Blast size‐specific flowcytometric ploidy assessment using FxCycle^TM Violet dye and its correlation with conventional cytogenetic ploidy in pediatric precursor B‐lineage acute lymphoblastic leukemia patients

Abstract

Introduction Numerical chromosomal abnormalities (aneuploidies), present in approximately 30%‐50% of pediatric precursor B‐lineage acute lymphoblastic leukemia (B‐ALL) patients, are commonly identified through a laborious conventional cytogenetic (CG) technique. Flow cytometry (FCM) can identify both physical and fluorescent properties of cells together, and by using fluorescent nucleic‐acid‐binding dyes, FCM can identify variations in total nucleic‐acid content of cells. FxCycleTM Violet dye (FxCV) is a selective DNA‐binding dye which permits simultaneous multiparametric immunophenotyping and cell‐cycle/ploidy assessment in a single assay. To date, only two studies have demonstrated the feasibility of FxCV‐aided FCM‐ploidy analysis in B‐ALL patients and only one of these studies have compared their results with CG‐ploidy. Methodology Blast size‐specific FCM‐ploidy was prospectively analyzed using FxCV‐dye in 109 pediatric B‐ALL patients, and the results were compared with concurrent CG‐ploidy status. Results FCM‐ploidy categorization was feasible in 98% of samples tested and the results were 82% concordant with CG‐ploidy status. We observed significant correlation between DNA content and blast size (r = .823, P < .001) and could demonstrate size differences between diploid vs low‐hyperdiploid (P = .025), diploid vs high‐hyperdiploid (P < .001) and low‐ vs high‐hyperdiploid blasts (P = .007). Conclusion FCM‐ploidy assessment using FxCV dye is a reliable assay and the results closely concur with CG‐based ploidy stratification and risk assessment. Using blast size‐assisted DNA content analysis, the results of FCM‐ploidy analysis can be further fine‐tuned.

Study of DNA ploidy in newly diagnosed multiple myeloma by flow cytometry and its correlation with disease prognosis and outcome

“…[21] Hypodiploid MM by DNA content flow cytometry is rarer, occurring in <2% of newly diagnosed MM patients; the previous studies have shown these patients to be unresponsive to therapy and to have short survival. [13,20] In recent studies, flow cytometric ploidy analysis is increasingly being used in the analysis of B-ALL with its correlation of cytogenetic-based ploidy stratification and risk assessment, [22] and other studies are being done to assess the potential role in the detection of measurable residual disease. [23] In summary, this is the first study from our center on ploidy analysis using flow cytometry which helped us in standardizing the method.…”

Flow Cytometric Ploidy Analysis in Acute Lymphoblastic Leukemia and Plasma Cell Myeloma

“…Immunohistochemistry-based cell cycle detection (iCCD) 53,54 • Geminin: Highest expression in S/G2/M phase and degrades after M phase • Chromatin licensing and DNA replication factor 1 (Cdt1): Highest expression in G1 phase and degrades before S phase • Gamma H2A.X: Marker for DNA double-strand breaks (DSBs) 55 Fluorescence in situ hybridization (FISH) 56 • Ploidy assessment by centromere and α-satellite DNA assessment probes Fluorescent ubiquitination-based cell cycle indicator (FUCCI) 57 • Fluorescence-labelled proteins tagged with Cdt 1 and Geminin • Time-lapse photographs are used to identify both temporality and spatial organization of cells in culture according to their stage in cell cycle Radioisotope-labeled DNA 58 • Incorporating radioisotope (now replaced by nucleoside analogues) into the DNA to calculate the time spent by a cell across the cell cycle stages Flow cytometry 2,3,8,9,52 Univariate • Using DNA binding dyes that enable only cellular DNA content assessment • Applicable in samples that have only/predominantly tumor cells…”

“…However, ploidy evaluation by FCM is not sensitive in assessing aneuploidies involving smaller chromosomes. 9 Role of DNA Index in B-Lineage Acute Lymphoblastic Leukemias Hyperdiploidy (51-65 chromosomes) is observed in nearly 30% of pediatric patients diagnosed with B-ALL and is associated with a favorable prognosis. [14][15][16] These hyperdiploid blasts are highly susceptible to apoptosis and hence require stringent conditions for their ex vivo survival.…”

“…11,13,[17][18][19][20][21] Although there is more than 95% concordance in hyperdiploidy identified by DI and karyotyping, not all B lymphoblasts with 51 to 65 chromosomes have DI 1.16. 8,9,22 In this context, it has been proposed to consider B lymphoblasts having 51 to 55 chromosomes and DI of 1.10 to 1.15 as "low DI-high hyperdiploid" (LDI-HHD). These patients with LDI-HHD have a poor prognosis as compared to patients with DI 1.16 and 51 to 65 chromosomes, that is, the "high DI-high hyperdiploid" category.…”

“…[24][25][26][27] Using blast sizespecific DI assessment, both hypodiploid and endoreduplicated hyperdiploid blasts in a sample can be identified in isolation. 9 Role of DNA Index in Plasma Cell Myeloma Malignant plasma cells in nearly 50% of patients with PCM are hyperdiploid due to trisomies involving odd-numbered chromosomes. [28][29][30][31][32] These hyperdiploid PCM patients have a better prognosis as compared to hypodiploid and IgH translocated PCM patients.…”