The use of monocyte subset repartitioning by flow cytometry for diagnosis of chronic myelomonocytic leukaemia

11071 Background: Monocytes are intrinsic members of the innate immune system and play an important role in immunity and inflammation. Human monocytes are subdivided into three populations depending on cell surface CD14 and CD16 expression: Classical (CD14++ CD16-), intermediate (CD14+CD16- ) and non-classical (CD14- CD16++). These populations have diverse functions and have been postulated to play both anti and pro-inflammatory roles in a variety of diseases including atherosclerosis, sarcoidosis and other rheumatological conditions. However, the prevalent monocyte populations in cancer have not as yet been identified. We aim to define the prevalent monocyte populations in non-small cell lung cancer as well as further characterising them using flow cytometry and Affymetrix technology. Methods: Blood was obtained from 24 newly diagnosed patients with advanced non-small cell lung cancer and 12 age matched healthy donors. Monocyte subpopulations were sorted using flow cytometry. Gene expression profiling was performed using Affymetrix Human U133 Plus 2.0 array. Results: The classical (CD14++CD16-) monocyte population is significantly increased in non-small cell lung cancer patients when compared to healthy donors (p<0.01). The intermediate (CD14+CD16+) and non-classical (CD14- CD16++) populations are unchanged. Analysis of the gene expression profile of the classical monocyte subset identified 265 up-regulated and 261 down-regulated genes in cancer patients compared to healthy donors (p<0.05, fold change >2). These genes were assigned to biological processes with gene ontology annotation. Functional annotation reveals a strong association of regulated genes with the G.O term “inflammation” (p=0.009). Among these inflammatory genes are a cluster of chemokines including CXCL2 and CXCR4. Conclusions: By identifying the prevalent monocyte subsets as well as characterising their function in cancer, there is the potential to target detrimental effects and promote their beneficial functions. As such, monocytes subsets may be used as a possible therapeutic target.

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Inflammation Response Cytokines IFN-γ and IL-10 Regulate Monocyte Subset Differentiation

Blood ◽

10.1182/blood-2019-129515 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3586-3586

Author(s):

Hui Zhong ◽

Weili Bao ◽

Yunfeng Liu ◽

Karina Yazdanbakhsh

Keyword(s):

