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.
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.
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.
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.
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.
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.
•AML leukemia stem cells (LSC) as defined by CD34/CD38 expression, light scatter and Side Population (SP).•In CD34 positive AML therapy resistant LSC are enriched in CD34 + CD38-/SP ...compartment.•Frequency of these LSC decreases with decreasing CD34%, with concomitant increase of normal CD34 + CD38- SP stem cells.•Frequency of putative LSC compartment is median 5.1 per million in these CD34 positive AML.•In CD34 negative AML putative LSC compartment has CD34-CD38+/SP phenotype.
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.
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.
Abstract
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.
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 × 10
6
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.
Abstract 1582
Although the originally defined CD34+CD38- leukemia stem cell (LSC)/leukemia initiating cell (LIC) in acute myeloid leukemia (AML) still serves as a lead in studies on the ...characterization of LSC/LIC, in more immuno-compromised mouse models, other stem cell immunophenotypes, i.e. CD34-CD38- and CD34-CD38+ turn out to have LSC/LIC activity (Taussig et al, Blood 2010; 115: 1976). Two functional LSC phenotypes offer the ability not only to restrict the LSC/LIC compartment to a lower frequency compartment, but also to include such CD34- negative immunophenotypes. These concern high activity of aldehyde dehydrogenase (ALDH) or high efflux of Hoechst 44432 (resulting in side population, SP). The present study deals with the possible overlap between CD34/CD38 and SP defined stem cell compartments. Based on our previous results (van der Pol, Haematologica 2003; 88: 983), we defined truly CD34 negative AML as AML with a very small CD34+ population only (usually≤1%), with particular scatter properties, and proven to be of normal origin. Using expression of cell surface markers present on LSC/LIC, but absent on HSC (van Rhenen et al, Leukemia 2007; 21: 1700; Blood 2007; 110: 2659), the LSC/LIC compartment in this type of leukemia (5 cases studied) was shown to be present in the CD34-CD38+ compartment, while the HSC was found in the CD34+CD38- compartment. When determining aberrant cell surface markers within the SP, the only malignant cells present in the SP population were CD34-CD38+. To assess the primitive character of the putative LSC/LIC sub-populations, cell sorting experiments were performed in which all aberrant marker positive SP cells along with marker positive non-SP cells (n=2) and all aberrant marker negative SP cells and marker negative non-SP cells (n=2), were assessed in the liquid culture stem cell assay. SP marker positive cells had 280- and 725-fold more stem cell activity compared to non-SP cells (14,500 versus 20 and 8,400 versus 30, respectively), while for marker negative cells only the SP fraction contained stem cell activity (8,000 and 8,200 colonies per million input cells). Even though the frequency of SP cells is far below that of non-SP cells, the absolute number of colonies was higher in SP than in non-SP cells. In CD34 positive leukemia, in contrast to CD34 negative AML, the SP fraction was largely filled with cells with all combinations of CD34 and CD38, which all fulfill the criteria (marker positivity) for malignancy. Liquid culture assay results were: for marker positive cells, colonies were formed only in the SP compartment in 2/3 cases studied (6,500 and 4,400 colonies per million input cells). In the remaining case colony formation was 195-fold higher in SP versus non-SP (4,100 versus 21 colonies per million input cells). For marker negative cells, in all three cases only the SP fraction produced colonies (5,300, 1,4500 and 90 per million input cells). Malignant character of marker positive cells was confirmed using FISH analysis, immediately after cell sorting prior to cell culturing, and at the end of the liquid culture on plucked colonies. These results show that both in CD34 negative and CD34 positive leukemia the SP fraction contains most primitive cells and has much higher clonogenic potency compared to non-SP cells. In CD34 negative AML, SP HSC are CD34+CD38- and SP LSC/LIC are CD34-CD38+. In CD34 positive AML, SP HSC are CD34+CD38- and SP LSC/LIC may be CD34+CD38-, but also CD34+CD38+, CD34-CD38+ and CD34-CD38-. The absence of CD34 negative engraftment, seen in earlier studies, likely results from relatively high immune competence of the NOD/SCID mice in those studies. The results suggest that CD34 negative leukemia may contain more immune sensitive LSC/LIC. Above that, CD34 negative AML is characterized by lower multidrug resistance (abstract Schuurhuis, Kelder et al, subm for this meeting). From these characteristics favorable outcome for the particular class of patients, defined as truly CD34 negative, can be predicted. This was indeed the case in our large study (394 patients): median overall survival not reached (>41 months) for CD34 negative patients (n=85) and 19 months for CD34 positive patients (n=309) (p=0.006). Our results warrant to consider CD34 negative patients as a completely different entity of AML patients, in terms of stem cell characteristics, which may evoke different therapeutic strategies compared to CD34 positive patients.
No relevant conflicts of interest to declare.
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