Mass screening for RHD gene fragments in RhD negative donors by minipool testing system using real-time PCR technology

Monika Pelc-Kłopotowska; Agnieszka Orzińska; Bogumiła Michalewska; Anna Walaszczyk; Jolanta Gawęda; Grzegorz Liszewski; Barbara Żupańska; Ewa Brojer

Vol 1, No 1 (2008)

Research paper

Published online: 2009-02-02

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Mass screening for RHD gene fragments in RhD negative donors by minipool testing system using real-time PCR technology

Monika Pelc-Kłopotowska, Agnieszka Orzińska, Bogumiła Michalewska, Anna Walaszczyk, Jolanta Gawęda, Grzegorz Liszewski, Barbara Żupańska, Ewa Brojer

Journal of Transfusion Medicine 2008;1(1):40-45.

Vol 1, No 1 (2008)

ORIGINAL PAPERS

Published online: 2009-02-02

Abstract

Background: RhD negative phenotype in Caucasians is caused mainly by a complete deletion of RHD gene. However, in some individuals the gene or its fragments are present but D protein is not expressed, has a low level of expression or lacks some epitopes. RhD negative patients transfused with some variants may be at risk of D immunization and non-invasive determination of fetal RHD may give false results.
Aim: Analysis of the minipool testing strategy and real time PCR typing for detection of RHD in RhD negative donors.
Material and methods: 2907 RhD negative donors typed by direct agglutination in tubes or microplates were studied. Plasma samples of 48 donors were pooled, DNA was extracted and tested by real-time PCR for exon 7, 10 and intron 4 of RHD. If the result was positive, further testing was performed to identify donors with RHD. RHD positive donors were investigated for exon 4 of RHD and RHDY and available donors were examined for common weak D and partial D types by SSP-PCR and by serological methods.
Results: RHD fragments were detected in 13/2907 RhD negative donors: in 7 - exon 7, 10 and intron 4; in 5 - exon 10 and in one - exon 7 and intron 4. In none was RHDY detected. In further analysis of available cases three donors were classified as D weak type 11 (2 cases) and DFR (1 case) and four as hybrid RHD-CE-D variants.
Conclusions: The RHD genotyping in DNA from donor plasma pools is useful for mass screening for RHD variants in RhD negative donors. RHD fragments were detected in 13/2907 (0.45%) Polish RhD negative donors.