Flow Cytometry ◽

Inflammatory Response ◽

Inflammatory Disease ◽

Disease Risk ◽

Conflicts Of Interest ◽

Response To Treatment ◽

Monocyte Subset ◽

Monocyte Differentiation ◽

Ifn Γ

Circulating monocytes comprise of a heterogeneous and functionally-diverse cell population which based on surface markers can be divided into three subsets: classical (CMo), intermediate (IMo), and non-classical monocytes/patrolling monocytes (PMo). The frequency/number, gene expression profile and activity of IMo and PMo significantly change in a variety of inflammatory diseases with the changes associated with disease risk and severity as well as response to treatment. While it is believed that CMo differentiate into IMo and that IMo further differentiate into PMo, there is paucity of data on the mechanisms that alter CMo to IMo/PMo differentiation profiles in these conditions. In addition, factors which induce human and mouse IMo and PMo differentiation have yet to be identified. To screen cytokine/chemokine candidates affecting IMo/PMo differentiation, human monocytes were isolated from healthy donors (HD) and cultured with candidates (22 cytokines/chemokines) for 3 days. On day 3, IMo/PMo marker expression was examined by flow cytometry focusing on candidate molecules that led to increased expression of markers (CD16, CX3CR1, CD11c, HO-1, HLA-DR) whose levels are normally found to be higher in IMo/PMo and to decrease in expression of markers (CD14, CD36, CCR2) which are expressed at higher level in CMo as measured by mean fluorescence intension (MFI). We found that of all molecules tested, only two , IFN-γ and IL-10, had significant effects: IFN-γ (10ng/ml) increased expression of CX3CR1 (20 fold), CD16 (60%), HLADR (15%) and inhibited CD36 (42% inhibition) and CD14 expression (45% inhibition) while IL-10 (10ng/ml) increased CD16 (3.3 fold), CD11c (45%), HO-1 (59%) and CX3CR1 (6 fold) expression and inhibited CCR2 (60% inhibition) expression. These data suggest that IFN-γ and IL-10, two key cytokines involved in sterile and infectious inflammation, induce IMo and PMo differentiation. To test whether the effect of IFN-γ and IL-10 in human in vitro cultures can be replicated in vivo, wildtype B6 mice were I.V. injected with IFN-γ or IL-10 for 3 days: IFN-γ, IL-10, or the same volume of PBS. The frequencies of monocyte subsets in blood (gated on CD45+Ly6G-CD11bhighCD115+ for total monocyte population and CMo/IMo/PMo based on Ly6C expression level) on day 4 were analyzed by flow cytometry. We found that IFN-γ (2.5μg/injection/mice twice/day) significantly increased IMo frequencies (from 12% to 35%) but decreased PMo frequencies (from 38% to 26%) while IL-10 (0.25μg/injection/mice twice/day) significantly induced PMo differentiation (from 38% to 63%) without effect on IMo frequencies. The data suggest that IFN-γ increases IMo frequency by simultaneously inducing CMo differentiation into IMo and inhibiting IMo differentiation into PMo. We have previously reported lower PMo frequency in patients with sickle cell disease (SCD), considered an inflammatory disease with altered immune profiles. To test whether altered differentiation programming of IMo/PMo may contribute to reduced PMo frequency in SCD, we analyzed the frequency of CMo/IMo/PMo at baseline and after IFN-γ or IL-10 injection to mimic an inflammatory response in AA mice (expressing normal human hemoglobin) and SS mice (expressing human SCD hemoglobin). We found significantly lower IMo frequency before treatment (AA vs SS:15.0% vs 9.2%) but also lower induction of IMo following IFN-γ treatment in SS mice (18%) relative to AA mice (35%), suggesting that IFN-γ inhibition of IMo differentiation into PMo in SCD is impaired. Furthermore, IL-10 was less effective in inducing PMo in SS as compared to AA mice (SS vs AA: 40% vs 60%). These data suggest that IFN-γ or IL-10-mediated monocyte differentiation in SCD is altered. Altogether, these data have unraveled a novel role for IFN-γ or IL-10, two key cytokines known to be induced during an inflammatory response, in monocyte differentiation, and suggest that IMo/PMo differentiation in a chronic inflammatory disease such as SCD may be defective due to altered response to IFN-γ and IL-10, opening up the potential for identification of novel therapeutic targets for IMo/PMo associated diseases including SCD. Disclosures No relevant conflicts of interest to declare.

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Practical limitations of monocyte subset repartitioning by multiparametric flow cytometry in chronic myelomonocytic leukemia

Blood Cancer Journal ◽

10.1038/s41408-019-0231-7 ◽

2019 ◽

Vol 9 (9) ◽

Cited By ~ 6

Author(s):

Prateek A. Pophali ◽

Michael M. Timm ◽

Abhishek A. Mangaonkar ◽

Min Shi ◽

Kaaren Reichard ◽

...

Keyword(s):

Flow Cytometry ◽

Chronic Myelomonocytic Leukemia ◽

Monocyte Subset ◽

Multiparametric Flow Cytometry ◽

Myelomonocytic Leukemia

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High Diagnostic Utility of Flow Cytometry Based Peripheral Blood Monocyte Subset Analysis, CD56 and CD2 Expression in Chronic Myelomonocytic Leukemia (CMML)

Blood ◽

10.1182/blood-2019-130499 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5437-5437

Author(s):

Xavier Calvo ◽

Ivonne Parraga ◽

Nieves Garcia-Gisbert ◽

Lourdes Florensa ◽

Sara Montesdeoca ◽

...

