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DNA Reference Reagents for Genotyping RH Variants.

Emilia Sippert1, Evgeniya Volkova1, Meagan Rippee-Brooks1

  • 1Office of Blood Research and Review, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland.

The Journal of Molecular Diagnostics : JMD
|March 17, 2024
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Summary

New DNA reference reagents for Rhesus (RH) blood group genotyping improve transfusion safety for patients with RH variants. These validated standards ensure accurate molecular testing for RH polymorphisms and alleles.

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Area of Science:

  • Immunology
  • Genetics
  • Biotechnology

Background:

  • Rhesus (RH) blood group variants can lead to Rh alloantibodies, necessitating precise red blood cell transfusions.
  • Current serologic reagents for RH variants lack specificity and are often in limited supply.
  • Accurate red blood cell genotyping assays are crucial for managing transfusions in patients with clinically significant RH variants.

Purpose of the Study:

  • To develop and validate well-characterized DNA reference reagents for Rhesus (RH) blood group genotyping.
  • To ensure the quality and accuracy of molecular tests for RH polymorphisms and alleles.
  • To provide reliable standards for the development and validation of RH genotyping assays.

Main Methods:

  • Production of eight lyophilized DNA reference reagents covering 21 RHD and RHCE polymorphisms.
  • International collaborative study involving 17 laboratories to evaluate DNA candidates using various molecular assays.
  • Utilized Sanger sequencing, next-generation sequencing, and third-generation sequencing for characterization.

Main Results:

  • Genotyping assays demonstrated 99.4% agreement for target RH polymorphisms and 87.9% for RH allele agreement.
  • Discordant RH allele results were often attributed to limited polymorphism coverage in specific genotyping methods.
  • Stability and degradation studies confirmed the suitability of these reagents as reference standards.

Conclusions:

  • Eight DNA reference reagents have been qualified for use in Rhesus (RH) blood group genotyping.
  • These reagents will enhance the accuracy and reliability of molecular testing for RH variants.
  • The validated standards support the development and analytical validation of RH genotyping assays for improved transfusion medicine.