REAL-TIME DEFORMABILITY CYTOMETRY (RT-DC) AS NOVEL DIAGNOSTIC TOOL IN MYELODYSPLASTIC SYNDROMES
(Abstract release date: 05/19/16)
EHA Library. Bulycheva E. 06/09/16; 132747; E1198
Disclosure(s): Travel support - TEVA GmbH
Research Support - Onconova
Ms. Ekaterina Bulycheva
Contributions
Contributions
Abstract
Abstract: E1198
Type: Eposter Presentation
Background
Myelodysplastic syndromes (MDS) are a group of disorders, which are often difficult to differentiate from reactive and toxic conditions causing cytopenia. Contractility and cytoskeletal structure of hematopoietic stem cells in MDS are known to differ from the healthy counterparts. Thus, detection of the cells with abnormal mechanical properties could be a promising diagnostic procedure for patients with potential MDS.
Aims
Therefore we aimed to evaluate if real-time deformability cytometry (RT-DC) can be a reliable diagnostic and prognostic tool for MDS patients, as well as to clarify molecular factors, which are associated with differential cell deformability.
Methods
We analyzed MACS-separated CD34+ cells from 19 patients with MDS (median age at BM sampling 65 years [range, 44-87]; m/f=13/6) and 8 age-matched healthy donors (62 years [range, 43-83]; m/f=3/5). Mechanical characterization was carried out using RT-DC at three flow rates according to previously described protocol (Otto O. et al., Nat Methods, 2015). With this technology, single cells are flowed through a microfluidic channel constriction and deformed without contact by shear stress. Statistical significance was assessed by 2-sided Kolmogorov-Smirnov test. Changes in cell cycle were evaluated by flow cytometry using propidium iodide staining. Molecular alterations were evaluated by targeted resequencing on a MiSEQ instrument using an amplicon assay (True Sight Myeloid Panel, Illumina) which covers 54 genes or gene hotspots related to myeloid neoplasms. Cytogenetic analysis was performed in all MDS patients. Overall survival (OS) was estimated using Cox proportionate regression models.
Results
Mean deformability for CD34+ cells of MDS patients was significantly lower than in control cells (0.0241 vs 0.0272, accordingly, p=0.018). MDS CD34+ cells underwent significantly less mitosis (2.4% vs 17.4%, p=0.002), arresting mostly in G1 phase (91.5% vs 66.7%, p<0.001). CD34+ сells from patients with mutations in RAS-pathway tended to be more deformable, whereas RUNX1-mutated cells appeared stiffer. Cytogenetics had no influence on mechanical properties. The deformability appears to be predictive for the OS starting from time of BM sampling, as 1-year OS in patients with more deformable cells (>median) was 72.9% vs 37.0% in patients with less deformable cells.
Conclusion
RT-DC provides a novel tool to reveal substantial differences in mechanical properties of CD34+ cells of MDS patients compared to healthy donors. This might allow incorporating this technique into diagnostic approaches of MDS patients.
Session topic: E-poster
Keyword(s): Diagnosis, Myelodysplasia, Myeloid malignancies
Type: Eposter Presentation
Background
Myelodysplastic syndromes (MDS) are a group of disorders, which are often difficult to differentiate from reactive and toxic conditions causing cytopenia. Contractility and cytoskeletal structure of hematopoietic stem cells in MDS are known to differ from the healthy counterparts. Thus, detection of the cells with abnormal mechanical properties could be a promising diagnostic procedure for patients with potential MDS.
Aims
Therefore we aimed to evaluate if real-time deformability cytometry (RT-DC) can be a reliable diagnostic and prognostic tool for MDS patients, as well as to clarify molecular factors, which are associated with differential cell deformability.
Methods
We analyzed MACS-separated CD34+ cells from 19 patients with MDS (median age at BM sampling 65 years [range, 44-87]; m/f=13/6) and 8 age-matched healthy donors (62 years [range, 43-83]; m/f=3/5). Mechanical characterization was carried out using RT-DC at three flow rates according to previously described protocol (Otto O. et al., Nat Methods, 2015). With this technology, single cells are flowed through a microfluidic channel constriction and deformed without contact by shear stress. Statistical significance was assessed by 2-sided Kolmogorov-Smirnov test. Changes in cell cycle were evaluated by flow cytometry using propidium iodide staining. Molecular alterations were evaluated by targeted resequencing on a MiSEQ instrument using an amplicon assay (True Sight Myeloid Panel, Illumina) which covers 54 genes or gene hotspots related to myeloid neoplasms. Cytogenetic analysis was performed in all MDS patients. Overall survival (OS) was estimated using Cox proportionate regression models.
Results
Mean deformability for CD34+ cells of MDS patients was significantly lower than in control cells (0.0241 vs 0.0272, accordingly, p=0.018). MDS CD34+ cells underwent significantly less mitosis (2.4% vs 17.4%, p=0.002), arresting mostly in G1 phase (91.5% vs 66.7%, p<0.001). CD34+ сells from patients with mutations in RAS-pathway tended to be more deformable, whereas RUNX1-mutated cells appeared stiffer. Cytogenetics had no influence on mechanical properties. The deformability appears to be predictive for the OS starting from time of BM sampling, as 1-year OS in patients with more deformable cells (>median) was 72.9% vs 37.0% in patients with less deformable cells.