Keyword(s):

Flow Cytometry ◽

Peripheral Blood ◽

Screening Test ◽

Molecular Data ◽

Youden Index ◽

Monocyte Subset ◽

Monocyte Subsets ◽

Clin Pathol ◽

Number Of Patients ◽

Classical Monocytes

INTRODUCTION The diagnosis of CMML according to WHO 2017 requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB). Establishing an accurate diagnostic is difficult since many clinical situations present persistent monocytosis. The presence of dysplasia is frequent but not always present and cytogenetic aberrations are infrequent in this disease (20-25% of cases). Although 85-90% of CMML patients present ≥1 mutation in TET2, SRSF2 or ASXL1, the use of NGS panels is not widespread. The study of PB monocyte subsets by flow cytometry (FC) has gained interest for CMML diagnosis. The increase of classical monocytes (Mo1) upper 94% presents a high sensitivity (Sn) and specificity (Sp) for CMML diagnosis (Sn 90.6, Sp 95.1; Selimoglu-Buet et al, Blood 2015). The 94% threshold was validated in two studies (Talati C et al, Blood 2017; Tarfi S et al, Blood Cancer J 2018). However, some controversies have recently appeared in the literature. Picot T detected the 95% cutoff as the one with the best Sn (100%) and Sp (97%) (Picot T et al, Front Oncol 2018). Hudson CA found that the presence of < 1.13% (Sn 100, Sp 96) of non-classical monocytes (Mo3) was the best predictor for CMML diagnosis (Hudson CA et al, Am J Clin Pathol 2018). With the exception of the study of Tarfi S, based on 47 CMML, the rest presented a very low number of patients (Talati C: 29; Picot T: 15; Hudson CA: 16) and therefore a bias could be expected specially when studying the Sn of the proposed methods. Moreover, the different series assessing the "monocyte assay" have no molecular data and therefore this could diminish the accuracy of the results since some patients may have received misdiagnoses. The aim of our study was to assess the Sn and Sp of different thresholds of Mo1 and Mo3 in a large series with well-annotated clinical, cytogenetic and molecular data. Moreover, we assessed whether the study of CD2 and CD56 monocyte expression in combination with the %Mo1 >94 test improves the detection of the disease. METHODS 50 CMML, 12 MDS, 11 MPN with ≥1x109/L monocytes and 79 reactive monocytosis with ≥1x109/L monocytes (N = 152) were prospectively studied from 02/2016 to 07/2019. We studied PB monocyte subsets by FC: Mo1 (CD14bright/CD16-), Mo2 (CD14bright/CD16+) and Mo3 (CD14dim or -/CD16bright). In addition, we assessed the expression of CD56 and CD2 in monocytes (positivity ≥ 20%). Finally, targeted NGS of the entire exonic sequence of 25 genes recurrently mutated in myeloid malignancies was performed (VAF sensitivity: 2%). Chi-Square or Fisher exact tests were used as appropriate. ROC curves were developed to explore optimal cutoffs in terms of sensitivity (Sn) and specificity (Sp). Moreover, we plotted the AUC of the subset of Mo1 and Mo3. Finally, the Youden index (YI) was used to detect the threshold of Mo1 and Mo3 with the best balance between Sn and Sp. RESULTS AND DISCUSSION The Sn and Sp of the Mo1>94% test in our series were similar to those reported by the French group (GFM). Our Sn and Sp were 90% and 92% respectively with a YI of 82. The Sn and Sp of the Mo1>93% were 94% and 84% with a YI of 78. Finally, the 95% cutoff proposed by Picot T et al showed a Sn of 81% and a Sp of 96% with a YI of 77. Therefore, the 94% cutoff presented the best balance between Sn and SP of the different thresholds assessed. The Mo3 threshold of 1.13% proposed by Hudson CA et al showed a Sn of 67% and a Sp of 95% with a YI of 62. The best Mo3 cutoff in our series was established in 3.18% with a Sn of 90% and Sp of 83%. The YI of this threshold was 73. The AUC for the percentage (%) of Mo1 (0.937, IC 95%: 0.89-0.99) was better than the AUC of the % of Mo3 (0.924, IC 95%: 0.88-0.97) reinforcing the use of %Mo1 as the item with the best discriminative power for CMML diagnosis. The AUC of the percentage of Mo1 population was similar to that reported by the GFM (Figure 1). The Sn and Sp for CD56 expression in monocytes was 67% and 91% respectively, while CD2 expression showed a Sn of 38% and a Sp of 99%. Finally, the presence of at least one of the following: Mo1 >94%, CD56+ or CD2+ presented the highest Sn (98%) and a Sp of 84%. This method may be a very good screening test due to the low false negative rate expected. This combined approach showed the best balance between Sn and Sp (YI: 82). CONCLUSIONS Our study supports the utility of the Mo1 >94% test as the best flow cytometry assay for establishing accurate diagnoses in CMML. The combined assay of Mo1, CD56 and CD2 may be of high utility as a screening test. Figure 1 Disclosures Bellosillo: Qiagen: Consultancy, Speakers Bureau; TermoFisher Scientific: Consultancy, Speakers Bureau.

