Applications of automated simultaneous double fluorescence (SDF). II. HLA class I phenotyping using...

HLA class I phenotyping was performed using T-lymphocyte populations isolated by immunomagnetic beads (IMBs) coated with monoclonal antibodies with specificity for CD2, CD4 or CD8. The results were compared to those obtained using density gradient-separated lymphocytes (PBL). The typing trays were read by the automated simultaneous double-fluorescence (SDF) technique previously established in our laboratory using an Astroscan 2100 system. The aims of the present study were to establish whether the advantages of IMB lymphocyte separation and automated plate reading by SDF were complementary and whether the results obtained by IMB-SDF and PBL-SDF were concordant. Similarity coefficients for paired results obtained by IMB-SDF and PBL-SDF varied between 0.825 using anti-CD8-coated IMBs and 0.914 using anti-CD4-coated IMBs with a consistent excess of stronger results observed with the PBL-SDF technique. The variations observed did not result in incorrect phenotype assignment but would significantly influence a cross-matching test. These results illustrate the feasibility of using IMB-separated lymphocytes for HLA phenotyping by SDF.