BRIEF REPORT How do we identify RHD variants using a practical molecular approach? Carine Prisco Arnoni, 1 Flavia Roche Moreira Latini, 1 Janaína Guilhem Muniz, 1 Diana Gazito, 1 Rosangela de Medeiros Person, 1 Tatiane Aparecida de Paula Vendrame, 1 José Augusto Barreto, 1 and Lilian Castilho 2 Serologic resolution of Rh discrepancies due to partial D or weak D phenotypes is a frequent problem encoun- tered during routine typing that can be solved by RHD genotyping because it provides better characterization of these variants. The objective of the current study was to develop algorithms for identification of D variants in multiethnic populations based on a logic sequence of molecular tests using a large number of atypical RhD specimens. Thus, a total of 360 blood samples with atypical D antigen expression were analyzed. A previ- ously published multiplex polymerase chain reaction (PCR) procedure was performed and depending on multiplex PCR analysis, the associated RHCE allele, and D variant frequency in our population, an algorithm was developed composed of six flow charts using spe- cific PCR–restriction fragment length polymorphism and/or specific exon sequencing. This strategy allowed the identification of 22 different variants with few assays and a much reduced cost. This study describes a simple and practical algorithm that we use to determine RHD genotypes in samples with unknown RHD. This strategy is relatively easy to implement and the algo- rithm can be adapted to populations with various ethnic backgrounds after an initial assessment of the type and frequency of D variants. Essentially, we demonstrate that sequencing of all RHD exons is not necessary for the identification of the majority of known D variants. T he majority of weak D phenotypes result from single-nucleotide polymorphisms (SNPs) in RHD encoding amino acid changes within either the membrane-spanning domains or the cytoplasmic loops of the protein. These changes can inter- fere with the integration of the RhD protein in the mem- brane leading to a reduced number of D antigen sites on red blood cells (RBCs). 1 Partial D, in contrast to weak D, is characterized by amino acid changes outside of the mem- brane that can alter or create new epitopes. 2 Therefore, individuals with partial D can make anti-D when stimu- lated by transfusion or pregnancy. In fact, many partial D are typed as D+ by direct agglutination and these individu- als will be identified only after anti-D formation. 3 RHD genotyping is useful for precise characterization of partial D and weak D types in donors and recipients and is a clinically important approach to prevent alloim- munization of recipients with partial D, usually typed as D+, when exposed to D+ RBCs or to some of the weak D RBCs typed as D– by serologic methods. However, RHD genotyping is not easy, because it is influenced by the size of the gene, by the presence of rearrangements with RHCE, 3 and by the high number of SNPs that are recog- nized in variants found among different ethnic groups. 4 A multitude of different primers and probes is avail- able for molecular genotyping of RHD making difficult the ABBREVIATION: SNP(s) = single-nucleotide polymorphism(s). From the 1 Colsan–Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil; and 2 INCTs–Hemocentro-Unicamp, Campinas, SP, Brazil. Address reprint requests to: Carine Prisco Arnoni, Colsan–Associação Beneficente de Coleta de Sangue, Avenida Jandira 1260, Indianópolis–CEP 04080-006, Brazil; e-mail: carine.arnoni@colsan.org.br. Received for publication July 26, 2013; revision received November 28, 2013, and accepted November 29, 2013. doi: 10.1111/trf.12557 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** ** TRANSFUSION 1