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The Use of Monocyte Subset Repartitioning By Flow Cytometry for Diagnosis of Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood-2020-140966 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 41-42

Author(s):

Aarya Murali ◽

Donna Cross ◽

Peter Mollee

Keyword(s):

Flow Cytometry ◽

Board Of Directors ◽

Real World ◽

Peripheral Blood ◽

Myeloproliferative Neoplasm ◽

Monocyte Subset ◽

Monocyte Count ◽

Advisory Committees ◽

Clin Pathol ◽

Blood Samples

Introduction Patients with chronic myelo-monocytic leukaemia (CMML) have been reported to have a relative predominance of classical or MO1 monocytes (CD14+/CD16-) at the expense of MO2 (CD14low/CD16+) and MO3 (CD14-/CD16+) monocytes (Selimoglu-Buet. Blood 2015). These authors suggested that an MO1 percentage cut-off of >94% could predict the diagnosis of CMML with high sensitivity and specificity (both >90%) from other causes of monocytosis. Since then, several independent groups have attempted to reproduce their protocol with variable results. Most recently, the Mayo Clinic (Pophali. Blood Cancer J. 2019) found that an MO1 cut off of > 94% in peripheral blood identified CMML with a sensitivity of 75% and a specificity of 95.4%. These figures were lower than previously reported - calling into question, the utility of flow cytometry to distinguish the aetiology of monocytosis in a real-world setting. Objective Our retrospective audit aimed to establish if monocyte subset repartitioning could be used to reliably diagnose CMML in a real-world sample of patients with monocytosis. Methods In this study, we assessed peripheral blood samples from 35 patients presenting with a monocytosis (absolute monocyte count > 1 x109/L) in a tertiary referral hospital in Brisbane, Australia between June 2015 and Sep 2019. The patients' final clinical diagnosis was extracted from the medical record by two clinicians (AM, PM) blinded to the results of the flow cytometry. Peripheral blood samples were subjected to MFC at a median of <24 hours (range, < 24 hours to 160 hours) after collection. Flow cytometry was performed using the BD FACS Canto II flow cytometer. Monocyte subsets were identified using Kaluza software (Beckman Coulter, USA). A CD45/ side scatter gate was set to locate the monocyte population and specific antibody combinations were used to identify and exclude other lineages; these were - CD24 to exclude granulocytes and B cells, CD16 to exclude neutrophils, CD2 to exclude T cells and CD56 to exclude NK cells. Based on the CD14 and CD16 expression, the monocytes were then divided into: MO1 (CD14+/CD16-), MO2 (CD14low/CD16+) and MO3 (CD14-/CD16+). Results are reported descriptively and Fishers Exact Test (IBM® SPSS® Statistics Version 26) was used to establish if there was any correlation between the percentage of classical monocytes and the diagnosis of CMML. Results Of the 35 patients included, 13 patients had CMML and four patients were diagnosed with another underlying myeloid neoplasm: one patient with myelodysplastic syndrome (MDS-RCMD), one patient with myeloproliferative neoplasm - not otherwise specified (MPN-NOS), one patient with multiple myeloma and one patient with acute myeloid leukaemia with myelodysplasia related changes (AML-MRC). Eighteen cases had non-clonal monocytosis. Six patients had a reactive monocytosis in the setting of autoimmune disease including: granulomatosis with polyangiitis (n=2), rheumatoid arthritis (n=1), IgG4 disease (n=1), mixed connective tissue disease (n=1) and polyarticular gout (n=1). Twelve cases of reactive monocytosis were due to infective and inflammatory causes. Among our 13 patients with CMML, seven cases (53.85%) had an MO1 percentage > 94%. In comparison, only four (18.18%) among the 22 cases of non-CMML were identified to have an MO1 percentage > 94%. There was no correlation between an MO1 percentage cut off of > 94% and a diagnosis of CMML (p = 0.057). Our study also examined the utility of an MO3 percentage cut off of < 1.13%, established by Hudson et al (Am J Clin Pathol 2018). Among our 13 cases of CMML, six patients (46.15%) were noted to have an MO3 percentage < 1.13%. Meanwhile, only eight, out of 22 non-CMML patients (36.36%) had MO3 percentage < 1.13%. There was no association between MO3 percentage and the diagnosis of CMML (p = 0.724). Conclusion Using the MO1 and MO3 percentage cut offs previously established, we were unable to reliably diagnose CMML. Given these findings, we suggest that more research with larger sample sizes is required before monocyte subset analysis can be applied in the clinical laboratory to discriminate reactive monocytosis from CMML. Disclosures Mollee: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees.

