Whole blood no-lyse no-wash micromethod for the quantitative measurement of monocyte HLA-DR.

Jordi Miatello, Valérie Faivre, Clémence Marais, Mégane Raineau, Didier Payen, Pierre Tissieres

Cytometry. Part B, Clinical cytometry 2024 Jan

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Monocyte (m)HLA-DR expression appears to be a potent marker of immunosuppression in critically ill patients. The persistence of low mHLA-DR expression is associated with an increased risk of nosocomial infections and mortality. To adapt this measurement to pediatric requirements and provide extensive 24/7 access, we have developed a whole blood no-lyse no-wash micromethod (MM) and compared it with the standardized method (SM). mHLA-DR was quantified by flow cytometry using Quantibrite™ Anti-HLA-DR PE/Monocyte PerCP-Cy™5.5 with either the SM performed in a diagnostic hematology laboratory using manufacturer protocol, or a whole blood no-lyse no-wash MM using an Attune flow cytometer located in the pediatric ICU. Median fluorescence intensity was measured in both techniques and converted to antibodies per cell (AB/C) calibrated with BD Quantibrite™ PE beads. Blood and Quantibrite™ reagent volume used with the MM was reduced by 5-fold compared to SM. In addition to Quantibrite™ Anti-Human HLA-DR PE/Monocyte PerCP-Cy™5.5, MM required anti-CD45 and anti-CD19 labeling. We determined the expression of mHLA-DR in 34 patients, 20 adults, and 14 children admitted to ICU. Correlation between MM and SM was excellent (Pearson's correlation: y = 0.8192x + 678.7, r = 0.9270, p < 0.0001). The estimated bias was 2467 ± 1.96 × 3307 AB/C; CI 95% [-4016; +8949]. The no-lyse no-wash whole blood microvolume method for measuring mHLA-DR expression allows for simplified sample preparation without compromising accuracy of the data. This method may simplify immune monitoring of critically ill patient by the deployment of a point of care method. © 2023 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.

Citation

Jordi Miatello, Valérie Faivre, Clémence Marais, Mégane Raineau, Didier Payen, Pierre Tissieres. Whole blood no-lyse no-wash micromethod for the quantitative measurement of monocyte HLA-DR. Cytometry. Part B, Clinical cytometry. 2024 Jan;106(1):58-63