Experiment Overview
| Repository ID: | FR-FCM-ZZM8 | Experiment name: | DNA ploidy analysis and six to seven color surface immunophenotyping in MM using FxCycle violet | MIFlowCyt score: | 84.00% |
| Primary researcher: | prashant tembhare | PI/manager: | prashant tembhare | Uploaded by: | prashant tembhare |
| Experiment dates: | 2014-06-04 - 2015-03-31 | Dataset uploaded: | Sep 2015 | Last updated: | Dec 2015 |
| Keywords: | [FxCycle Violet] [Simultaneous six color surface staining & DNA ploidy ] [multiple myeloma] | Manuscripts: |
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| Organizations: |
Tata Memorial Centre, Hematopathology Laboratory, Mumbai, MAHARASHTRA (India)
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| Purpose: | To standardize an easy flow cytometric DNA ploidy and simultaneous six color immunophenotyping method that can be used in small number of tumor cells identified using specific immunophenotype. This method will help in ploidy analysis in cases with low tumor cells like monoclonal gammopathy of undtermined significance or multiple myeloma with small number of plasma cells in the bone marrow sample. | ||||
| Conclusion: | FxCycle Violet (FCV) based DNA-ploidy method is a sensitive and easy method for simultaneous evaluation of up to seven color immunophenotyping & DNA analysis. It is useful in DNA-ploidy evaluation of minute tumor population in cases like residual disease in multiple myeloma (MM) and MM precursor conditions like MGUS. | ||||
| Comments: | DNA ploidy is determined by calculating the DNA Index i.e. a ratio of geometric mean of G0/G1 peak of tumor cells (isolated with a specific immunophenotype) to the geometric mean of G0/G1 peak of patient's own lymphocytes present in the sample. Hence, patient's own lymphocytes represent the reference diploid cells to calculate DNA index. Gating approach: Doublets were excluded using FSC height versus area as doublets typically have disproportionately higher FSC area than FSC height. This does not affect cells in G2 phase as cells in G2 ohase typically have proportionately high FSC area and FSC height. Next, cell debris present in the sample were excluded using viability gate in SSC versus FSC dot plot. tumor cells and lymphocytes were gated using expression of surface markers. FCV unstained cells: Depending on the age of sample after collection and duration of fixation after ideal 10 minutes, FCV might not stain few cells or stain weakly. These cells might form a small trail of wealy stained cells. However, as this forms a continuous trail towards negative region i.e sub-G1 region, these does not interfere with the ploidy analysis. these can be easily excluded from the analysis. | ||||
| Funding: | Not disclosed | ||||
| Quality control: | DNA ploidy and simultaneous six color immunophenotyping using FxCycle-Violet. | All daily routine quality measures for flow cytometer were performed. Fresh samples (within 12 hours of collection) were processed and were acquired at low rate to get good CV. Compensation: Generic compensation was performed using lymphocytes and tube specific or label specific was performed during analysis i.e. post acquisition compensation. Debris of dead cells were excluded using a gate based on the SSC and FSC. DI was calculated as a ratio of geometric mean of G0/G1 peak of tumor cells to G0/G1 peak of patient own lymphocytes present in the sample. Thus, this method avoids use of normal cells from other individuals. | |||
Experiment variables
| Timepoints | |
|---|---|
| · At diagnosis only | 7C MGUS FCV 321_M5_390.LMD · 7C MM FCV 457_M5_534_Hyper..LMD · 7C MM FCV 475_M5_561_Hyper..LMD · 7C MM FCV 513_504455_near Hyper.LMD · 7C MM FCV_801_M5_934_diploid.LMD · 7C MM FCV_825_M5_964_Diploid.LMD |
| Sample Type | |
|---|---|
| · Bone Marrow Aspirate | 7C MGUS FCV 321_M5_390.LMD · 7C MM FCV 457_M5_534_Hyper..LMD · 7C MM FCV 475_M5_561_Hyper..LMD · 7C MM FCV 513_504455_near Hyper.LMD · 7C MM FCV_801_M5_934_diploid.LMD · 7C MM FCV_825_M5_964_Diploid.LMD |
