Performance evaluation of quantiBRITE phycoerythrin beads

• Published in: Cytometry (New York, N.Y.) Vol. 45; no. 4; pp. 250 - 258
• Main Authors: Pannu, Kulwinder K., Joe, Edward T., Iyer, Sujata B.
• Format: Journal Article
• Language: English
• Published: New York John Wiley & Sons, Inc 01.12.2001
• Subjects: antibodies bound per cell; Antibodies, Monoclonal - immunology; Antigens, CD - analysis; Antigens, Differentiation, T-Lymphocyte - analysis; CD4; CD4 Antigens - analysis; CD4-Positive T-Lymphocytes - cytology; CD4-Positive T-Lymphocytes - immunology; CD69; flow cytometer; Flow Cytometry - methods; Flow Cytometry - standards; Humans; interlaboratory standardization; Lectins, C-Type; Lymphocyte Activation; Microspheres; Phycoerythrin; QuantiBRITE; quantitative calibration; Reproducibility of Results; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0196-4763; 1097-0320
• DOI: 10.1002/1097-0320(20011201)45:4<250::AID-CYTO10021>3.0.CO;2-T

Background The performance of QuantiBRITE phycoerythrin (PE) beads to standardize quantitation in terms of antibodies bound per cell (ABC) was evaluated by measuring precision, variation across multiple instruments, and variation across time. Methods For CD4 quantitation, whole blood was stained with a two‐color CD4 reagent using a no‐wash/no‐lyse format. For CD69 quantitation, whole blood was activated with either phorbol myristate acetate (PMA) or CD3 beads and then stained with a three‐color CD69 reagent using a lyse‐no‐wash format. Results Across 20 normal donors, the mean CD4 ABC was 51,000. Within‐assay precision on quantitation of CD4 ABC on T cells had a coefficient of variance (CV) of <1.0%. Across multiple flow cytometers, quantitation of CD4 ABC had a CV of <5.0%. Within‐donor CV on CD4 ABC on 20 donors across 2 months ranged from 1.3% to 3.2%. Within‐assay precision on quantitation of CD69 on T cells activated with either PMA or CD3 beads had a CV of <3.0%. Within‐donor CV of CD69 ABC across 1 month ranged from 2% to 18% on PMA‐activated samples and from 7% to 24% on CD3 bead‐activated samples. Conclusions Our results indicate that the QuantiBRITE PE beads provide a useful tool for standardized analysis across labs. When used in conjunction with 1:1 conjugates of PE‐to‐monoclonal antibody, the QuantiBRITE PE beads provide a simple yet robust means of quantitating expression levels in terms of ABC. Cytometry 45:250–258, 2001. © 2001 Wiley‐Liss, Inc.