Immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes. A report from the IMDSFlow working group.

Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117+ erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84-94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86-97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage.

Fast and accurate prediction of positive and negative urine cultures by flow cytometry.

BACKGROUND: Urinary tract infection (UTI) is a widespread infectious disease in humans. Urine culture, a huge workload in the microbiology laboratory, is still the standard diagnostic test for UTI, but most of the cultures are negative. A reliable screening method could reduce unnecessary cultures and quicken reporting of negative results. METHODS: We evaluated the usefulness of a flow cytometry (FC) screening method in the prediction of positive urine culture to reduce the number of urine cultures. The urine specimens sent to the laboratory for culture were tested with the flow cytometer Accuri C6. FC bacterial counts were compared to standard urine culture results to assess the best cut-off values. RESULTS: Two hundred nine urine samples were included, of which 79 (37.8 %) were culture positive. On comparing the culture and the FC data in the ROC curve, the FC bacterial counts of ≥10(6) bacteria/mL provided a reliable screening for bacteriuria with a sensitivity and specificity of 99 and 58 %, respectively. All negative FC results (<10(6) bacteria/mL) showed a negative predictive value of 99 % with a negative likelihood ratio of 0.02. The FC bacterial counts of ≥10(8)/mL showed a positive predictive value of 99 % with a positive likelihood ratio of 60.9. CONCLUSIONS: Counting bacteria in human urine samples by the FC is a fast, accurate and cost-effective screening method for bacteriuria. Our results showed that FC is able to rule out UTI, which can lead to a substantial reduction (36 %) of urine cultures. It also demonstrated that this method predicts positive cultures accurately.

Identification of a small subpopulation of candidate leukemia-initiating cells in the side population of patients with acute myeloid leukemia.

In acute myeloid leukemia (AML), apart from the CD34(+)CD38(-) compartment, the side population (SP) compartment contains leukemic stem cells (LSCs). We have previously shown that CD34(+)CD38(-) LSCs can be identified using stem cell-associated cell surface markers, including C-type lectin-like molecule-1 (CLL-1), and lineage markers, such as CD7, CD19, and CD56. A similar study was performed for AML SP to further characterize the SP cells with the aim of narrowing down the putatively very low stem cell fraction. Fluorescence-activated cell sorting (FACS) analysis of 48 bone marrow and peripheral blood samples at diagnosis showed SP cells in 41 of 48 cases that were partly or completely positive for the markers, including CD123. SP cells in normal bone marrow (NBM) were completely negative for markers, except CD123. Further analysis revealed that the SP fraction contains different subpopulations: (a) three small lymphoid subpopulations (with T-, B-, or natural killer-cell markers); (b) a differentiated myeloid population with high forward scatter (FSC(high)) and high sideward scatter (SSC(high)), high CD38 expression, and usually with aberrant marker expression; (c) a more primitive FSC(low)/SSC(low), CD38(low), marker-negative myeloid fraction; and (d) a more primitive FSC(low)/SSC(low), CD38(low), marker-positive myeloid fraction. NBM contained the first three populations, although the aberrant markers were absent in the second population. Suspension culture assay showed that FSC(low)/SSC(low) SP cells were highly enriched for primitive cells. Fluorescence in situ hybridization (FISH) analyses showed that cytogenetically abnormal colonies originated from sorted marker positive cells, whereas the cytogenetically normal colonies originated from sorted marker-negative cells. In conclusion, AML SP cells could be discriminated from normal SP cells at diagnosis on the basis of expression of CLL-1 and lineage markers. This reveals the presence of a low-frequency (median, 0.0016%) SP subfraction as a likely candidate to be enriched for leukemia stem cells.

Relationship between CD34/CD38 and side population (SP) defined leukemia stem cell compartments in acute myeloid leukemia.

Leukemic stem cells (LSCs), defined by CD34/CD38 expression, are believed to be essential for leukemia initiation and therapy resistance in acute myeloid leukemia. In addition, the side population (SP), characterized by high Hoechst 33342 efflux, reflecting therapy resistance, has leukemia initiating ability. The purpose of this study is, in both CD34-positive and CD34-negative AML, to integrate both types of LSC compartment into a new more restricted definition. Different CD34/CD38/SP defined putative LSC and normal hematopoietic compartments, with neoplastic or normal nature, respectively, were thus identified after cell sorting, and confirmed by FISH/PCR. Stem cell activity was assessed in the long-term liquid culture stem cell assay. SP fractions harbored the strongest functional stem cell activity in both normal and neoplastic cells in both CD34-positive and CD34-negative AML. Overall, inclusion of SP fraction decreased the size of the putative CD34/CD38 defined LSC compartment by a factor >500. For example, for the important CD34+CD38- LSC compartment, the median SP/CD34+CD38- frequency was 5.1 per million WBC (CD34-positive AML), and median SP/CD34-CD38+ frequency (CD34-negative AML) was 1796 per million WBC. Improved detection of LSC may enable identification of therapy resistant clones, and thereby identification of novel LSC specific, HSC sparing, therapies.

