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Original ArticleOpen Access
Application of Flow Cytometric Beads for Simultaneous CD4 and CD8 Determinations in HIV-1 Infected Thalassemia Patients
Tiensiwakul P 
,
Boonmongkol P ,
Nonsee N ,
Bunchontevakul S ,
Desudchit P
,
Boonmongkol P ,
Nonsee N ,
Bunchontevakul S ,
Desudchit P 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/100WBC 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 assay for CD4 and CD8 determinations.
Keywords: CD4-positive T-lymphocytes, CD4 lymphocyte count, CD4-CD8 ratio, CD8-positive T-lymphocytes,
Erythroblasts, Flow cytometry, HIV-1, Thalassemia
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/100WBC 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 assay for CD4 and CD8 determinations.
Keywords: CD4-positive T-lymphocytes, CD4 lymphocyte count, CD4-CD8 ratio, CD8-positive T-lymphocytes,
Erythroblasts, Flow cytometry, HIV-1, Thalassemia
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