Flow cytometric analysis of whole blood lysis, three anticoagulants, and five cell preparations.
We studied the effects of anticoagulants and cell preparation methods on lymphocyte forward-angle scatter (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 (WB) 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 lower 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.
Laboratory of Cellular and Molecular Biology, Food and Drug Administration, National Institutes of Health, Bethesda, Maryland 20892.
P H
PH
Carter
S
S
Resto-Ruiz
G C
GC
Washington
S
S
Ethridge
A
A
Palini
R
R
Vogt
M
M
Waxdal
T
T
Fleisher
P D
PD
Noguchi
G E
GE
Marti
D015703:Antigens, CD / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D001774:Blood Chemical Analysis; D001793:Blood Preservation; D018826:CD13 Antigens; D019343:Citric Acid; D004492:Edetic Acid; D005434:Flow Cytometry; D005947:Glucose / Q000031:analogs & derivatives; D006461:Hemolysis; D006493:Heparin; D006649:Histocompatibility Antigens / Q000032:analysis; D006801:Humans; D017493:Leukocyte Common Antigens; D008027:Light; D018950:Lipopolysaccharide Receptors; D011419:Propidium; D012542:Scattering, Radiation
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D006649:Histocompatibility Antigens; D018950:Lipopolysaccharide Receptors; C002113:acid citrate dextrose; D019343:Citric Acid; D011419:Propidium; D006493:Heparin; D004492:Edetic Acid; D017493:Leukocyte Common Antigens; D018826:CD13 Antigens; D005947:Glucose