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ELN IMDS Flow Working Group Validation of the Monocyte Assay for Chronic Myelomonocytic Leukemia Diagnosis By Flow Cytometry

Blood ◽

10.1182/blood-2021-145159 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2602-2602

Author(s):

Orianne Wagner-Ballon ◽

Peter Bettelheim ◽

Jeroen Lauf ◽

Frauke Bellos ◽

Matteo G. Della Porta ◽

...

Keyword(s):

Flow Cytometry ◽

Multicenter Study ◽

Working Group ◽

Complete Blood Count ◽

High Sensitivity ◽

Chronic Myelomonocytic Leukemia ◽

Monocyte Subset ◽

Global Correlation ◽

Skilled Operator ◽

Myelomonocytic Leukemia

Abstract Introduction It was proposed that peripheral blood (PB) monocyte subset analysis evaluated by flow cytometry, hereafter referred to as "monocyte assay", could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay required a large multicenter validation. Methods PB and/or bone marrow (BM) samples from adult patients displaying monocytosis were assessed with the "monocyte assay" by ten ELN iMDS Flow working group centers (6 equipped with BD FACSCanto™ II (BD Biosciences), 3 with Navios™ (Beckman Coulter) and one with BD™ LSRII (BD Biosciences)) with harmonized protocols. The corresponding files were reanalyzed in a blind fashion by a skilled operator and the cMo (CD14 ++CD16 -) percentages obtained by both analyses were compared. Information regarding age, gender, complete blood count, marrow cytomorphology, cytogenetics and molecular analysis was collected. Confirmed diagnoses were collected when available as well as follow-up for CMML patients. Results The comparison between cMo percentages from 267 PB files provided by the 10 centers and the centralized cMo percentages showed a good global significant correlation (r=0.88; p<0.0001; FigA) with no bias (FigB). Confirmed diagnoses were available for 212 files, namely 101 CMML according to the WHO criteria, 99 reactive monocytosis, and 12 MPN with monocytosis. A phenotype in favor of CMML, either classical with accumulation of cMo ≥94% or a bulbous aspect (FigC), was observed respectively in 81 and 14 patients. Hence, a total of 95 out of the 101 CMML patients translated into a sensitivity of 94% (FigD). Assessment of C reactive protein counts were available in seven of the 14 patients with the characteristic bulbous profile and correlated with an inflammatory state, showing a median of 93.0 [7.0-157.4] mg/L. Conversely, a phenotype not in favor of CMML (FigC) was observed in 83 of the 99 patients with reactive monocytosis and in 10 of 12 patients with MPN with monocytosis, leading to a 84% specificity (FigD). We established a Receiver Operator Curve (ROC) and again obtained a 94% cut-off value of cMo with an area under the ROC curve (AUC) of 0.865 (FigE). The second aim of this multicenter study was to assess the feasibility of the monocyte assay on 117 BM samples provided by 7 out of the 10 ELN centers, 43 of which being paired to PB samples. The comparison between cMo percentages provided by the 7 centers and the centralized cMo percentages showed a lower global significant correlation compared to PB samples (r=0.74; p<0.0001; FigF) with a slight underestimation of cMo percentage by the participating centers (FigG). The comparison between PB and BM samples cMo% obtained by centralized reanalysis showed an excellent global correlation (r=0.93; p<0.0001; FigH) with a higher percentage in the marrow (FigI). Seventy-nine files were associated to a confirmed diagnosis, as expected mostly CMML (n=69), only seven reactive monocytosis and three MPN with monocytosis. Thus, we determined a sensitivity of the "monocyte assay" on BM samples of 87% (a phenotype in favor of CMML being observed in 60 out of the 69 CMML with 6 bulbous aspect profiles) and a specificity of 80% (a phenotype not in favor of CMML being observed in 5 of the 7 patients with reactive monocytosis and in 3 of the 3 patients with MPN with monocytosis). Conclusions This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership.

