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. 2024 Jan;106(1):58-63.
doi: 10.1002/cyto.b.22142. Epub 2023 Sep 13.

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

Jordi Miatello  1   2   3 , Valérie Faivre  4   5 , Clémence Marais  1   2   3 , Mégane Raineau  2   3 , Didier Payen  6 , Pierre Tissieres  1   2   3
Affiliations
Free article

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

Jordi Miatello et al. Cytometry B Clin Cytom. 2024 Jan.
Free article

Abstract

Background: 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).

Methods: 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.

Results: 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].

Conclusions: 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.

Keywords: HLA-DR; flow cytometry; immunosuppression; monocytes; sepsis.

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