Purpose Natural killer (NK) cells are traditionally identified by flow cytometry using a combination of markers (CD16/CD56/CD3), because a specific NK-cell marker is still missing. Here we ...investigated the utility of CD314, CD335 and NKp80, compared to CD16/CD56/CD3, for more robust identification of NK-cells in human blood, for diagnostic purposes. Methods A total of 156 peripheral blood (PB) samples collected from healthy donors (HD) and patients with diseases frequently associated with loss/downregulation of classical NK-cell markers were immunophenotyped following EuroFlow protocols, aimed at comparing the staining profile of total blood NK-cells for CD314, CD335 and NKp80, and the performance of distinct marker combinations for their accurate identification. Results NKp80 showed a superior performance (vs. CD314 and CD335) for the identification of NK-cells in HD blood. Besides, NKp80 improved the conventional CD16/CD56/CD3-based strategy to identify PB NK-cells in HD and reactive processes, particularly when combined with CD16 for further accurate NK-cell-subsetting. Although NKp80+CD16 improved the identification of clonal/tumor NK-cells, particularly among CD56 - cases (53%), aberrant downregulation of NKp80 was observed in 25% of patients, in whom CD56 was useful as a complementary NK-cell marker. As NKp80 is also expressed on T-cells, we noted increased numbers of NKp80 + cytotoxic T-cells at the more advanced maturation stages, mostly in adults. Conclusion Here we propose a new robust approach for the identification of PB NK-cells, based on the combination of NKp80 plus CD16. However, in chronic lymphoproliferative disorders of NK-cells, addition of CD56 is recommended to identify clonal NK-cells, due to their frequent aberrant NKp80 - phenotype.
A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cytometry (FCM) ...marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1+ and TRBC1− monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1+/TRBC1− ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ families, whereas highly-variable TRBC1+/TRBC1− ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10−4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ+ leukemia/lymphoma patients.
Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor β chain (TRBC1) expression for assessing Tαβ-cell clonality has been recently validated. However, its utility for the ...diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαβ cells (i.e., T-LGL normal-counterpart) show broader TRBC1
/TRBC1
ratios vs. total Tαβ cells. We compared the distribution and absolute counts of TRBC1
and TRBC1
Tαβ-LGL in blood containing polyclonal (
= 25) vs. clonal (
= 29) LGL. Overall, polyclonal TRBC1
or TRBC1
Tαβ-LGL ranged between 0.36 and 571 cells/μL (3.2-91% TRBC1
cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/μL (<0.9% or >96% TRBC1
cells). Among the distinct TCRVβ families, the CD28
effector-memory and terminal-effector polyclonal Tαβ cells ranged between 0 and 25 TRBC1
or TRBC1
cells/μL and between 0 and 100% TRBC1
cells, while clonal LGL ranged between 32 and 5515 TRBC1
or TRBC1
cells/μL, representing <1.6% or >98% TRBC1
cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαβ-LGL suspected of T-LGLL based on altered percentages of TRBC1
Tαβ cells. However, in the absence of lymphocytosis or in the case of TαβCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαβ-LGL-expressing individual TCRVβ families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.
For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the ...effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of therapy emerged as an independent unfavorable prognostic factor for DFS in childhood BCP-ALL in the discovery cohort—hazard ratio (95% confidence interval) of 2.50 (1−9.66); p = 0.05—together with the BM MRD status (p = 0.031). Further investigation of the predictive value of the combination of these two variables (%EC within stromal cells and MRD status at day +78) allowed classification of BCP-ALL into three risk groups with median DFS of: 3.9, 3.1 and 1.1 years, respectively (p = 0.001). These results were confirmed in two validation cohorts of childhood BCP-ALL (n = 74) (p = 0.001) and adult BCP-ALL (n = 40) (p = 0.004) treated at different centers. In summary, our findings suggest that an imbalanced EC/MSC ratio in BM at day +78 of therapy is associated with a shorter DFS of BCP-ALL patients, independently of their MRD status. Further prospective studies are needed to better understand the pathogenic mechanisms involved.