Flow cytometric analysis of viable bacteria in urine samples of febrile patients at the emergency department.

BACKGROUND: Fast and reliable diagnostics are important in febrile patients admitted to the emergency department. Current urine diagnostics are fast but moderately reliable or reliable but time consuming. Flow cytometry (FC) is a new promising technique in the diagnostics of complicated urinary tract infections by counting bacteria in urine samples. The aim of this study is to improve the FC method by counting only viable bacteria. METHODS: Urine was obtained from 135 consecutive febrile patients at the emergency department. According to protocol regular diagnostic urine tests were performed. In addition, FC counting of viable and non-viable bacteria was executed after staining with thiazole orange and propidium iodide. All test results were compared to the results of urine culture (≥ 105 colony forming units/mL). RESULTS: At a cut-off value of 2.01 × 105 viable bacteria/mL the sensitivity was 100% and specificity was 78.4% (AUC-value 0.955 on ROC-curve). Spearman correlation test exhibited a higher correlation for flow cytometric counting of only viable bacteria than counting of all bacteria (0.59 vs. 0.37). Using ROC-curves, the AUC-values for FC counting of all bacteria, only viable bacteria and Gram staining were respectively 0.935, 0.955, and 0.968 (P > 0.05). CONCLUSION: FC counting of only viable bacteria can predict quickly and reliably positive and negative urine cultures in febrile patients admitted to the emergency department. It can help to improve the speed and accuracy of the diagnostic procedure at the emergency department. © 2017 Clinical Cytometry Society.

Accurate and fast urinalysis in febrile patients by flow cytometry.

BACKGROUND: The urine culture is worldwide accepted as the gold standard in diagnosing urinary tract infections, but is time consuming and costly, other methods are fast but moderately reliable. We investigated whether counting the number of bacteria by flow cytometry could be a fast and accurate method to analyze urine samples in febrile patients at the emergency department (ED). METHODS: Urine samples were obtained from 140 febrile patients at the ED. Urinalysis was performed according to standard procedures. Flow cytometric analysis for bacteria was performed with the Accuri C6 flow cytometer. Diagnostic values were determined at various cut-off points by using urine culture as the gold standard. RESULTS: The highest diagnostic accuracy of urinalysis of bacteria was obtained with flow cytometric analysis (AUC of 0.96). The best cut-off value for bacteria counted by flow cytometry based on the ROC-curve was 3.72 × 106 bacteria/mL, this resulted in a sensitivity of 94.7% and a specificity of 88.2%. CONCLUSIONS: Counting bacteria by flow cytometry has the highest diagnostic accuracy and is superior to other methods in urinalysis in febrile patients in the ED when using urine culture as the gold standard.

Assessment of the normal or leukemic nature of CD34+ cells in acute myeloid leukemia with low percentages of CD34 cells.

BACKGROUND AND OBJECTIVES: The percentages of CD34+ cells in the bone marrow of patients with acute myeloid leukemia (AML) vary widely. Especially in the low range (<5% CD34+ cells), the nature (normal or malignant) of the CD34+ cells is uncertain. Since only in a minority of cases are molecular techniques applicable, in this study we explored a multiparameter approach using phenotypic and functional characteristics to discriminate normal CD34+ cells from malignant ones. DESIGN AND METHODS: CD34+ cells from 24 AML patients with <5% CD34+ cells and from 3 patients with >50% CD34+ cells were studied immunophenotypically for aberrant phenotypes, CD133 and CD90 expression and for P-glycoprotein activity. RESULTS: In the low (0.02-0.7%) CD34+ range, our approach offered strong evidence for a normal origin of the CD34+ cells in 18/19 cases, which was confirmed by interphase fluorescent in situ hybridization on sorted CD34+ cells in 3 cases, which had concomitant presence of cytogenetic abnormalities in the CD34- blasts. In contrast, in the intermediate (1.6-3.5%) CD34+ range, the CD34+ cells appeared as normal in only 1/5 cases. In the high (51-67%) CD34+ range, as expected the majority of CD34+ cells were malignant, although in 2/3 cases a small subpopulation (i.e. 0.15% and 0.20%) of CD34+ cells were of normal origin. INTERPRETATION AND CONCLUSIONS: Our multiparameter approach enabled us to define the nature of CD34+ cells in AML. This has implications for studies dealing with the characterization of primitive malignant cells. Moreover, it enabled identification of truly CD34 negative AML, which would be eligible for CD34-based immunological purging of autologous stem cell transplants.

Rationale for the clinical application of flow cytometry in patients with myelodysplastic syndromes: position paper of an International Consortium and the European LeukemiaNet Working Group.

An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

Implementation of flow cytometry in the diagnostic work-up of myelodysplastic syndromes in a multicenter approach: report from the Dutch Working Party on Flow Cytometry in MDS.

