Consistent, multi‐instrument single tube quantification of <scp>CD</scp>20 in antibody bound per cell based on <scp>CD</scp>4 reference

Degheidy, Heba; Abbasi, Fatima; Mostowski, Howard; Gaigalas, Adolfas K.; Marti, Gerald E.; Bauer, Steven R.; Wang, Lili

doi:10.1002/cyto.b.21253

Cited by 13 publications

(19 citation statements)

References 26 publications

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“…1.29.1) assumes that the labeled antibody against the unknown and labeled anti-CD4 antibody have similar effective fluorescence per antibody values (so called effective F/P). Several studies support the assumption that different antibodies against different antigens with the same fluorophore label can have similar effective F/P values if these antibodies are produced by the same manufacturer (Wang et al, 2006;Degheidy et al, 2014Degheidy et al, , 2015. However, this assumption has not been verified with results of unknown antibodies with the same fluorophore label produced by different manufacturers.…”

Section: Commentary Background Informationmentioning

confidence: 98%

“…Once the ABC scales are put in place, users can then quantify test biomarkers relative to the CD4 reference marker using test antibodies (with the same fluorophore labels and high binding affinities and specificities) against the test biomarkers. Three published studies (Wang et al, 2006;Degheidy et al, 2014Degheidy et al, , 2015 have provided evidence supporting the assumption that different antibodies against different antigens with the same fluorophore label can have similar effective fluorescence per antibody values if the antibodies are produced by the same manufacturer.…”

Section: Introductionmentioning

confidence: 93%

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Quantitative Flow Cytometry Measurements in Antibodies Bound per Cell Based on a CD4 Reference

et al. 2016

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Multicolor flow cytometer assays with fluorescently labeled antibodies are routinely used in clinical laboratories to measure the cell number of specific immunophenotypes and to estimate expression levels of specific receptors/antigens either on the cell surface or intracellularly. The cell number and specific receptors/antigens serve as biomarkers for pathological conditions at various stages of a disease. Existing methods and cell reference materials for quantitative expression measurements have not yet produced results that are of wide clinical interest or are instrument-independent across all fluorescence channels. This unit details a procedure for quantifying surface and intracellular biomarkers by calibrating the output of a multicolor flow cytometer in units of antibody bound per cell (ABC). The procedure includes (1) quality control of the flow cytometer, (2) fluorescence intensity calibration using hard dyed microspheres assigned with fluorescence intensity values, (3) compensation for fluorescence spillover between adjacent fluorescence channels, and (4) application of a biological reference calibrator to establish an ABC scale. The unit also points out current efforts for quantifying biomarkers in a manner that is independent of instrument platforms and reagent differences.

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Section: Commentary Background Informationmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 93%

Quantitative Flow Cytometry Measurements in Antibodies Bound per Cell Based on a CD4 Reference

et al. 2016

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“…Cytometer setup and tracking beads (CST, BD Biosciences) were used for daily quality control and setup of initial PMT voltages. On the first day of experiment, PMT voltage of the PE channel was optimized to ensure the MedFI of unstained cells is approximately 2.5 times above the electron noise of the PE channel according to previously published protocol [45]. QuantiBrite PE beads were then run; the resulting MedFI values of the four PE intensity beads were recorded and used as target values to ensure consistency between experiments performed at different days.…”

Section: Cytometer Calibration and Cross Experiments Harmonizationmentioning

confidence: 99%

Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis

et al. 2021

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In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories. To date, the lack of adequate reference materials, and the complexity of the cytometer instrumentation have resulted in few standards. This study was designed to evaluate CD19 expression in three potential biological cell reference materials and provide a preliminary assessment of their suitability to support future development of CD19 reference standards. Three commercially available human peripheral blood mononuclear cells (PBMCs) obtained from three different manufacturers were tested. Variables that could potentially contribute to the differences in the CD19 expression, such as PBMCs manufacturing process, number of healthy donors used in manufacturing each PBMC lot, antibody reagent, operators, and experimental days were included in our evaluation. CD19 antibodies bound per cell (ABC) values were measured using two flow cytometry-based quantification schemes with two independent calibration methods, a single point calibration using a CD4 reference cell and QuantiBrite PE bead calibration. Three lots of PBMC from three different manufacturers were obtained. Each lot of PBMC was tested on three different experimental days by three operators using three different lots of unimolar anti-CD19PE conjugates. CD19 ABC values were obtained in parallel on a selected lot of the PBMC samples using mass spectrometry (CyTOF) with two independent calibration methods, EQ4 and bead-based calibration were evaluated with CyTOF-technology. Including all studied variabilities such as PBMC lot, antibody reagent lot, and operator, the averaged mean values of CD19 ABC for the three PBMC manufacturers (A,B, and C) obtained by flow cytometry were found to be: 7953 with a %CV of 9.0 for PBMC-A, 10535 with a %CV of 7.8 for PBMC-B, and 12384 with a %CV of 16 for PBMC-C. These CD19 ABC values agree closely with the findings using CyTOF. The averaged mean values of CD19 ABC for the tested PBMCs is 9295 using flow cytometry-based method and 9699 using CyTOF. The relative contributions from various sources of uncertainty in CD19 ABC values were quantified for the flow cytometry-based measurement scheme. This uncertainty analysis suggests that the number of antigens or ligand binding sites per cell in each PBMC preparation is the largest source of variability. On the other hand, the calibration method does not add significant uncertainty to the expression estimates. Our preliminary assessment showed the suitability of the tested materials to serve as PBMC-based CD19+ reference control materials for use in quantifying relevant B cell markers in B cell lymphoproliferative disorders and immunotherapy. However, users should consider the variabilities resulting from different lots of PBMC and antibody reagent when utilizing cell-based reference materials for quantification purposes and perform bridging studies to ensure harmonization between the results before switching to a new lot.

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“…This is a convenient way to calibrate and track the day-to-day performance of the flow cytometer or to ensure crossinstrument standardization [9,12]. Practical solutions to accomplish standardization have been described e lsewhere [13,14].…”

Section: Instrument Set-up Maintenance and Performancementioning

confidence: 99%

Best Practices in Performing Flow Cytometry in A Regulated Environment: Feedback from Experience Within The European Bioanalysis Forum

Strate¹,

Longdin²,

Geerlings³

et al. 2017

Bioanalysis

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Flow cytometry is a powerful tool that can be used for the support of (pre)clinical studies. Although various white papers are available that describe the set-up and validation of the instrumentation (the flow cytometer) and validation of flow cytometry methods, to date no guidelines exist that address the requirements for performing flow cytometry in a regulated environment. In this manuscript, the European Bioanalysis Forum presents additional practice guidance on the use of flow cytometry in the support of drug development programs and addresses areas that are not covered in the previous publications. The concepts presented here are based on the consensus of discussions in the European Bioanalysis Forum Topic Team 32, in meetings in Barcelona, Limelette and multiple telephone conferences.

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Consistent, multi‐instrument single tube quantification of CD20 in antibody bound per cell based on CD4 reference

Cited by 13 publications

References 26 publications

Quantitative Flow Cytometry Measurements in Antibodies Bound per Cell Based on a CD4 Reference

Quantitative Flow Cytometry Measurements in Antibodies Bound per Cell Based on a CD4 Reference

Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis

Best Practices in Performing Flow Cytometry in A Regulated Environment: Feedback from Experience Within The European Bioanalysis Forum

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