Statistical criteria to establish optimal antibody dilution in flow cytometry analysis
Article first published online: 31 JAN 2007
Copyright © 2007 Clinical Cytometry Society
Cytometry Part B: Clinical Cytometry
Volume 72B, Issue 3, pages 223–226, May 2007
How to Cite
Collino, C. J. G., Jaldin-Fincati, J. R. and Chiabrando, G. A. (2007), Statistical criteria to establish optimal antibody dilution in flow cytometry analysis. Cytometry, 72B: 223–226. doi: 10.1002/cyto.b.20158
- Issue published online: 9 APR 2007
- Article first published online: 31 JAN 2007
- Manuscript Accepted: 28 SEP 2006
- Manuscript Revised: 12 SEP 2006
- Manuscript Received: 3 MAY 2006
- SECyT (Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba)
- CONICET (Consejo de Investigaciones Científicas y Tecnológicas de la República Argentina)
- Agencia Nacional de Ciencia de la República Argentina. Grant Number: FONCyT: BID 1201/OC-AR PICT N° 05-13945
- statistical criteria;
- unpaired t-test;
- antibody titration;
- flow cytometry;
- mean peak of channel fluorescence (MPCF)
In direct techniques of flow cytometry, the optimal antibody dilution or titer point is established from the plateau area of the antibody titration curve. However, the plateau area is defined without any statistical criteria, which may lead to an incorrect selection of antibody dilution. Herein, we report statistical criteria to establish the optimal antibody dilution for CD14, CD8, CD4, and CD3 analysis by flow cytometry in peripheral whole blood.
The unpaired t-test (two-tail P value) was used as statistical criteria to analyze the titration curve of the following monoclonal antibody panels: CD14-FITC, CD8-FITC, CD4-RD1, and CD3-PC5.
Using the unpaired t-test (two-tail P value), the plateau area from the antibody titration curve was fitted when two consecutive antibody volumes showed mean peak of channel fluorescence (MPCF) values not significantly different. When the antibody was used at volume corresponding to that of the antibody titration point, the flow cytometry analysis of whole blood samples with different density of cell antigens can be correctly discriminated.
This statistical criteria allows the fitting of the plateau area of MPCF versus antibody volume and consequently, to define the optimal antibody dilution. © 2007 Clinical Cytometry Society