Using droplet digital PCR to screen for rare blood donors: Proof of principle

Dezan, Marcia Regina; Peron, Ana Cláudia; Oliveira, Théo Gremen Mimary de; Oliveira, Vítor Hugo de; Gomes, Carolina; Salles, Nanci Alves; Rocha, Vanderson; Mendrone‐Júnior, Alfredo; Dinardo, Carla Luana

doi:10.1016/j.transci.2020.102882

Cited by 1 publication

(1 citation statement)

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“…Because the detection of rare entities by ddPCR is a highly sensitive alternative method, use of this method to search for rare alleles in blood donors may enable screening for rare erythrocyte phenotypes. In addition, ddPCR can also be employed with multiple samples at the same time, thereby greatly reducing the difficulty and cost of testing while still ensuring efficiency ( Dezan et al, 2020 ). Therefore, ddPCR is widely used in mutation screening of populations ( Boone et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

SLC44A2 Frequency, a New TaqMan Real-Time Polymerase Chain Reaction Method for HNA-3A/3B Genotyping, and a New Application of Droplet Digital PCR

Wang

Chen

et al. 2022

Front. Genet.

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Background: Human neutrophil antigen-3A (HNA-3A) and human neutrophil antigen-3B (HNA-3B) are generated by a single-nucleotide polymorphism (rs2288904, c.461G > A) in exon 7 of the choline transporter-like protein-2 gene (CTL2, also known as SLC44A2). Antibodies to HNA-3 can be generated following blood transfusion or other factors resulting in exposure to HNA-3 antigens. These antibodies can cause transfusion-related acute lung injury (TRALI) or neonatal alloimmune neutropenia (NAIN). This study describes a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) method to screen for the HNA-3 genotype using specific primers and probes designed to detect allelic polymorphisms. Considering the high sensitivity and accuracy of droplet digital PCR (ddPCR) in the identification of the rare SLC44A2*2 allele, we used this technique to identify blood donors with the rare HNA-3B antigen and calculate the allele frequency of SLC44A2 in mixed populations with different proportions.Methods: DNA samples purified from 208 donors in northwest China were subjected to TaqMan real-time PCR to detect allelic polymorphisms in SLC44A2. The results were confirmed by Sanger sequencing. The rare HNA-3B antigen was detected by ddPCR. SLC44A2 frequency was determined by two-channel ddPCR.Results: The genotypes of all DNA samples were detected by the TaqMan real-time PCR using specific probes for HNA-3, and the results were consistent with the Sanger sequencing results in respect to the HNA-3A and HNA-3B polymorphisms. The allele frequencies of SLC44A2*1 and SLC44A2*2 in the 208 donors in northwest China were 64.9% (95% confidence interval [CI], 59%–70.8%) and 35.1% (95% CI, 29.2%–41%), respectively. The ratio of SLC44A2*2 alleles was accurately detected in all blood pools by ddPCR but not by TaqMan real-time PCR. This allowed for the SLC44A2 frequency in the population to be accurately inferred.Conclusion: This new method of detecting SLC44A2 alleles was highly sensitive and specific, as confirmed by Sanger sequencing. ddPCR using the designed probes resulted in successful detection of the rare HNA-3B antigen. Furthermore, we successfully detected the rare HNA-3B antigen and inferred the SLC44A2 frequency by ddPCR using the probes that we designed.

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Section: Introductionmentioning

confidence: 99%