Flow cytometry (FC) is recognized as an important tool in the diagnosis of myelodysplastic syndromes (MDS) especially when standard criteria fail. A working group within the Dutch Society of Cytometry aimed to implement FC in the diagnostic work-up of MDS. Hereto, guidelines for data acquisition, analysis and interpretation were formulated. Based on discussions on analyses of list mode data files and fresh MDS bone marrow samples and recent literature, the guidelines were modified. Over the years (2005-2011), the concordance between the participating centers increased indicating that the proposed guidelines contributed to a more objective, standardized FC analysis, thereby ratifying the implementation of FC in MDS.

The new violet laser dye, Krome Orange, allows an optimal polychromatic immunophenotyping based on CD45-KO gating.

BACKGROUND: Polychromatic immunophenotyping improves characterization of leukocyte subpopulations and their malignant counterparts. However, the lack of various fluorochrome-labeled monoclonal antibodies (MoAbs) hinders the formation of multi-color panels. CD45 appears to be an important MoAb for immunophenotyping of these cells. Plotted against the side scatter, CD45 provides immunological cell differentiation and the ability to recognize various normal and malignant leukocyte subpopulations. CD45 is commonly used and labeled with various fluorochromes and as a result, is incorporated in multi-color panels as a conjugate of less available fluorochromes, such as the violet laser dyes. However, these dyes (e.g. Pacific Orange/PO) often possess low fluorescence intensity, which may be too weak to differentiate between populations. The new organic dye Krome Orange (KO, emission at 528 nm) appears to be a more intense violet laser dye, serving as an alternative to PO. METHODS: Intensities of CD45 conjugated with FITC, PE, ECD, PE-Cy5, PE-Cy7, PO and KO were tested in different cell sources. Various lineage markers were sequentially back gated on CD45-KO to identify subpopulations. A 10-color MoAb panel for determination of aberrancies in small cell samples was composed to test specificity of CD45-KO. CONCLUSIONS: We showed in various fixed and unfixed cells from different sources that KO is a suitable fluorochrome with a significantly higher quantum yield than PO and is even brighter than other violet laser dyes (e.g. Pacific Blue). CD45-KO/SS enables us to distinguish and characterize various normal and malignant leukocyte subpopulations. By using a 10-color MoAb panel to screen on aberrancies, we showed that CD45-KO provides reliable immunophenotyping within small amounts of cells and thereby improves the quality of 10-color stainings.

Chemotherapeutic treatment of bone marrow stromal cells strongly affects their protective effect on acute myeloid leukemia cell survival.

Bone marrow stromal cells (BMSCs) have been found to support leukemic cell survival; however, the mechanisms responsible are far from elucidated yet. Therefore, the effect of BMSCs on both proliferation and apoptosis characteristics of acute myeloid leukaemia (AML) cells was investigated as well as the effect of BMSCs exposure to chemotherapy on the stromal supportive capacity. Leukemic HL-60 and primary AML cells were either untreated or treated with cytarabine and subsequently cultured for 3-4 days, in the presence or absence of untreated or cytarabine-treated BMSCs. The effect on proliferation and apoptosis was investigated with flow cytometry using CFSE labeling and Syto16 and 7AAD staining. BMSCs were found to maintain cytarabine-exposed primary AML cells by protection against spontaneous apoptosis. Accordingly, an increase in phosphorylated-AKT and Bcl-2 expression was found. Concomitant exposure of BMSCs to cytarabine resulted in a dose-dependent decrease of protective capacity of BMSCs. Thus, inhibition of spontaneous apoptosis of leukemic cells mediated by phosphorylation of AKT/Bcl-2 pathway results in protection of leukemic cells by BMSCs, which decreases after BMSCs exposure to chemotherapy. Targeting both the tumor cells and intervening in their interaction with the bone marrow microenvironment may thus affect clinical outcome in AML.

New approaches for the detection of minimal residual disease in acute myeloid leukemia.

The detection of minimal residual disease (MRD) in patients with acute leukemia has been studied for about 15 years by different groups in both the United States and Europe. It has been found that MRD detection can be performed using molecular and immunophenotypic aberrancies that are present in the leukemic clone at diagnosis and not in normal bone marrow. When performing MRD assessments after chemotherapy, it is possible to identify patients at risk for relapse. This review is not an overview of all MRD studies, but rather discusses the possibilities for optimizing MRD detection, the use of flow cytometry versus polymerase chain reaction techniques, and the implications for future patient treatment. When informative, we compare literature on MRD in acute myeloid leukemia (AML) with information from MRD studies in acute lymphoblastic leukemia. Finally, we address the promising detection of AML stem cells, the likely cells of origin in AML, for prediction of clinical outcome and guidance of future therapies.

The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.

In CD34(+) acute myeloid leukemia (AML), the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously, we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population, containing the CD34(+)CD38(-)CLL-1(+) cells, does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission, both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future.