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

Abstract

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 6 19.1%). The mean ABC value for B-CLL B-cells stained with the same conjugate was 21,700 (CV 6 42.0%). Using the CD4 T-cell as a biologic calibrator for FITC conjugate, the mean ABC value for CD20-FITC on normal B-cells was 199,300. CD20-FITC staining on 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 6 10.4)%, and it did not differ significantly in CLL samples.Conclusion: The expression of CD20 on normal and B-CLL lymphocytes can be 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. q

Toward quantitative fluorescence measurements with multicolor flow cytometry

“…The procedure for whole blood staining was described previously (12). Briefly, the whole blood washed with 13 PBS was stained with labeled antibodies for 30 min at room temperature.…”

“…The upper part of Figure 1b shows five containers with a population of standard cells (dotted line) at the bottom of each container. In this case, the biological standard will be cells with a known number of specific protein antigen, e.g., normal or lyophilized T lymphocytes with CD4 receptors (3,12). The biological standard is incubated with antibodies for that specific protein antigen.…”

HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20low expressing tumor cells that resist rituximab-mediated lysis

“…29,31,32 This may explain the poor response to rituximab of B-cell malignancies expressing low CD20 levels such as B-cell chronic lymphocytic leukemia B-CLL. The number of CD20 molecules on B-CLL was reported to be in the order 22,000 molecules per cell, 33 which is 300 to 600-fold lower than observed in lymphoma. 29,[33][34][35] Recently, a panel of fully human antibodies including ofatumumab (HuMax-CD20), HuMab-2C6 and HuMab-7D8, were generated in human Ig transgenic mice.…”

“…The number of CD20 molecules on B-CLL was reported to be in the order 22,000 molecules per cell, 33 which is 300 to 600-fold lower than observed in lymphoma. 29,[33][34][35] Recently, a panel of fully human antibodies including ofatumumab (HuMax-CD20), HuMab-2C6 and HuMab-7D8, were generated in human Ig transgenic mice. This group of human antibodies represents a panel of CD20 mAbs that bind to a unique membrane-proximal CD20 epitope, including the small and large extracellular loop.…”

“…The CD20 antibodies-bound-per cell (CD20-ABC) ranged from 7,000 to 135,000 and is comparable to CLL samples and low expressing lymphoma samples. 29,[33][34][35] The only variable parameter between these clones is the CD20 expression level and interpretation of results is, therefore, not complicated by differences in expression levels of complement regulatory proteins (CD46, CD55, CD59). 29 The clones were subjected to rituximab-and HuMab-7D8-induced CDC, which demonstrated that rituximab required an approximately 5-6 times greater CD20 expression to induce maximum cell lysis ( Figure 1B).…”