Transfusion associated graft-versus-host disease (GVH) due to blood from HLA-homozygous related and unrelated donors has been previously described. Identification of the serological typing of "blanks" in cadaver donor organs in the absence of family genotyping may be important in the differential diagnosis of GVH disease in recipients receiving simultaneous solid organ transplant and bone marrow infusion. This report describes a flow Cytometry-based typing approach to assess homozygosity of HLA-Class I (A or B Locus). A combination of fluorochrome labeled, A or B Locus allospecific Monoclonal antibodies (One Lambda Inc., CA), and a kit of standard quantitative fluorochrome labeled microbead's (Flow Cytometry Standards Corp., PR) were used to determine the Molecules of Equivalent Soluble Fluorochrome (MESF) of HLA-Class I in normal laboratory volunteers (LV) and donor marrows. A three color flow cytometry technique was also introduced to look at the distribution of HLA-class I MESF within subpopulations of lymphocytes. Six (of seven) LV that were determined to be HLA (A2,A-) or (B8,B-) or (B12,B-) by serology were confirmed to be HLA (A2,A2), (B8,B8) or (B12,B12) respectably, homozygous by flow Cytometry since they had doubled the amount of MESF-HLA-A2, B8 or B12 when compared to 27 HLA-A2, B8 and B12 heterozygous (P<0.001). Two of the six were confirmed HLA (A2,A2) and HLA (B12,B12) by family genotyping, the other 4 genotypes were not available for confirmation. One HLA (B8,B-) was within the range of heterozygosity, which confirmed that the second B antigen is not determined. The simultaneous three color analysis revealed the existence of a bimodal distribution of HLA-Class I in the T cell compartment (CD3 positive cells) of peripheral blood lymphocytes. We found more MESF HLA-Class I on CD8 positive cells than on CD4 positive cells for heterozygous (P=0.04) and for homozygous (P=0.04). There was an association of the CD8 and CD4 peaks with the overall bimodal distribution of HLA-Class I MESF Quantitation. In conclusion, HLA typing by flow cytometry/MESF methodology is a rapid procedure which may serve in the confirmation of homozygosity for "blank" antigens as determined by serology.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology