Application of flow cytometric beads for simultaneous CD4 and CD8 determinations in HIV-1 infected thalassemia patients.

Background: CD4 and CD8 are determined by either a dual step assay using a calculation of absolute lymphocytes obtained from routine CBC or by a single step assay by including cytometric beads and fluorescent microspheres in the sample so that an absolute cell count can be made, simultaneously. Since thalassemia is common in Thailand, and a number of nucleated red blood cells (NRBC) are observed in peripheral blood of thalassemia patients with severe anemia, we speculated that NRBC in HIV-1 infected thalassemia patient with severe anemia might cause an error in CD4 and CD8 determinations in the dual step assay.

Objective: Comparing cytometric beads in three-color-lyse-no-wash in the single step assay in CD4 and CD8 with dual step assay by calculation using absolute lymphocytes obtained from routine CBC in HIV-1 infected thalassemia patients with severe anemia.

Material And Method: Simultaneous screening of alpha (SEA type)- and beta thalassemia using a multiplex PCR was done. In the thalassemia patients with severe anemia having 13-1,392 NRBC/100 WBC, it found significant differences (p < 0.001-0.002, paired-t-test) of the means of cytometric bead CD4 and CD8 and the means of NRBC corrected CD4 and CD8 as compared to the means of NRBC uncorrected CD4 and CD8 in dual step determinations. In the thalassemia patients with lesser severe anemia, having less than 10 NRBC/100 WBC, there were no significant differences (p > 0.05, paired-t-test) of the means of cytometric bead CD4 and CD8 and the means of NRBC corrected CD4+ and CD8+ as compared to the means of NRBC uncorrected CD4 and CD8 in dual step assay. In comparison of CD4 and CD8 determinations in HIV-1 infected thalassemia patients with severe anemia having more than 10 NRBC/100 WBC, there were significant differences (p < 0.002, paired-t-test) of the means of cytometric bead CD4 and CD8 and the means of NRBC corrected CD4 and CD8 as compared to the means of NRBC uncorrected CD4 and CD8 in dual step assay.

Conclusion: Results indicated that the NRBC in HIV-1 infected or uninfected thalassemia with severe anemia having more than 10 NRBC/100 WBC do cause an error in CD4 and CD8 determinations in dual step in routine assay. Therefore, either cytometric beads application in the single step or the conventional calculation using NRBC corrected absolute lymphocytes in three-color-lyse-no-wash assay is essentially needed in the flow cytometric assayfor CD4 and CD8 determinations.