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

Abstract Identification of underlying cytogenetic (CG) aberrancies plays a significant role in risk stratification of hematological malignancies. These abnormalities can be due to aberrancies that affect the number or structure of chromosomes. Numerical chromosomal abnormalities are called aneuploidies, which result from either gain or loss of whole chromosomes. Ploidy assessment by CG is a laborious and less sensitive technique. With the aid of fluorescent nucleic acid binding dyes, the total DNA content and different phases of the cell cycle specific to any population of interest can be deciphered and analyzed by flow cytometry (FCM). DNA index (DI), a parameter derived by FCM DNA analysis, is equivalent to conventional CG-based ploidy assessment. In this study, the technical aspects and implications of FCM DNA assessment among patients diagnosed with acute lymphoblastic leukemia and plasma cell myeloma are discussed.


Concept of Flow Cytometric Cell Cycle Analysis
The cells in G0/G1 (pre-replicative), S (replicative), and G2/M (post-replicative) phases differ by their intracellular DNA content.[4][5] Fluorescent Nucleic Acid Binding Dyes These dyes emit fluorescence upon binding to their nucleic acid targets. 6To be eligible for FCM DNA analysis, a fluorescent dye must fulfill the following prerequisites 6 : • It must have specific binding to nucleic acids.
• It must acquire strong fluorescence after binding to its nucleic acid target.• The dye's fluorescence emission must be stoichiometric to the target cell's nucleic acid content.• The dye must have preferential binding to either DNA or RNA.Dyes that have an equal affinity to both DNA and RNA are less preferred.However, undesired but minimal RNA binding in DNA preferential dyes can be overcome by RNAse treatment.
Table 1 Sequential stages of cell cycle and intracellular events at each stage 53 Interphase Phase Intracellular events G0 • Dormant stage between two cell cycles • Cells perform their physiological function, but there is no synthesis of RNA and ribosomal protein machinery needed for DNA synthesis

Technique Brief Description
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

Components of DNA Histogram
In FCM, the cell cycle is analyzed as a frequency histogram where the fluorescence intensity of DNA binding dye and the number of cells is plotted across the X-axis and Y-axis, respectively. 4In a cell cycle histogram, the fluorescence intensity of DNA binding dyes is expressed in a linear scale.The linear scale simplifies interpretation of DNA histogram as each unit in this scale represents an equal interval in increments of fluorescence (i.e., DNA content per cell).As the data is well spread in a linear scale, individual phases of cell cycle can be grossly identified by visual inspection itself.Also, the linear scale facilitates identification of artefacts and sub-G0/G1 peaks, which might be readily missed in a log-scale.Due to the stoichiometric binding of DNA-specific dyes, all cells in any given phase of the cell cycle are expected to have the same amount of DNA and the "expected" cell cycle must be as the green, blue, and red colored bars depicted in ►Fig. 1.However, due to cell-to-cell variability in dye binding, a gaussian curve with normal distribution is generated for each phase of the cell cycle, and a multimodal histogram is generated as depicted in ►Fig. 1.
Following are the components of a DNA histogram.The G1/G0 curve represents cells in G0/G1 phases of the cell cycle.Using DNA-specific dyes, a distinction between G0-and G1phase cells is not feasible as both G0-and G1-phase cells have the same DNA content.In contrast to G0 cells, which have only DNA, cells in the G1 phase also have RNA (refer ►Table 1).Combined use of RNA binding dyes like pyronin Y along with a DNA-specific dye (having a different emission spectrum) can aid distinction between G0-and G1phase cells.The G2/M curve represents cells in the G2/M phase of cell cycle.In theory, the G2/M-phase cells must have exactly double the DNA content of cells in the G0/G1 phase.However, the extensive DNA-protein interactions in the G2/ M-phase cells result in extensive chromatin compaction, which hinders stoichiometric nucleic acid binding by the dyes.Due to this, the dye fluorescence intensity of cells in the G2/M phase is 1.97 times that of the cells in the G0/G1 phase. 7The S curve is a broad-based histogram between G0/ G1 and G2/M curves.This curve reflects the gradual increase in the DNA content of a cell until it reaches a content equivalent to a cell in the G2/M phase.
In a DNA histogram, the "origin point" indicates the point on x-axis where the fluoresce of the DNA binding dye starts.In a typical DNA histogram, the peak staring from this origin point has the least DNA content, that is, the G0/G1 peak.This origin point on the x-axis must be clearly defined in the DNA histogram as it facilitates data normalization for comparing DNA histograms from different experiments or samples or different cell populations within the same sample.The origin point is also the end result of variations in sample age, sample processing, and measurement techniques.The exact start and end points of G1/G0, S, and G2/M histograms are always arbitrary by manual gating.To identify the correct proportion of cells representing each phase of a cell cycle, mathematical modeling-based curve fitting algorithms are available (Dean-Jett model of 1974, Fox-modified Dean-Jett model of 1980, Watson's pragmatic model of 1987, etc.).