In recent years the volume and complexity of flow cytometry data has increased substantially. This has led to a greater number of identifiable cell populations in a single measurement. Consequently, ...new gating strategies and new approaches for cell population definition are required. Here we describe how the EuroFlow Lymphoid Screening Tube (LST) reference data base for peripheral blood (PB) samples was designed, constructed and validated for automated gating of the distinct lymphoid (and myeloid) subsets in PB of patients with chronic lymphoproliferative disorders (CLPD). A total of 46 healthy/reactive PB samples which fulfilled pre-defined technical requirements, were used to construct the LST-PB reference data base. In addition, another set of 92 PB samples (corresponding to 10 healthy subjects, 51 B-cell CLPD and 31 T/NK-cell CLPD patients), were used to validate the automated gating and cell-population labeling tools with the Infinicyt software.
An overall high performance of the LST-PB data base was observed with a median percentage of alarmed cellular events of 0.8% in 10 healthy donor samples and of 44.4% in CLPD data files containing 49.8% (range: 1.3–96%) tumor cells. The higher percent of alarmed cellular events in every CLPD sample was due to aberrant phenotypes (75.6% cases) and/or to abnormally increased cell counts (86.6% samples). All 18 (22%) data files that only displayed numerical alterations, corresponded to T/NK-cell CLPD cases which showed a lower incidence of aberrant phenotypes (41%) vs B-cell CLPD cases (100%). Comparison between automated vs expert-bases manual classification of normal (r2 = 0.96) and tumor cell populations (rho = 0.99) showed a high degree of correlation.
In summary, our results show that automated gating of cell populations based on the EuroFlow LST-PB data base provides an innovative, reliable and reproducible tool for fast and simplified identification of normal vs pathological B and T/NK lymphocytes in PB of CLPD patients.
Myelodysplastic syndromes and acute myeloid leukemia (AML) are heterogeneous disorders in which conflicting results in apoptosis and multidrug resistance (MDR) have been reported. We have evaluated ...by multiparameter flow cytometry the expression of apoptosis- (APO2.7, bcl-2, and bax) and MDR-related proteins P-glycoprotein (P-gp), multidrug resistance protein (MRP), and lung resistance protein (LRP) specifically on bone marrow (BM) CD34+ cells, and their major CD32-/dim and CD32+ subsets, in de novo AML (n=90), high-risk myelodysplastic syndrome (n=9), and low-risk myelodysplastic syndrome (n=21) patients at diagnosis, and compared with normal BM CD34+ cells (n=6). CD34+ myeloid cells from AML and high-risk myelodysplastic syndrome patients displayed higher expression of bcl-2 (P <0.0001) and lower reactivity for APO2.7 (P=0.002) compared with low-risk myelodysplastic syndrome and normal controls. Similar results applied to the two predefined CD34+ myeloid cell subsets. No significant differences were found in the expression of P-gp, MRP, and LRP between low-risk myelodysplastic syndrome patients and normal BM, but decreased expression of MRP (P <0.03) in AML and high-risk myelodysplastic syndromes and P-gp (P=0.008) in high-risk myelodysplastic syndromes were detected. Hierarchical clustering analysis showed that low-risk myelodysplastic syndrome patients were clustered next to normal BM samples, whereas high-risk myelodysplastic syndromes were clustered together and mixed with the de novo AML patients. In summary, increased resistance to chemotherapy of CD34+ cells from both AML and high-risk myelodysplastic syndromes would be explained more appropriately in terms of an increased antiapoptotic phenotype rather than a MDR phenotype. In low-risk myelodysplastic syndromes abnormally high apoptotic rates would be restricted to the CD34- cell compartments.
Summary
The standardized EuroFlow protocol, including CD19 as primary B‐cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B‐cell precursor acute ...lymphoblastic leukaemia (BCP‐ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP‐ALL patients treated with CD19‐targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19‐positive, whereas this was 81% in patients that became (partially) CD19‐negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.
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