This report was originally presented at an NCCLS Subcommittee meeting on Flow Cytometry, August 6–7, 1990, Philadelphia, PA, and in abstract form at the Fifth Annual Clinical Applications of Cytometry Meeting at Charleston, SC, September 1990. Use of trade names is for identification only and does not constitute endorsement by the Food and Drug Administration, Public Health Service, or the U. S. Department of Health and Human Services.
Flow cytometric analysis of whole blood lysis, three anticoagulants, and five cell preparations†
Article first published online: 8 MAR 2005
Copyright © 1992 Wiley-Liss, Inc.
Volume 13, Issue 1, pages 68–74, 1992
How to Cite
Carter, P. H., Resto-Ruiz, S., Washington, G. C., Ethridge, S., Palini, A., Vogt, R., Waxdal, M., Fleisher, T., Noguchi, P. D. and Marti, Gerald. E. (1992), Flow cytometric analysis of whole blood lysis, three anticoagulants, and five cell preparations. Cytometry, 13: 68–74. doi: 10.1002/cyto.990130111
- Issue published online: 21 JUN 2005
- Article first published online: 8 MAR 2005
- Manuscript Accepted: 10 AUG 1991
- Manuscript Received: 6 MAY 1991
- Ethylenediaminetetracetic acid (EDTA);
- acid citrate dextrose;
- cell viability
We studied the effects of anticoagulants and cell preparation methods on lymphocyte forward-angle s catter (FSC), autofluorescence, and immunofluorescent staining for CD45, CD14, and CD13. Blood samples collected in ethylenediaminetetracetic acid (EDTA), heparin, and acid citrate dextrose (ACD) were processed by using conventional Hypaque-Ficoll (HF) separation and four whole blood (WR) lysis techniques: Immuno-lyse, Q-Prep, FACS Lyse, and Gen Trak Lysis. Lymphocytes prepared by using three of the four whole blood methods gave FCS values comparable to those isolated by HF, while one method (FACS Lyse) gave consistently lover values. Autofluorescence values were comparable by all methods except Immuno-lyse, which showed consistently higher values in blood stored for 24 h with any anticoagulant. Immunofluorescent values for CD45-stained cells were quite consistent across all methods, and among the whole blood methods, FACS Lyse and Q-Prep uniformly gave the highest purity of CD45-positive cells in the lymphocyte light scatter gates. Additionally, propidium iodide (PI) analyses of CD45-stained whole blood, and analyzed without lysis, confirmed that ACD and heparin were superior to EDTA for maintaining viable leucocytes overnight. Future studies should focus on other commonly used reagents, a wide variety of abnormal samples, and cell viability.