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

Abstract

AbstractBackgroundMonocyte (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).MethodsmHLA‐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.ResultsWe 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].ConclusionsThe 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.