Conditions in blood sampling procedures that extend the ex vivo stability of eosinophil activity markers in peripheral blood from allergic patients and healthy controls.
BACKGROUND
Serum-ECP, EG2-epitope on intracellular ECP and surface expression of CD9 and CD11b in peripheral blood eosinophils (PBE) are considered to be markers that mirror clinical parameters in allergic inflammation.


OBJECTIVE
The aim was to investigate the impact of the blood sampling procedure on PBE markers and to identify optimal conditions for extended pre-analysis storage.


METHODS
Blood, from healthy individuals and patients with allergic rhinitis/asthma, was collected in tubes with EDTA, citrate, or without anti-coagulant. The expression of EG2-epitope, CD9, and CD11b were analyzed in eosinophils and neutrophils after 1, 5, and 24 hours of storage at +4 degrees C, according to the FOG-method and flow cytometry. In vitro stimulation with fMLP/PMA was used for metabolic activity analysis and CD11b mobilization. Following a 1-hour clotting period at +20 to 22 degrees C, samples were stored at +4 degrees C and serum-ECP levels were measured.


RESULTS
The EG2-epitope, serum-ECP, and CD9 were stable in samples from both healthy controls and allergic patients at all storage conditions. The EG2-epitope, serum-ECP and PBE count were significantly increased in the patient group, whereas no differences were observed in the expression of CD9 or CD11b. Both granulocytes and monocytes retained their metabolic activity for 24 hours. Neutrophils in citrate-blood increased their ability to respond to fMLP, as compared with EDTA-blood.


CONCLUSIONS
In vitro analysis of selected activity markers and functional tests could be performed on granulocytes from both healthy individuals and allergic patients after 24 hours storage at +4 degrees C. The anticoagulant citrate seems to be preferable to EDTA when monocytes or CD11b expression are analyzed.
Halldén
G
G
Department of Laboratory Medicine, Karolinska Hospital, Stockholm, Sweden.
Nopp
A
A
Ihre
E
E
Peterson
C
C
Lundahl
J
J
D000328:Adult; D015703:Antigens, CD / Q000032:analysis / Q000378:metabolism; D001249:Asthma / Q000276:immunology*; D015415:Biomarkers / Q000032:analysis; D001798:Blood Proteins / Q000032:analysis; D001800:Blood Specimen Collection; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000276:immunology*; D000939:Epitopes; D005260:Female; D006801:Humans; D007958:Leukocyte Count; D007962:Leukocytes / Q000378:metabolism; D016177:Macrophage-1 Antigen / Q000032:analysis; D008297:Male; D008562:Membrane Glycoproteins; D008875:Middle Aged; D009504:Neutrophils / Q000276:immunology; D012221:Rhinitis, Allergic, Perennial / Q000276:immunology*; D012260:Ribonucleases; D060245:Tetraspanin 29
D015703:Antigens, CD; D015415:Biomarkers; D001798:Blood Proteins; C554869:CD9 protein, human; D047091:Eosinophil Granule Proteins; D000939:Epitopes; D016177:Macrophage-1 Antigen; D008562:Membrane Glycoproteins; D060245:Tetraspanin 29; D012260:Ribonucleases