Bicentric evaluation of stabilizing sampling tubes for assessment of monocyte HLA‐DR expression in clinical samples

Abstract

Background Diminished expression of human leukocyte antigen DR on circulating monocytes (mHLA‐DR), measured by standardized flow cytometry procedure, is a reliable indicator of immunosuppression in severely injured intensive care unit patients. As such, it is used as stratification criteria in clinical trials evaluating novel immunostimulating therapies. Preanalytical constraints relative to the short delay between blood sampling and flow cytometry staining have nevertheless limited its use in multicentric studies. The objective of the present work was to compare mHLA‐DR expression between whole blood samples simultaneously drawn in EDTA or Cyto‐Chex BCT tubes. Methods In two university hospitals, mHLA‐DR was assessed in fresh whole blood from septic patients (n = 12) and healthy donors (n = 6) simultaneously sampled on EDTA and Cyto‐Chex BCT tubes. Staining was performed immediately after sampling and after blood storage at room temperature. Results We confirmed that samples collected in Cyto‐Chex tube had substantially enhanced stability for mHLA‐DR results (48–72 h) over those collected in EDTA. On baseline values, despite good correlation between tubes (r = 0.98, p < 0.001), mHLA‐DR expression was systematically lower with Cyto‐Chex BCT. Conclusion The present reports confirms the potential of Cyto‐Chex BCT tubes to stabilize mHLA‐DR expression before staining and extends the work of Quadrini et al. [Cytometry B 2021;100:103‐114]. In centers without rapid access to flow cytometry facilities, it enables to tolerate delays in mHLA‐DR staining. However, a 30% gap exists between results obtained with EDTA and Cyto‐Chex BCT tubes. As current thresholds for clinical decisions were obtained with EDTA samples, further studies are needed to confirm clinical thresholds with Cyto‐Chex BCT tubes.

Monocyte HLA‐DR expression as an enrollment biomarker in sepsis clinical trials: Evaluation of two sampling tubes and definition of respective clinical thresholds

“…Recently, stabilizing sampling tubes (i.e., Cyto-Chex BCT tubes) were reported to stabilize mHLA-DR expression up to 72 h after sampling when stored at room temperature (Quadrini et al, 2021). We next confirmed these results in a small group of 12 COVID-19 patients (Hamada et al, 2022). However, although nicely…”

“…correlated, we observed a systematic À30% difference between mHLA-DR results obtained from Cyto-Chex BCT tubes versus standard-EDTA samples (Hamada et al, 2022). As current thresholds used in clinical practice have all been established with EDTA samples, it thus remains of utmost importance to further compare mHLA-DR values obtained with these two different sampling tubes in ICU patients presenting with wide range of mHLA-DR expression.…”

HLA-DR expression on monocytes and outcome of anti-CD19 CAR T-cell therapy for large B-cell lymphoma

“…Based on mHLA-DR at D−7 before CAR T-cell infusion, we defined 2 distinct groups of patients, using the threshold of 13 500 Ab/c, which was determined as the fifth percentile in a HV cohort and in accordance with the literature ( supplemental Figure 3 ). 24 , 29 , 35 , 36 , 37 , 38 , 39 Among the 103 patients, only 87 had mHLA-DR measured at D−7, and 10 (11%) had a low mHLA-DR D−7 (<13 500 Ab/c), whereas 77 (89%) had an mHLA-DR D−7 ≥13 500 Ab/c. We then compared these 2 groups of patients, considering clinical and biological values at D−7 before infusion.…”

“…Lower and upper values of reference intervals (ie, 13 500 and 35 200 Ab/c) were calculated according to international recommendations as the 5th and 95th percentiles of HV values ( supplemental Figure 3 ), in accordance with previously reported reference values, 24 , 29 , 35 , 36 , 37 , 38 and with the normal values from the routine immunology laboratory (ie, 13 500 Ab/c). 39 …”

Concomitant Assessment of Monocyte HLA-DR Expression and Ex Vivo TNF-α Release as Markers of Adverse Outcome after Various Injuries—Insights from the REALISM Study

“…These conditions may limit the accessibility of the technique in centers that do not have rapid access to flow cytometry facilities [ 32 ]. Recent data nevertheless suggest the possibility to measure mHLA-DR after blood collection in stabilizing sampling tubes allowing to quantify mHLA-DR expression up to 72 h after sampling and storage at room temperature [ 31 , 33 ]. Alternatively, a bedside protocol for flow cytometry measurement of mHLA-DR was recently proposed [ 34 ].…”