Method for DNA Ploidy Analysis Along with Immunophenotyping for Rare Populations in a Sample using FxCycle Violet

Prashant Tembhare1, Yajamanam Badrinath1, Sitaram Ghogale1, Papagudi Ganesan Subramanian1

1 Hematopathology Laboratory, Advanced Centre for Treatment Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.38
DOI:  10.1002/cpcy.15
Online Posting Date:  April, 2017
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The clinical use of flow cytometric DNA ploidy assay has been extended towards stratifying the risk of diseases, such as monoclonal gammopathies or B cell acute lymphoblastic leukemia, and to detect circulating tumor cells, both of which require detection of minute cell populations. This unit describes a protocol for determining DNA ploidy in fixed samples with simultaneous surface immunophenotyping. It is an easy method for simultaneous 6‐ to 8‐color immunophenotyping and DNA content analysis using FxCycle Violet (FCV; DAPI) dye. This protocol is a one‐step modification of routine multicolor immunophenotyping that includes surface staining followed by fixation and then DNA staining with FCV. It utilizes mature lymphocytes from the sample as an internal control for determination of DNA index. It is a sensitive method that allows DNA‐ploidy determination and cell cycle analysis in a rare tumor population as low as 100 events, as well as DNA ploidy determination in various subsets of hematopoietic cells in the same sample based on their immunophenotype. © 2017 by John Wiley & Sons, Inc.

Keywords: DNA ploidy; FxCycle Violet; DAPI; flow cytometry; 6‐ to 8‐color immunophenotyping

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Table of Contents

  • Significance Statement
  • Basic Protocol 1:  
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:  

  • Sample to be processed: Any unfixed sample like peripheral blood (PB), bone marrow aspirate (BM), fine needle aspirate (FNA), body fluids within 24 to 48 hr of collection can be processed for the DNA‐ploidy using this method (this protocol has been standardized using EDTA anticoagulated bone marrow aspirate and peripheral blood samples)
  • Red cell lysing reagent (see recipe)
  • Phosphate buffer saline (PBS) solution with 0.5% albumin (PBS‐A; see recipe)
  • Antibodies: Antibodies conjugated with fluorochromes that can be excited with blue and red laser depending upon the instrument; antibodies conjugated with fluorochromes excited with violet laser should be avoided due to significant spectral overlap with FCV (see Table 6.38.1 for details on the antibodies used)
  • Medium‐A of Fix and Perm kit (Invitrogen, cat. no. GAS002S100)
  • Reconstituted FCV (see recipe)
  • Instrument quality control beads and set up beads: CS&T Beads (Becton Dickinson, cat. no. 642412), flowcheck beads (BC instrument, cat. no. A63493), and SPHERO Rainbow Calibration Particles (Spherotech, cat. no. RCP‐30‐5A)
  • 12 × 75–mm polystyrene tubes
  • 15‐ml polypropylene tubes (Tarsons, cat. no. 546021)
  • Centrifuge
  • Pipets
  • Vortex
  • Neubauer chamber
  • Flow cytometer equipped with a 405‐nm violet laser, 488‐nm argon‐ion lasers, and a 635‐nm red laser, as a fluorescence excitation source like FACS Canto‐II (BD), Navios (BC): this protocol has been used on both the instruments.
  • Flow cytometry analysis software: e.g., Kaluza, version 1.3 (Beckman Coulter)
Table 6.8.1   MaterialsDetails for Antibodies Used for B cell Precursor Acute Lymphoblastic Leukemia (B‐ALL) and Multiple Myeloma (MM) a

Fluorochrome FITC PE ECD/PE‐CF594 PC5.5 PC7 APC APC‐Alexa Fluor 700 APC‐Alexa Fluor 750
B cell precursor acute lymphoblastic leukemia antibody and DNA ploidy panel
Antibody/reagent FxCycleViolet CD58 CD86 CD25 CD34 CD10 CD19 CD45 CD38
Catalog number F_10347 IM1218U 555658 562 403 347 203 A46527 IM2470 A71117 A86049
Clone AICD58 2331(FUN‐1) M‐A251 8G12 ALB1 J3‐119 J.33 LS198‐4‐3
Isotype control IgG2a IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1
Company Invitrogen BC BD BD BD BC BC BC BC
Multiple Myeloma and DNA ploidy panel
Antibody/reagent FxCycle Violet x CD81 CD19 CD27 CD56 CD138 CD45 CD38
Catalog number F_10347 x IM2579 A07770 B21444 A 51078 A87787 A71117 A86049
Clone x x JS64 J3‐119 1A4CD27 N901 (HLD‐A6) B‐A38 J.33 LS198‐4‐3
Isotype control x x M IgG1 M IgG1 M IgG1 M IgG1 M IgG1 M IgG1 M IgG1
Company Invitrogen x BC BC BC BC BC BC BC

 aAbbreviations: BC, Beckman Coulter; BD, BD Biosciences.
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Literature Cited

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