Analysis of the temperature affects on leukocyte surface antigen expression

Flow cytometric analysis of leukocyte surface antigens has been used to characterize infectious and septic processes in patients. We wanted to investigate how the sampling and processing temperature, the anticoagulant used, and the storage of the sample influence leukocyte immunophenotyping. Four blood samples, two using acid citrate dextrose and two using heparin as an anticoagulant, were taken from five intensive‐care unit patients with severe sepsis and five healthy volunteers. The samples were collected, stored, and processed either at +4°C or at room temperature (RT). The samples were processed for flow cytometric analysis within 1 hr of collection or after 6 or 24 hr storage. The surface antigens of interest were neutrophilic CD11b and CD64, monocytic CD11b, CD14, CD40, CD64, CD80 and HLA‐DR, and lymphocytic CD69 (separately in CD4+ and CD8+ T cells, B cells, and natural killer cells). The fluorescence intensities were higher at RT than at +4°C. During storage the intensities increased at RT, but at +4°C there were only minor changes. The effects were similar with both anticoagulants studied. According to our results, flow cytometric analysis of leukocyte surface antigen expressions should be performed using +4°C temperature throughout the process and within 6 hr. J. Clin. Lab. Anal. 25:118–125, 2011. © 2011 Wiley‐Liss, Inc.

[1]  J. Hoffmann.  Neutrophil CD64: a diagnostic marker for infection and sepsis , 2009, Clinical chemistry and laboratory medicine.

[2]  J. Nuutila,et al.  CRP/CD11b ratio: a novel parameter for detecting gram-positive sepsis. , 2009, Human immunology.

[3]  G. Theodorou,et al.  Prognostic value of phagocytic activity of neutrophils and monocytes in sepsis. Correlation to CD64 and CD14 antigen expression , 2008, Clinical and experimental immunology.

[4]  H. Joensuu,et al.  Expression of CD11b/CD18 adhesion molecules on circulating phagocytes—a novel aid to diagnose infection in patients with cancer , 2008, Supportive Care in Cancer.

[5]  S. Blecher,et al.  Expression of cell surface receptors and oxidative metabolism modulation in the clinical continuum of sepsis , 2008, Critical care.

[6]  N. Mehta,et al.  CD40 and CD80/86 act synergistically to regulate inflammation and mortality in polymicrobial sepsis. , 2008, American journal of respiratory and critical care medicine.

[7]  C. Coopersmith,et al.  Lymphocyte phenotyping to distinguish septic from nonseptic critical illness. , 2008, Journal of the American College of Surgeons.

[8]  J. Nuutila,et al.  Simultaneous quantitative analysis of FcgammaRI (CD64) expression on neutrophils and monocytes: a new, improved way to detect infections. , 2007, Journal of immunological methods.

[9]  H. Kautiainen,et al.  Monocyte CD14 and soluble CD14 in predicting mortality of patients with severe community acquired infection , 2007, Scandinavian journal of infectious diseases.

[10]  C. Roussos,et al.  Neutrophil CD64 expression and serum IL-8: sensitive early markers of severity and outcome in sepsis. , 2006, Cytokine.

[11]  B. Davis,et al.  Neutrophil CD64 is an improved indicator of infection or sepsis in emergency department patients. , 2006, Archives of pathology & laboratory medicine.

[12]  C. S. Scott,et al.  Simultaneous determination of membrane CD64 and HLA-DR expression by blood neutrophils and monocytes using the monoclonal antibody fluorescence capability of a routine haematology analyser. , 2006, Journal of immunological methods.

[13]  C. Meisel,et al.  Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: a multicenter standardized study. , 2005, Clinical chemistry.

[14]  H. Kautiainen,et al.  Peripheral blood phagocyte CD14 and CD11b expression on admission to hospital in relation to mortality among patients with community-acquired infection , 2005, Inflammation Research.

[15]  B. Davis,et al.  Impact of preanalytical variables on granulocytic surface antigen expression: A review , 2005, Cytometry. Part B, Clinical cytometry.

[16]  T. Doco‐Lecompte,et al.  A standardized procedure for quantitation of CD11b on polymorphonuclear neutrophil by flow cytometry: potential application in infectious diseases. , 2004, Clinical and laboratory haematology.

[17]  J. Kirby,et al.  Neutrophil chemotaxis and receptor expression in clinical septic shock , 2004, Intensive Care Medicine.

[18]  Sobhy M. Mostafa,et al.  Is low monocyte HLA-DR expression helpful to predict outcome in severe sepsis? , 2003, Intensive Care Medicine.

[19]  H. Kautiainen,et al.  Cellular markers of systemic inflammation and immune suppression in patients with organ failure due to severe acute pancreatitis. , 2001, Scandinavian journal of gastroenterology.

[20]  J. Silver,et al.  Parameters influencing membrane CD14 expression and soluble CD14 levels in sepsis. , 2001, European journal of medical research.

[21]  D. Bianchi,et al.  Premature infants respond to early-onset and late-onset sepsis with leukocyte activation. , 2000, The Journal of pediatrics.

[22]  R. Rabin,et al.  Neutrophil CD11b expression as a diagnostic marker for early-onset neonatal infection. , 1998, The Journal of pediatrics.

[23]  M. Ahern,et al.  Effects of ex vivo manipulation on the expression of cell adhesion molecules on neutrophils. , 1995, Journal of immunological methods.

[24]  H. Repo,et al.  Anticoagulant selection influences flow cytometric determination of CD11b upregulation in vivo and ex vivo. , 1995, Journal of immunological methods.

[25]  H. Repo,et al.  Flow cytometric determination of CD11b upregulation in vivo. , 1993, Journal of immunological methods.

[26]  L. Leino,et al.  CD11b is a calcium-dependent epitope in human neutrophils. , 1992, Biochemical and biophysical research communications.