Conclusion
RT-DC provides a novel tool to reveal substantial differences in mechanical properties of CD34+ cells of MDS patients compared to healthy donors. This might allow incorporating this technique into diagnostic approaches of MDS patients.
Session topic: E-poster
Keyword(s): Diagnosis, Myelodysplasia, Myeloid malignancies
Abstract: E1198
Type: Eposter Presentation
Background
Myelodysplastic syndromes (MDS) are a group of disorders, which are often difficult to differentiate from reactive and toxic conditions causing cytopenia. Contractility and cytoskeletal structure of hematopoietic stem cells in MDS are known to differ from the healthy counterparts. Thus, detection of the cells with abnormal mechanical properties could be a promising diagnostic procedure for patients with potential MDS.
Aims
Therefore we aimed to evaluate if real-time deformability cytometry (RT-DC) can be a reliable diagnostic and prognostic tool for MDS patients, as well as to clarify molecular factors, which are associated with differential cell deformability.
Methods
We analyzed MACS-separated CD34+ cells from 19 patients with MDS (median age at BM sampling 65 years [range, 44-87]; m/f=13/6) and 8 age-matched healthy donors (62 years [range, 43-83]; m/f=3/5). Mechanical characterization was carried out using RT-DC at three flow rates according to previously described protocol (Otto O. et al., Nat Methods, 2015). With this technology, single cells are flowed through a microfluidic channel constriction and deformed without contact by shear stress. Statistical significance was assessed by 2-sided Kolmogorov-Smirnov test. Changes in cell cycle were evaluated by flow cytometry using propidium iodide staining. Molecular alterations were evaluated by targeted resequencing on a MiSEQ instrument using an amplicon assay (True Sight Myeloid Panel, Illumina) which covers 54 genes or gene hotspots related to myeloid neoplasms. Cytogenetic analysis was performed in all MDS patients. Overall survival (OS) was estimated using Cox proportionate regression models.
Results
Mean deformability for CD34+ cells of MDS patients was significantly lower than in control cells (0.0241 vs 0.0272, accordingly, p=0.018). MDS CD34+ cells underwent significantly less mitosis (2.4% vs 17.4%, p=0.002), arresting mostly in G1 phase (91.5% vs 66.7%, p<0.001). CD34+ сells from patients with mutations in RAS-pathway tended to be more deformable, whereas RUNX1-mutated cells appeared stiffer. Cytogenetics had no influence on mechanical properties. The deformability appears to be predictive for the OS starting from time of BM sampling, as 1-year OS in patients with more deformable cells (>median) was 72.9% vs 37.0% in patients with less deformable cells.
Conclusion
RT-DC provides a novel tool to reveal substantial differences in mechanical properties of CD34+ cells of MDS patients compared to healthy donors. This might allow incorporating this technique into diagnostic approaches of MDS patients.
Session topic: E-poster
Keyword(s): Diagnosis, Myelodysplasia, Myeloid malignancies
Type: Eposter Presentation
Background
Myelodysplastic syndromes (MDS) are a group of disorders, which are often difficult to differentiate from reactive and toxic conditions causing cytopenia. Contractility and cytoskeletal structure of hematopoietic stem cells in MDS are known to differ from the healthy counterparts. Thus, detection of the cells with abnormal mechanical properties could be a promising diagnostic procedure for patients with potential MDS.
Aims
Therefore we aimed to evaluate if real-time deformability cytometry (RT-DC) can be a reliable diagnostic and prognostic tool for MDS patients, as well as to clarify molecular factors, which are associated with differential cell deformability.
Methods
We analyzed MACS-separated CD34+ cells from 19 patients with MDS (median age at BM sampling 65 years [range, 44-87]; m/f=13/6) and 8 age-matched healthy donors (62 years [range, 43-83]; m/f=3/5). Mechanical characterization was carried out using RT-DC at three flow rates according to previously described protocol (Otto O. et al., Nat Methods, 2015). With this technology, single cells are flowed through a microfluidic channel constriction and deformed without contact by shear stress. Statistical significance was assessed by 2-sided Kolmogorov-Smirnov test. Changes in cell cycle were evaluated by flow cytometry using propidium iodide staining. Molecular alterations were evaluated by targeted resequencing on a MiSEQ instrument using an amplicon assay (True Sight Myeloid Panel, Illumina) which covers 54 genes or gene hotspots related to myeloid neoplasms. Cytogenetic analysis was performed in all MDS patients. Overall survival (OS) was estimated using Cox proportionate regression models.
Results
Mean deformability for CD34+ cells of MDS patients was significantly lower than in control cells (0.0241 vs 0.0272, accordingly, p=0.018). MDS CD34+ cells underwent significantly less mitosis (2.4% vs 17.4%, p=0.002), arresting mostly in G1 phase (91.5% vs 66.7%, p<0.001). CD34+ сells from patients with mutations in RAS-pathway tended to be more deformable, whereas RUNX1-mutated cells appeared stiffer. Cytogenetics had no influence on mechanical properties. The deformability appears to be predictive for the OS starting from time of BM sampling, as 1-year OS in patients with more deformable cells (>median) was 72.9% vs 37.0% in patients with less deformable cells.
Conclusion
RT-DC provides a novel tool to reveal substantial differences in mechanical properties of CD34+ cells of MDS patients compared to healthy donors. This might allow incorporating this technique into diagnostic approaches of MDS patients.
Session topic: E-poster
Keyword(s): Diagnosis, Myelodysplasia, Myeloid malignancies
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