Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B-cells

被引:27
|
作者
Wang, Lili
Abbasi, Fatima
Gaigalas, Adolfas K.
Vogt, Robert F.
Marti, Gerald E.
机构
[1] Natl Inst Stand & Technol, Biochem Sci Div, Gaithersburg, MD 20899 USA
[2] US FDA, Ctr Biol Evaluat & Res, NIH, Gaithersburg, MD 20899 USA
[3] CDC, Div Sci Lab, Atlanta, GA 30341 USA
关键词
CD20; CD4; QuantiBRITE (TM) PE quantification kits; ABC value; MESF value; RM (TM) 8640; B-cell chronic lymphocytic leukemia;
D O I
10.1002/cyto.b.20140
中图分类号
R446 [实验室诊断]; R-33 [实验医学、医学实验];
学科分类号
1001 ;
摘要
Background: Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes as potential disease biomarkers. CD20 expression in B-cell chronic lymphocytic leukemia (B-CLL) is one of the best examples of such a biomarker, but results from the use of different QFC methods vary considerably. Methods: We measured CD20 expression on normal and B-CLL B-cells, using FITC and PE conjugates from the same monoclonal antibody (Mab). As a biological control and calibrator, we also measured CD4 expression on T-cells with FITC and PE Mab. Calibration curves were constructed using the CLSI (formerly NCCLS) consensus guidelines for QFC. Calibration with QuantiBRITE (TM) PE-labeled microspheres and the use of unimolar PE conjugates provided direct measurement of antibody bound per cell (ABC) for CD4 and CD20. Calibration for FITC conjugates was based on molecules of equivalent soluble fluorochrome (MESF), as determined by NIST RM 8640 microsphere standards. These MESF values were then converted to ABC, using the CD4 T-cell as a biologic calibrator, to normalize FITC and PE results for CD20 expression. Results: On normal B cells, the mean ABC value for unimolar CD20-PE conjugate was 143,500 (CV +/- 19.1%). The mean ABC value for B-CLL B-cells stained with the same conjugate was 21,700 (CV +/- 42.0%). Using the CD4 T-cell as a biologic calibrator for FITC conjugate, the mean ABC value for CD20FITC on normal B-cells was 199,300. CD20-FITC staining an B-CLL cells was generally too weak for accurate quantification. On normal T-cells, the mean ABC value for CD4 unimolar PE conjugate was (36,800 +/- 10.4)%, and it did not differ significantly in CLL samples. Conclusion: The expression of CD20 on normal and B-CLL lymphocytes can he quantified in ABC units using unimolar CD20-PE conjugates. In addition, CD4 expression on T-cells can be used as a biological calibrator to quantify CD20-FITC ABC, with reasonable agreement between the two conjugates with different fluorochromes. Issues regarding the accuracy of MESF microsphere calibrators and effective F/P ratios for FITC conjugates will require additional laboratory studies. (c) 2006 International Society for Analytical Cytology.
引用
收藏
页码:410 / 415
页数:6
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