Institute of Integrative Biology of the Cell, CNRS, CEA, Paris-Saclay University, Gif-sur-Yvette, France.
Paediatric Intensive Care and Neonatal Medicine, AP-HP, Paris-Saclay University, Bicêtre Hospital, Le Kremlin-Bicêtre, France.
Cytometry B Clin Cytom. 2024 Jan;106(1):58-63. doi: 10.1002/cyto.b.22142. Epub 2023 Sep 13.
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.
单核细胞 (m)HLA-DR 表达似乎是危重病患者免疫抑制的有力标志物。低 mHLA-DR 表达的持续存在与医院获得性感染和死亡率增加有关。为了适应儿科的需求并提供广泛的 24/7 访问，我们开发了一种全血不裂解、不洗涤微方法 (MM)，并将其与标准化方法 (SM) 进行了比较。
使用流式细胞术通过 Quantibrite™ Anti-HLA-DR PE/Monocyte PerCP-Cy™5.5 定量 mHLA-DR，SM 采用制造商方案在诊断血液学实验室中进行，或者使用位于儿科 ICU 的 Attune 流式细胞仪进行全血不裂解、不洗涤 MM。在两种技术中测量中荧光强度，并转换为与 BD Quantibrite™ PE 珠校准的每细胞抗体 (AB/C)。与 SM 相比，MM 减少了 5 倍的血液和 Quantibrite™ 试剂用量。除了 Quantibrite™ Anti-Human HLA-DR PE/Monocyte PerCP-Cy™5.5 之外，MM 还需要抗 CD45 和抗 CD19 标记。
我们确定了 34 名入住 ICU 的患者（20 名成人和 14 名儿童）的 mHLA-DR 表达。MM 与 SM 之间的相关性非常好（Pearson 相关性：y=0.8192x+678.7，r=0.9270，p<0.0001）。估计偏差为 2467±1.96×3307 AB/C；95%CI [−4016；+8949]。
用于测量 mHLA-DR 表达的无裂解、无洗涤全血微体积方法允许简化样品制备，而不会影响数据的准确性。这种方法通过部署即时护理方法，可能会简化对危重病患者的免疫监测。
