Premium
A novel and easy F xCycle ™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six‐color immunophenotyping
Author(s) -
Tembhare Prashant,
Badrinath Yajamanam,
Ghogale Sitaram,
Patkar Nikhil,
Dhole Nilesh,
Dalavi Pooja,
Kunder Nikesh,
Kumar Ashok,
Gujral Sumeet,
Subramanian P.G.
Publication year - 2016
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.22803
Subject(s) - immunophenotyping , flow cytometry , population , propidium iodide , microbiology and biotechnology , coefficient of variation , aneuploidy , biology , ploidy , cytometry , pathology , chemistry , medicine , chromosome , genetics , chromatography , apoptosis , environmental health , programmed cell death , gene
Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B‐cell acute lymphoblastic leukemia (B‐ALL) and multiple myeloma (MM). Current methods of flow‐cytometric (FC) DNA‐ploidy evaluation are either technically difficult or limited to three‐ to four‐color immunophenotyping and hence, challenging to evaluate DNA‐ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six‐ to seven‐color immunophenotyping and DNA‐ploidy using a dye–FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra‐assay variation for FCV was studied. Using this six‐color immunophenotyping & FCV‐protocol DNA‐ploidy was determined in bone‐marrow samples from 124 B‐ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1‐peak with FCV (mean‐CV 4.1%) was slightly higher than PI (mean‐CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra‐assay variation was very low with CV of 0.005%. In B‐ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near‐hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near‐hyperdiploidy in 8% and remaining 30% were diploid. FCV‐based DNA‐ploidy method is a sensitive and easy method for simultaneous evaluation of six‐color immunophenotyping and DNA analysis. It is useful in DNA‐ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions. © 2015 International Society for Advancement of Cytometry