A whole-blood flow cytometric assay for leukocyte CD11b expression using fluorescence signal triggering.
A flow cytometric assay for measurements of leukocyte CD11b expression in whole blood has been developed and evaluated. The method is based on triggering of the flow cytometer by a fluorescent pan leukocyte marker, RPE-CD45. This enabled flow cytometric analysis in whole blood, and avoidance of in vitro artefacts related to cell purification and hemolysis. Our methodological evaluation suggested the following routine procedure: sampling with sodium citrate as the anticoagulant, sample incubation at 22 degrees C, and mild sample fixation with 0.5% formaldehyde saline. The latter provided good sample stability during 24 h. Moreover, the assay provided good assay reproducibility, low labelling antibody consumption, and minimal sample manipulation (< 30 min) and acquisition time demands. The assay seems to reflect the CD11b expression of circulating leukocytes, and is also suitable for studies of agonist stimulated CD11b expression in leukocyte subpopulations in vitro. When full CD11b responsiveness to agonist stimulation is desired, samples should be incubated at 37 degrees C, but this also elevated CD11b expression in unstimulated samples. The present whole-blood technique is thus suitable for analyses of CD11b expression for both research and clinical routine laboratory use. The assay can easily be modified for measurements of other leukocyte antigens by use of other specific fluorescent antibodies.
Department of Laboratory Medicine, Karolinska Hospital, Stockholm, Sweden.
N
N
Li
G
G
Halldén
P
P
Hjemdahl
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000328:Adult; D000925:Anticoagulants / Q000494:pharmacology; D016477:Artifacts; D001793:Blood Preservation; D002469:Cell Separation; D005260:Female; D005404:Fixatives / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005557:Formaldehyde / Q000494:pharmacology; D006461:Hemolysis; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis*; D007962:Leukocytes / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D016177:Macrophage-1 Antigen / Q000032:analysis* / Q000096:biosynthesis; D008297:Male; D008875:Middle Aged; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D015203:Reproducibility of Results; D013696:Temperature; D013917:Thrombin / Q000494:pharmacology
D000925:Anticoagulants; D005404:Fixatives; D016177:Macrophage-1 Antigen; D009842:Oligopeptides; D005557:Formaldehyde; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000244:Adenosine Diphosphate; C034585:glycyl-prolyl-arginyl-proline; D017493:Leukocyte Common Antigens; D013917:Thrombin