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The presence of94% classical monocytes (MO1, CD14++/CD16-) in peripheral blood (PB) has an excellent performance for the diagnosis of chronic myelomonocytic leukemia (CMML). However, the monocyte gating strategy is not well defined. The objective of the study was to compare monocyte gating strategies and propose an optimal one.This is a prospective, single center study assessing monocyte subsets in PB. First, we compared monocyte subsets using 13 monocyte gating strategies in 10 samples. Then we developed our own 10 color tube and tested it on 124 patients (normal white blood cell counts, reactive monocytosis, CMML and a spectrum of other myeloid malignancies). Both conventional and computational (FlowSOM) analyses were used.Comparing different monocyte gating strategies, small but significant differences in %MO1 and percentually large differences in %MO3 (nonclassical monocytes) were found, suggesting that the monocyte gating strategy can impact monocyte subset quantification. Then, we designed a 10-color tube for this purpose (CD45/CD33/CD14/CD16/CD64/CD86/CD300/CD2/CD66c/CD56) and applied it to 124 patients. This tube allowed proper monocyte gating even in highly abnormal PB. Computational analysis found a higher %MO1 and lower %MO3 compared to conventional analysis. However, differences between conventional and computational analysis in both MO1 and MO3 were globally consistent and only minimal differences were observed when comparing the ranking of patients according to %MO1 or %MO3 obtained with the conventional versus the computational approach.The choice of monocyte gating strategy appears relevant for the monocyte subset distribution test. Our 10-color proposal allowed satisfactory monocyte gating even in highly abnormal PB. Computational analysis seems promising to increase reproducibility in monocyte subset quantification.

High-quality data on bone marrow involvement (BMI) assessed by flow cytometry (FC) in follicular lymphoma (FL) is lacking.We set up a prospective protocol with a 10-color tube and acquisition of 500.000 leukocytes on a Nav flow cytometer for evaluation of BMI in FL by FC.FC was compared with a combination of histopathology and IGH gene rearrangement, which were considered the gold standard. We also compared BMI by FC with PET.Fifty-two patients were included (median 67 years, 54% female). BMI by FC was seen in 35 (67%), with a median involvement of 1.2% (interquartile range: 0.3%-7%) of leukocytes. Comparison with the gold standard revealed two false negatives and two false positives (potentially true involvement undetected by the gold standard). BMI by PET was seen in 14/46 (30%). Immunophenotype of FL in the bone marrow was highly heterogeneous. The most common phenotypic abnormality was dim expression of CD19 (0.5 log loss in 30% of patients). CD10 was negative in 13 (37%) and incompletely positive (overlap with the negative population) in a further 8 (28%) while entirely positive only in 14 (48%). Other abnormalities (loss of CD20, gain or loss of CD79b, expression of CD43, and substantial loss of CD45) were rare. Computational analysis by means of FlowSOM confirmed the heterogeneous phenotype, with FL from different patients clustering in unrelated metaclusters.BMI by FL was frequent and immunophenotype was heterogeneous. However, this protocol enabled detection of FL in bone marrow in the vast majority of patients with bone marrow involvement by the gold standard.

Immunohistochemicaland flow cytometric assessment of PD-L1 expression on tumor cells and immunecells might suppose a great benefit to identify patients undergoing immunotherapies. However, the use of different techniques and antibodies cancomplicate valid comparisons between results obtained in different trials, and even more because of small biopsy specimens can result infalse-negative PD-L1 evaluation. Herein we describe the accurate identification of PD-L1 on peripheralblood myeloid derived suppressor cells (MDSCs) in such a case.

Background Multiple myeloma (MM) is characterized by malignant plasma cells in the bone marrow with an associated immunosuppressive microenvironment. PD-L1 suppresses immune response and is overexpressed in MM plasma cells and MDSCs. PD-L1 plays an important role in tumor immune evasion and drug resistance. PD-L1 is considered a therapeutic target, yet some MM individuals do not respond to treatments with PD-L1 or PD-1 inhibitors. The aim of this study was to design a screening assay to identify PD-L1+ MDSCs using flow cytometry. Methods Using no-lyse no-wash methods, samples were labeled with Vybrant DyeCycle Violet Stain to identify nucleated cells and with antibodies to identify MDSCs. PD-L1 surface expression was studied ± PMA stimulation. Kinetics of surface expression of PD-L1 were studied over time. PD-L1 cell surface and cytoplasmic expression were compared. Competitive experiments with Durvalumab were studied. Samples were analyzed using Attune NxT Flow Cytometer. Results PD-L1 surface expression increased after stimulation, but showed variation among subjects. PD-L1 surface expression was highest after 1-5 minute stimulation. Cytoplasmic levels of PD-L1 were undetectable. Co-incubation with Durvalumab resulted in variable inhibitory PD-L1 profiles. Conclusions Since cytoplasmic PD-L1 is not found, PD-L1 may reveal steric changes in response to stimulation, which may be associated with a PD-L1 immunoregulatory mechanism affecting therapies. Assessment of PD-L1 folding may help to develop better treatment strategies or to predict therapy resistance. No-lyse no-wash methodologies show promise as an emerging strategy to model conformational changes in the target site. For research use only.

Trisomy 12 chronic lymphocytic leukemia (CLL) is phenotypically different from the rest of CLL cytogenetic subgroups. However, it is unknown whether this is also the case for trisomy 12 CLL-phenotype monoclonal B-cell lymphocytosis (MBL).We analyzed the expression of several markers in a series of 89 cytogenetically characterized MBL (including 17 trisomy 12 cases). Additionally, we compared the expression of these markers between trisomy 12 MBL, trisomy 12 CLL and a series of cases with trisomy 12 but fulfilling only 3 of the 5 Moreau CLL diagnostic criteria.CD20, CD22 and CD79b were expressed more intensely in trisomy 12 MBL than in non-trisomy 12 MBL and there was a trend towards a dimmer expression of CD43 in trisomy 12 MBL. There were no differences between trisomy 12 CLL and trisomy 12 MBL. Expression of CD20 was dimmer and that of CD43 brighter in trisomy 12 MBL than in cases with trisomy 12 and 3 Moreau criteria.In this study, trisomy 12 and non-trisomy 12 MBL were phenotypically different, but the differences are similar to those described between trisomy 12 and non-trisomy 12 CLL, suggesting that they are a distinct subgroup within CLL, given that the differences can already be found at its precursor stage. © 2017 International Clinical Cytometry Society.