Analytical Requirements for Measuring Monocytic Human Lymphocyte Antigen DR by Flow Cytometry: Application to the Monitoring of Patients with Septic Shock

Abstract

The concept of immunoparalysis has recently been proposed for explaining the failure of 20 years of clinical trials using antiinflammatory drugs in sepsis (1)(2)(3). Immunoparalysis is characterized mainly by the paralysis of monocytic functions. In particular, because of decreased expression of HLA-DR, antigen-presenting capacity is severely depressed (4)(5). Recent clinical studies have confirmed that among a large panel of activation markers expressed on different leukocyte populations (i.e., neutrophils, lymphocytes, and monocytes), decreased HLA-DR expression on monocytes constitutes a reliable marker of immunoparalysis and seems to correlate with an increased risk for fatal outcome (6)(7)(8). Nevertheless, little work has been devoted to analytical aspects related to its measurement by flow cytometry. Indeed, flow cytometry cannot yet be considered a standardized tool, and many variables must be taken into account for ensuring the technical quality of results (9). This is especially required in clinical research when clinicians and immunologists are assessing the potential value of a new marker, as is presently the case for monitoring septic patients, and it is particularly true for the determination of HLA-DR, which is considered a rapidly up- or down-regulated marker (5)(10). The present study was designed to determine whether different protocol procedures could lead to discrepant results for HLA-DR measurements. After establishing a reliable protocol, the second objective was to demonstrate immunoparalysis in monitoring patients with septic shock. Samples of peripheral blood were collected in EDTA anticoagulant tubes. Staining was performed on whole blood using PC5-labeled CD45, fluorescein isothiocyanate (FITC)-labeled CD14 (Immunotech), and phycoerythrin-labeled HLA-DR (clone L243; Becton Dickinson). Samples were lysed manually by use of FACS lysing solution (Becton Dickinson) or the automated Q-Prep system (Beckman-Coulter). Cells were analyzed on a Coulter EPICS XL flow cytometer (System II software; Beckman-Coulter). After debris was …