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

IF 2.3 3区医学 Q3 MEDICAL LABORATORY TECHNOLOGY Cytometry Part B: Clinical Cytometry Pub Date : 2023-09-13 DOI:10.1002/cyto.b.22142

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

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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.

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用于定量测量单核细胞 HLA-DR 的全血无溶剂免洗微量法。

背景：单核细胞 (m)HLA-DR 表达似乎是重症患者免疫抑制的有效标志。低 mHLA-DR 表达的持续存在与院内感染和死亡风险的增加有关。方法：使用 Quantibrite™ 抗 HLA-DR PE/单核细胞 PerCP-Cy™5.5 流式细胞仪对 mHLA-DR 进行定量，在血液学诊断实验室中使用制造商提供的 SM 方法，或在儿科重症监护室中使用 Attune 流式细胞仪进行全血无溶剂免洗 MM 方法。两种技术都测量了中位荧光强度，并转换成用BD Quantibrite™ PE珠校准的每细胞抗体（AB/C）。与 SM 相比，MM 使用的血液和 Quantibrite™ 试剂量减少了 5 倍。除了使用 Quantibrite™ Anti-Human HLA-DR PE/Monocyte PerCP-Cy™5.5，MM 还需要抗 CD45 和抗 CD19 标记：我们测定了入住重症监护室的 34 名患者（20 名成人和 14 名儿童）的 mHLA-DR 表达。MM和SM之间的相关性非常好（Pearson相关性：y = 0.8192x + 678.7，r = 0.9270，p 结论：MM和SM之间的相关性非常好（Pearson相关性：y = 0.8192x + 678.7，r = 0.9270，p）：采用免洗全血微量法测定 mHLA-DR 表达可简化样本制备，同时不影响数据的准确性。这种方法可简化重症患者的免疫监测，是一种定点护理方法。

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来源期刊

Cytometry Part B: Clinical Cytometry 医学-病理学

CiteScore

6.80

自引率

32.40%

发文量

审稿时长

>12 weeks

期刊介绍： Cytometry Part B: Clinical Cytometry features original research reports, in-depth reviews and special issues that directly relate to and palpably impact clinical flow, mass and image-based cytometry. These may include clinical and translational investigations important in the diagnostic, prognostic and therapeutic management of patients. Thus, we welcome research papers from various disciplines related [but not limited to] hematopathologists, hematologists, immunologists and cell biologists with clinically relevant and innovative studies investigating individual-cell analytics and/or separations. In addition to the types of papers indicated above, we also welcome Letters to the Editor, describing case reports or important medical or technical topics relevant to our readership without the length and depth of a full original report.