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Differential modulatory effects of Propofol and Sevoflurane anesthesia on blood monocyte HLA-DR and CD163 expression during and after cardiac surgery with cardiopulmonary bypass: a preliminary randomized flow cytometry study

Perfusion ◽

10.1177/0267659119848295 ◽

2019 ◽

Vol 35 (1) ◽

pp. 48-56

Author(s):

Silverio Sbrana ◽

Anna Nunziata ◽

Simona Storti ◽

Dorela Haxhiademi ◽

Annamaria Mazzone ◽

...

Keyword(s):

Flow Cytometry ◽

Cardiac Surgery ◽

Cardiopulmonary Bypass ◽

Lactate Dehydrogenase ◽

Blood Monocyte ◽

Compensatory Mechanism ◽

Monocyte Subset ◽

C Reactive Protein ◽

Reactive Protein ◽

Hla Dr

Introduction: The increase of the anti-inflammatory CD163highHLA-DRlow blood monocyte subset is one of the mechanisms dampening inflammation during cardiac surgery with cardiopulmonary bypass. We evaluated the effect of two different anesthetic protocols, intravenous Propofol infusion or Sevoflurane-gas administration, on the perioperative frequency of this subset. Methods: Blood from patients (Propofol = 11, Sevoflurane = 13) undergoing minimally invasive mitral valve surgery was drawn preoperatively (T1), before declamping (T2), at 6 (T3), 24 (T4), 48 (T5), and 72 hours (T6) after declamping. C-reactive protein, haptoglobin, and lactate dehydrogenase were measured. A hemolytic index, as C-reactive protein/haptoglobin ratio, was introduced. Monocyte expression of HLA-DR, CD163, and the CD163highHLA-DRlow subset fraction was quantified by flow cytometry. Baseline-referred variations of plasmatic and cellular data at T2 were normalized for clamping times. Subsequent time-point variations were normalized for the final cardiopulmonary bypass times. Results: Variations of hemolytic index and lactate dehydrogenase were higher with Propofol at T3 (p = 0.004 and p = 0.02, respectively) when compared with Sevoflurane. At T2, the down-modulation of CD163 was higher with Propofol (p = 0.005). Starting from T3, the up-regulatory trend of CD163 was basically higher with Propofol, although not significantly. Propofol induced higher increments of HLA-DR low fractions, at T2 (p = 0.04) and, to a lesser extent, at T4 (p = 0.06). Starting from T3, the CD163highHLA-DRlow subset variations were higher with Propofol, especially at T4 and T6. Conclusion: Propofol seems to induce a higher postoperative fraction of the CD163highHLA-DRlow monocyte subset. This could represent either a compensatory mechanism dampening the higher inflammatory condition observed with Propofol at T2 or a consequence of a higher postoperative Propofol-induced hemolysis.

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