ψ. Our goal was to evaluate the performance of a molecular screening tool for identifying active RHD alleles among Brazilian blood donors classified as D– C+ and/or E+.

Defining the clinical relevance of red blood cell autoantibodies by Monocyte Monolayer Assay

The Monocyte Monolayer Assay (MMA) is an in vitro simulation of red blood cell (RBC) alloantibody behavior. It has been classically applied to predict the risks of post‐transfusion hemolytic reactions when transfusing incompatible RBC units. Quantifying erythrophagocytosis by MMA may be an interesting option for situations where there is doubt whether a RBC autoantibody is mediating significant hemolysis. Here, we present three situations involving RBC autoantibodies in which the MMA was decisive for clarifying the diagnosis and choosing the best clinical treatment.

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

To examine the accuracy of fetal RHD genotype and RHD pseudogene determination in a multiethnical population.

Successful management of neonatal alloimmune thrombocytopenia in the second pregnancy: a case report

ABSTRACT Neonatal alloimmune thrombocytopenia is a serious disease, in which the mother produces antibodies against fetal platelet antigens inherited from the father; it is still an underdiagnosed disease. This disease is considered the platelet counterpart of the RhD hemolytic disease of the fetus and newborn, yet in neonatal alloimmune thrombocytopenia the first child is affected with fetal and/or neonatal thrombocytopenia. There is a significant risk of intracranial hemorrhage and severe neurological impairment, with a tendency for earlier and more severe thrombocytopenia in subsequent pregnancies. This article reports a case of neonatal alloimmune thrombocytopenia in the second pregnancy affected and discusses diagnosis, management and the clinical importance of this disease.

Massive autoimmune hemolysis documented by monocyte monolayer assay in a multiply transfused patient using reticulocytes isolated by simple centrifugation in microhematocrit tubes: ASSAY TO DOCUMENT AUTOIMMUNE HEMOLYSIS

A 34-year-old man who was positive for the human immunodeficiency virus was admitted to the hospital with intense anemia and hemodynamic instability. His hemoglobin concentration at admission was 1 g/dL, and he had a mean corpuscular volume of 110 fL/cell and an absolute reticulocyte count of 12,000/mm. He received urgent transfusion of 5 units of Images of the Monocyte Monolayer Assay performed to picture the autoimmune RBC destruction. (A) Negative control. (B,C) The patient’s results reveal that the RBCs gathered inside the macrophages in an aspect resembling a pearl necklace.