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Variables in the Quantification of CD4 in Normals and Hairy Cell Leukemia Patients



Gerald E. Marti, William G. Stetler-Stevenson, Fatima Abbasi, Gregory A. Jasper, Robert J. Kreitman, Lili Wang


Background: Quantitative flow cytometry (QFCM) is being applied in the clinical flow cytometry laboratory. Quantitative normal T-cell CD4 expression represents a biologic standard and quality control agent. However, Low levels of CD4 expression were detected in normal T-cells in Hairy Cell Leukemia (HCL) samples. Methods: The QuantiBrite System® was used to determine the level of CD4 expression (mean antibody bound per cell, ABC) in fresh and shipped HCL blood and fresh normal donor blood (NDB). The effect of shipping specimens, lysing reagent, cell preparation method and antibody lot were evaluated. Results: Shipped HCL specimens (n = 69) had a significantly lower mean CD4 ABC of 38,788 (CV = 9.1%) compared to fresh specimens (n = 105) CD4 value of 40,330 (CV = 8.4%) (p < 0 .05). Consistent differences in CD4 ABC based upon antibody lot were observed in fresh HCL and NDB samples. Stain/lyse and lyse/stain methods using NH4Cl lyse were compared in NDB using identical samples and antibodies. The NDB CD4 ABC values obtained with the lyse (NH4Cl )/stain method (45,562, 3.7% CV) were lower than those obtained with the stain/lyse (NH4Cl) method (49,955, 3.3% CV) with p<0.001. In addition, with the stain/lyse method NDB CD4 ABC values in specimens prepared using FACS Lyse (46,445, 2.2% CV) were lower than the values when NH4Cl lyse was used (49,955, 3.3% CV) (p<0.001). Conclusions: Technical variables such as shipped verses fresh samples, method of sample preparation, lysing reagent and antibody lot impact significantly on QFCM.
Cytometry Part B-Clinical Cytometry


Quantitative flow cytometry (QFCM), CD4 expression, Biological standard and control, anti-CD4 PE unimolar conjugate, QuantiBRITE PE beads, Sample preparation method, Lysing reagent, Antibody lot, Hairy Cell Leukemia.


Marti, G. , Stetler-Stevenson, W. , Abbasi, F. , Jasper, G. , Kreitman, R. and Wang, L. (2011), Variables in the Quantification of CD4 in Normals and Hairy Cell Leukemia Patients, Cytometry Part B-Clinical Cytometry, [online], (Accessed April 14, 2024)
Created January 2, 2011, Updated October 12, 2021