Resolution of RHCE Haplotype Ambiguities in Transfusion Settings

Area of Science:

• Immunohematology
• Molecular Biology
• Genetics

Background:

• Red blood cell (RBC) transfusion is limited by alloimmunization, necessitating precise blood group typing.
• The Rh system, particularly RhCE, presents challenges due to serological phenotyping limitations.
• Existing molecular methods for RHCE analysis are complex for routine use.

Purpose of the Study:

• To develop an efficient reticulocyte isolation method for RhCE haplotype analysis.
• To establish an RT-PCR sequencing protocol for comprehensive RHCE allele and haplotype identification.
• To resolve ambiguous RhCE typing results in a clinical setting.

Main Methods:

• Tested two reticulocyte isolation protocols: size/density-based and antigen-specific sorting.
• Implemented an RHCE-specific RT-PCR followed by allele-specific sequencing of cDNA.
• Analyzed EDTA blood samples processed up to 48 hours post-collection.

Main Results:

• Antigen-specific reticulocyte sorting proved most efficient.
• The combined RT-PCR sequencing protocol accurately determined RHCE cDNA haplotypes.
• All tested samples showed concordance between RhCE phenotype and haplotype sequencing.
• Two previously ambiguous samples were resolved, including the identification of a novel RHCE allele (RHCE*03 c.340C>T).

Conclusions:

• Efficient reticulocyte isolation and RT-PCR sequencing enable robust RhCE haplotype analysis.
• This method improves the resolution of complex RhCE cases in immunohematology.
• The findings contribute to more accurate blood group typing and transfusion safety.