title: A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping.
abstract: 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.
authors.affiliation: Hematopathology Laboratory, Tata Memorial Center Hospital, Parel, Mumbai, 400012, India.
authors.forename: Prashant
authors.initials: P
authors.lastname: Tembhare
authors.affiliation: Hematopathology Laboratory, Tata Memorial Center Hospital, Parel, Mumbai, 400012, India.
authors.forename: Yajamanam
authors.initials: Y
authors.lastname: Badrinath
authors.affiliation: Hematopathology Laboratory, Tata Memorial Center Hospital, Parel, Mumbai, 400012, India.
authors.forename: Sitaram
authors.initials: S
authors.lastname: Ghogale
authors.affiliation: Hematopathology Laboratory, Tata Memorial Center Hospital, Parel, Mumbai, 400012, India.
authors.forename: Nikhil
authors.initials: N
authors.lastname: Patkar
authors.affiliation: Hematopathology Laboratory, Tata Memorial Center Hospital, Parel, Mumbai, 400012, India.
authors.forename: Nilesh
authors.initials: N
authors.lastname: Dhole
authors.affiliation: Hematopathology Laboratory, Tata Memorial Center Hospital, Parel, Mumbai, 400012, India.
authors.forename: Pooja
authors.initials: P
authors.lastname: Dalavi
authors.affiliation: Hematopathology Laboratory, Tata Memorial Center Hospital, Parel, Mumbai, 400012, India.
authors.forename: Nikesh
authors.initials: N
authors.lastname: Kunder
authors.affiliation: Hematopathology Laboratory, Tata Memorial Center Hospital, Parel, Mumbai, 400012, India.
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Hematopathology Laboratory, Tata Memorial Center Hospital, Parel, Mumbai, 400012, India.
authors.forename: Sumeet
authors.initials: S
authors.lastname: Gujral
authors.affiliation: Hematopathology Laboratory, Tata Memorial Center Hospital, Parel, Mumbai, 400012, India.
authors.forename: P G
authors.initials: PG
authors.lastname: Subramanian
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D015703:Antigens, CD / Q000032:analysis / Q000235:genetics / Q000276:immunology; D001853:Bone Marrow / Q000276:immunology / Q000473:pathology; D002467:Cell Nucleus / Q000648:ultrastructure; D002645:Chickens; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D004912:Erythrocytes / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D009101:Multiple Myeloma / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology / Q000473:pathology; D011003:Ploidies; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology / Q000473:pathology; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes
keywords: DNA ploidy; FxCycle-violet; cell cycle; simultaneous multicolor immunophenotyping