Leukocyte immunophenotyping: methodological aspects

Introduction

Flow cytometric analysis of leukocyte surface receptors has been performed and shown beneficial, for example to characterize infectious and septic patients [1, 2]. For many surface antigens the results may vary depending on the sampling temperature, the anticoagulant used and the storage of the sample before analysis [3]. In order to obtain reliable data on leukocyte immunophenotyping for patient diagnostic purposes, we wanted to carry out a thorough evaluation on these methodological issues with a wide range of leukocyte surface antigens.

Methods

Four blood samples, two using acid citrate dextrose (ACD) and two using heparin as an anticoagulant, were taken from five ICU patients with severe sepsis and from five healthy volunteers. The patients and the healthy volunteers were combined into one study population (n = 10). The samples were taken, processed and stored either at +4°C or at room temperature. The surface antigen staining and flow cytometry were performed immediately after sample collection or after 6 or 24 hours at the above-mentioned temperatures. Antibodies of interest were for monocytes and neutrophils CD11b and CD64, for monocytes CD14, CD40, CD80, human leukocyte antigen (HLA)-DR, and for lymphocytes (CD4⁺ and CD8⁺ T cells, B cells, and NK cells) CD69. The flow cytometry analysis was done in three different time points, after 1, 6 or 24 hours of sampling. Inter-assay standardization and fluorescence quantifications were performed using microspheres.

Results

The fluorescence intensities were higher at room temperature compared with +4°C and they increased after storage (Figure 1). The effect was observed especially for CD11b-antigen in monocytes and neutrophils and for HLA-DR in monocytes.

Figure 1

CD11b fluorescence intensity for neutrophils (n = 10).

Conclusions

According to our results, flow cytometry leukocyte analysis using CD antigens should be performed using +4°C temperature throughout the measurement including sample collection, preparation and storage, and the analysis should be performed within 6 hours.