Testing for the D zygosity with three different methods revealed altered Rhesus boxes and a new weak D type

Area of Science:

• Genetics
• Immunology
• Molecular Biology

Background:

• Accurate Rhesus factor (Rh) typing is vital for predicting hemolytic disease of the newborn (HDN).
• Distinguishing D+/D+ from D+/D- partners is critical for Rh-negative mothers with anti-D antibodies.
• Identifying the hybrid Rhesus box in fathers aids in estimating HDN risk.

Purpose of the Study:

• To develop and evaluate a novel Polymerase Chain Reaction-Sequence Specific Priming (PCR-SSP) method for detecting the hybrid Rhesus box.
• To compare the efficacy of the new PCR-SSP method against existing PCR-SSP and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) techniques.
• To assess the reliability of these methods in classifying various D blood group phenotypes, including D-negative, D-positive, and weak D variants.

Main Methods:

• A new PCR-SSP assay incorporating an internal amplification control was designed.
• The novel method was validated against two established PCR-based techniques (PCR-SSP and PCR-RFLP).
• Testing was performed on a cohort of 83 D-positive, 13 D-negative, and 37 weak D blood samples.

Main Results:

• The RHD deletion was consistently detected in all D-negative and weak D samples across all tested methods.
• Eighty-two out of 83 D-positive samples yielded concordant results among the three methods, indicating high agreement.
• The PCR-RFLP method's internal control failed in two weak D samples (type 4.0 and a novel type 29), suggesting potential interference from downstream Rhesus box polymorphisms.
• The novel weak D type 29 sample exhibited an altered downstream Rhesus box, while weak D type 4.0 showed no amplification with primers for both upstream and downstream Rhesus boxes.

Conclusions:

• A reliable PCR-SSP method with an internal control has been established for detecting the hybrid Rhesus box.
• Polymorphisms within the downstream Rhesus box can impede accurate RHD detection using certain PCR-based methods.
• The findings highlight the importance of robust molecular techniques for precise RhD genotyping, especially in cases of weak D phenotypes.