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Analysis of the temperature affects on leukocyte surface antigen expression
Joel Jämsä1, Virva Huotari, Eeva-Riitta Savolainen
1Department of Anaesthesiology, Division of Intensive Care Medicine, Oulu University Hospital, University of Oulu, Oulu, Finland. joel.jamsa@oulu.fi
Insights
For accurate leukocyte immunophenotyping, perform flow cytometry at +4°C and within 6 hours. Room temperature processing and longer storage significantly alter results, impacting infectious and septic process characterization.
Area of Science:
- Immunology
- Clinical Pathology
Background:
- Flow cytometry is crucial for analyzing leukocyte surface antigens in infectious and septic conditions.
- Standardization of pre-analytical variables is essential for reliable immunophenotyping results.
Purpose of the Study:
- To evaluate the impact of sampling temperature, anticoagulant type, and storage duration on leukocyte immunophenotyping.
- To determine optimal conditions for preserving leukocyte surface antigen expression for flow cytometry.
Main Methods:
- Blood samples from sepsis patients and healthy volunteers were collected using heparin or acid citrate dextrose.
- Samples were processed at +4°C or room temperature (RT) and analyzed immediately, or after 6 or 24 hours of storage.
- Expression levels of key surface antigens (e.g., CD11b, CD64, CD14, HLA-DR, CD69) on neutrophils, monocytes, and lymphocytes were quantified.
Main Results:
- Higher fluorescence intensities were observed at RT compared to +4°C.
- Storage at RT led to increased fluorescence intensities, while +4°C resulted in minimal changes.
- The effects of temperature and storage were consistent regardless of the anticoagulant used.
Conclusions:
- Flow cytometric analysis of leukocyte surface antigens is significantly influenced by pre-analytical conditions.
- +4°C temperature throughout the process and analysis within 6 hours are recommended for optimal and reliable results.
Abstract:
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.
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