Colsan- Associação Beneficente de Coleta de Sangue, São Paulo, Brazil.
Hemocentro- São José do Rio Preto, São José do Rio Preto, Brazil.
Vox Sang. 2019 Nov;114(8):869-875. doi: 10.1111/vox.12851. Epub 2019 Oct 6.
Weak D phenotypes with very low antigen densities and DEL phenotype may not be detected in RhD typing routine and could be typed as D-negative, leading to D alloimmunization of D-negative recipients. The present study aimed to investigate the presence of RHD-positive genotypes in blood donors typed as D-negative by an automated system using the solid-phase methodology as a confirmatory test.
Two screenings were performed in different selected donor populations. For the first screening, we selected 1403 blood donor samples typed as D-negative regardless of the CE status, and in the second screening, we selected 517 donor samples typed as D-negative C+ and/or E+. RhD typing was performed by microplate in an automated equipment (Neo-Immucor®), and the confirmatory test was performed by solid-phase technique using Capture R® technology. A multiplex PCR specific to RHD and RHDψ was performed in a pool of 6 DNA samples. Sequencing of RHD exons was performed in all RHD-positive samples, and a specific PCR was used to identify the D-CE(4-7)-D hybrid gene.
No weak D type was found in either screening populations. Additionally, 353 (18·4%) D-negative samples presented previously reported non-functional RHD genes, 2 samples had a DEL allele, and 6 samples demonstrated new alleles, including one novel DEL allele. Our study identified six new RHD alleles and showed that the inclusion of a confirmatory test using serological methodology with high sensitivity can reduce the frequency of weak D samples typed as D-negative.
抗原密度非常低的弱 D 表型和 DEL 表型在 RhD 定型常规中可能无法检测到，可能被定型为 D 阴性，从而导致 D 阴性受者的 D 同种免疫。本研究旨在通过固相方法学的自动化系统对被定型为 D 阴性的献血者进行 RHD 阳性基因型检测，以调查其存在情况，该方法学作为一种确认性试验。
在不同的选定献血者群体中进行了两次筛选。第一次筛选中，我们选择了 1403 例无论 CE 状态如何均被定型为 D 阴性的献血者样本；在第二次筛选中，我们选择了 517 例被定型为 D 阴性 C+和/或 E+的献血者样本。RhD 定型采用微板自动化仪器（Neo-Immucor®）进行，确认性试验采用固相技术（Capture R®）进行。在 6 个 DNA 样本的混合池中进行了针对 RHD 和 RHDψ 的多重 PCR。对所有 RHD 阳性样本进行 RHD 外显子测序，并使用特定的 PCR 来鉴定 D-CE(4-7)-D 杂合基因。
在两次筛选人群中均未发现弱 D 型。此外，353 例（18.4%）D 阴性样本存在先前报道的无功能 RHD 基因，2 例样本存在 DEL 等位基因，6 例样本表现出新的等位基因，包括一个新的 DEL 等位基因。本研究鉴定了 6 个新的 RHD 等位基因，并表明采用高灵敏度血清学方法学进行确认性试验的纳入可以降低被定型为 D 阴性的弱 D 样本的频率。
