Flow Cytometric DNA Ploidy Analysis in Haemato-Lymphoid Neoplasms: An Analysis of 132 Cases

Abstract

Background: FxCycle^TM Violet (FCV) based flow cytometric (FCM) DNA ploidy analysis is a rapid and simple tool that can substantiate in characterizing the biological behaviour across the spectrum of haematological malignancies and correlates with cytogenetic studies. Materials and Methods: In this prospective study, we performed simultaneous immunophenotyping with FCV based on ploidy analysis in n=132 consecutive new samples, comprising n=110 samples of haemato-lymphoid neoplasms, including acute leukemias (n=67, 60.9%), CML with myeloid blast crisis (n=1, 0.9%), MDS with excess blasts (n=2, 1.8%), mature B cell/ T cell neoplasms (n=37, 33.7%), multiple myeloma (n=3, 2.7%) along with n=22 normal samples. The FCM DNA data was compared with corresponding conventional karyotyping results, wherever available. Results: In FCM ploidy analysis (n=110), the overall DNA index (DI) ranged from 0.81 to 2.17 and S-Phase fraction (SPF) from 0.1-31.6%. Diploidy was seen in n = 90 (81.8%), low-hyperdiploidy in n = 10 (9.1%), high-hyperdiploidy in n = 7 (6.4%) with one case each (0.9% each) having near-tetraploidy, high-hypodiploidy and low-hypodiploidy. The DI of all viable cell populations in normal samples ranged from 0.96-1.05. Conventional karyotyping was performed in n=76/110 cases (70%) with n= 11/76 (15%) culture failures. The modal chromosome number ranged from 45 to 63. A concordance of 95.4% (n=62/65) was noted with corresponding FCM DI. Conclusion: FCV-based ploidy is a sensitive technique that provides complementary information and ascertains a strong correlation with conventional cytogenetics across all haemato-lymphoid neoplasms. It can detect aneuploidy in all B-ALL and myeloma cases, even in hemodiluted samples with cytogenetic culture failure; supplement the diagnoses of erythroleukemia, and provide a useful screen for a higher grade lymph node disease in lymphoma cases with SPF > 3%.

Keywords: Cytogenetics; DNA ploidy; FxCycle™ violet; Karyotyping; S-phase fraction.

Figure 1

[A] Sequential gating strategy in a case of B-cell chronic lymphoproliferative disorder (B-CLPD) with immunoprofile of chronic lymphocytic leukemia (CLL). Initially “singlets” were selected using forward scatter (FSC)-Area (A) and height (H); “All cells” that are viable, are then selected from singlets using side scatter (SSC)-A and FSC-A; CD45-SSC-A on all viable cells showing granulocytes in magenta, monocytes in orange, lymphocytes in brown; CD19/SSC-A on all viable cells showing all CD19 positive “B-cells” in green and CD19 negative “T-Cells” in blue. [B] Gated B-cells showing kappa restricted clonal abnormal B-Cells in red expressing CD5, CD20, CD23, CD200 (and also CD43, CD79b and negative for CD10, CD11c, CD38, CD103, CD123 not shown here) and normal B-Cells in mustard expressing CD19, CD20, Lambda (and Kappa not shown here) and negative for CD5 (also negative for CD10, CD11c, CD38, CD43, CD79b, CD103 and CD123 not shown here). [C] FxCycle based DNA ploidy analysis showing histograms of FxCycle-A on linear scale for normal diploid granulocytes, monocytes, normal B-cells and monocytes, along with Kappa restricted abnormal B-cells. The DNA index: Median fluorescence intensity (MFI) of G₀G₁ of abnormal kappa restricted B-Cells divided by MFI G₀G₁ normal T-cells= 49,822/48,822= 1.02 with S-Phase fraction of 0.6%.