Endothelial Expression of Duffy Antigens in Patients with Fyb-GATA Homozygosity and Suspected Anti-Fy3 Antibody Formation: A Case Series

BACKGROUND The Duffy blood group consists of 6 different antigens expressed on a glycoprotein with 7 transmembrane domains, and is carried on the surface of both erythroid and non-erythroid tissues. The majority individuals of African ethnicity do not express the Duffy glycoprotein on their erythroid cells but appear to be primarily susceptible to alloimmunization against only the FY1 (Fya) antigen. The accepted explanation for this finding is that these individuals are homozygotes for a variant in the FY2 (Fyb) antigen in which a GATA-box mutation in the promoter region specifically prevents erythroid expression; the uninhibited expression of the Fyb antigen on non-erythroid tissues protects against alloimmunization. Occasionally, however, patients with FYB-GATA homozygosity develop clinically significant antibodies against other Duffy antigens, such as FY3 (Fy3), despite the fact that these antigens do not have known antithetical alleles. A mechanistic explanation for this observation is lacking.

METHODS From a single center, four adult sickle cell patients with anti-Fy3 were identified, with antibody specificity verified at a reference laboratory. All four individuals had previously undergone human erythrocyte antigen genotyping using the IDCORE XT^TM flow bead PCR assay and were found to be FYB-GATA homozygotes, with a predicted RBC phenotype of Fy(a-b-). The development of apparent anti-Fy3 antibodies in the post-transfusion plasma of these individuals prompted the analysis of stored tissue that had been collected for other diagnostic purposes (gall bladder, liver, breast and bone marrow, respectively) for endothelial expression of Duffy antigens. This analysis was performed via IHC using the anti-Duffy monoclonal antibodies 2C3 and MIMA-29. The 2C3-mAb recognizes the Fy6 antigen, a linear epitope on the first extracellular domain region of the Duffy glycoprotein adjacent to the antithetical Fy^a/Fy^b antigens, while the MIMA29-mAb binds to a region in extracellular domain 4 which is thought to carry the Fy³ antigen. IgG isotype antibodies were used as a negative control, and Duffy-antigen expressing RBCs were used as a positive control.

RESULTS Both 2C3-mAb and MIMA29-mAb stained Duffy-positive RBCs. No staining was apparent on the fixed bone marrow biopsy with either antibody and may have reflected the paucity of endothelial tissue in the sample. On the gall bladder, liver, and breast biopsies, 2C3-mAb stained endothelium strongly while while MIMA-29 mAb staining was negative. No staining with IgG antibody negative controls was observed in any sample.

CONCLUSION The three individuals in this series with interpretable results appeared to have only partial expression of Duffy antigens on non-erythroid tissue. While this expression included the extracellular domain that carries the antithetical Fya/Fyb antigens, it does not include the domain that carries the Fy3 antigen. As there is no known antithetical antigen to Fy3, the most likely explanation is that Fy3 is a conformational antigen that, possibly due to tissue-specific differences in tertiary structure, is only expressed on erythroid cells. It may therefore be concluded that at least some individuals with a Fy(a-b-) phenotype secondary to FYB-GATA homozygosity, while protected against sensitization to the Fyb antigen, remain susceptible to sensitization against the Fy3 antigen.

Lee:Janssen R&D: Consultancy.