Clinically Relevant Parameters Derived from FCM DNA analysis
In patients diagnosed with B-lineage acute lymphoblastic leukemia (B-ALL) and plasma cell myeloma (PCM), the DNA index (DI) and S-phase fraction (SPF) are the two parameters of clinical relevance obtained by FCM DNA analysis.Flow Cytometric DNA Analysis Bommannan 527

DNA Index
The DI is calculated as the ratio of the G0/G1 phase of malignant cells to that of the G0/G1 phase of normal diploid control cells. 42][13] The advantages of ploidy assessment by DI are that the technique is more rapid and sensitive than CG, and can aid in ploidy identification even in samples with less than 1% cells of interest. 8This technique can identify cell population-specific ploidy even in samples with markedly heterogeneous cellular composition (e.g., bone marrow).However, ploidy evaluation by FCM is not sensitive in assessing aneuploidies involving smaller chromosomes. 95][16] These hyperdiploid blasts are highly susceptible to apoptosis and hence require stringent conditions for their ex vivo survival.This is reflected in frequent metaphase failures and/or poor morphology of chromosomes during conventional CG evaluation of these patients.Under ambient culture conditions used for conventional karyotyping, the normal diploid cells in the sample can overwhelm the hyperdiploid blasts and result in pseudodiploidy.Ploidy assessment by DI is effective in overcoming these limitations of conventional CG-based ploidy assessment.
The relevance of DI in pediatric patients with B-ALL was observed as early as 1985. 178][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. 8,9,22In 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. 23In fact, patients with DI 1.24 (corresponding 58 modal chromosomes) have the best prognosis. 23►Fig. 2 demonstrates the FCM DNA analysis histograms of individual patients with diploid (►Fig.2a-c) and hyperdiploid (►Fig.2d-f) B lymphoblasts.
Hypodiploidy (<46 chromosomes) is observed in less than 5% of pediatric B-ALL patients. 15In this category, patients with 31 to 39 chromosomes (low hypodiploid) and 24 to 29 chromosomes (near haploid) have dismal prognosis. 15,24,25ndoreduplication is a phenomenon where a cell undergoes DNA replication without undergoing cell division. 24his results in the cell having multiple copies of its chromosomes within a single nucleus.Although endoreduplication is a physiological process in certain human cells (e.g., megakaryocytes), its occurrence in blasts is pathological and complicates ploidy assessment. 24Nearly 65% of patients with near haploidy and 45% of patients with low hypodiploidy undergo endoreduplication. 24Due to this phenomenon, CG can misinterpret endoreduplicated near-haploid and low-hypodiploid lymphoblasts as hyperdiploid (50-60 chromosomes) and near-triploid (up to 78 chromosomes) lymphoblasts, respectively. 245][26] Hence, misinterpretation of low hypodiploidy as near triploidy might not affect the patient's prognosis.However, misclassification of endoreduplicated near haploidy as high hyperdiploidy is detrimental. 245][26][27] Using blast sizespecific DI assessment, both hypodiploid and endoreduplicated hyperdiploid blasts in a sample can be identified in isolation. 99][30][31][32] These hyperdiploid PCM patients have a better prognosis as compared to hypodiploid and IgH translocated PCM patients. 33,34As plasma cells are terminally differentiated, conventional CG results in normal diploid metaphases (possibly of myeloid origin) in nearly 70% of PCM samples. 34In this context, a multiparametric FCM DI analysis can identify ploidy in both normal and malignant plasma cells. 10,35Using normal plasma cells as the reference population, malignant plasma cells with DI of 0.95 to 1.05, 1.06 to 1.50, 1.51 to 1.70, and greater than 1.71 are considered diploid, hyperdiploid, near tetraploid, and tetraploid, respectively. 35Using lymphocytes as diploid control, an alternative cutoff of 0.95 (hypodiploid), 0.95 to 1.05 (diploid), 1.06 to 1.15 (near hyperdiploid), and 1.16 (hyperdiploid) to define DI-based ploidy has also been described. 10

S-Phase Fraction of Malignant Cells
In a cell cycle, cells in the S phase are those that undergo active DNA replication (refer to ►Table 1).As cells in the S phase eventually result in mitosis (i.e., physical doubling of the cells), the SPF reflects the proliferative capability of the tumor.

Role of S-Phase Fraction Assessment in Acute Lymphoblastic Leukemias
The relevance of SPF assessment in acute leukemias dates back to 1980 when the presence of greater than 6% lymphoblasts in the S phase was associated with poor prognosis among pediatric patients diagnosed with ALL. 36This 6% cutoff was established based on the median SPF (range of 1-60% and mean of 9%) among 65 ALL samples with greater than 80% lymphoblasts, irrespective of their B-or T-lineage origin. 36The prognostic relevance of this 6% SPF was further confirmed by contemporary literature from both the Dutch and British groups. 11,36However, further literature regarding the relevance of SPF in pediatric patients with ALL is sparse and has not claimed any prognostic relevance. 18,37,38ue to the emergence of more robust risk assessment modalities, SPF is no longer used as a prognostic tool in any present-day pediatric ALL treatment protocols.

Role of S-Phase Fraction Assessment in Plasma Cell Myeloma
The proliferative capability of plasma cells, as indicated by the plasma cell labeling index (PCLI), is a time-tested prognostic factor in patients diagnosed with plasma cell dyscrasias. 39Initial PCLI identification assay using radiolabeled thymidine incorporation followed by autoradiography was both technically challenging and hazardous.In 1985, PCLI by a smear-based immunofluorescence assay utilizing an anti-5-Bromo-2'-deoxyuridine (BrdU) antibody was introduced.1][42][43][44] Although this BrdU-based PCLI technique is safer and relatively easier than the radiolabeled thymidine technique, it is not widely utilized in routine practice. 39n 1994, Orfäo et al described an FCM-based CD38/propidium iodide dual staining technique to analyze the DNA content of plasma cells in the bone marrow of patients diagnosed with PCM. 45 According to that study conducted among 120 treatment naïve PCM patients, those patients with 3% malignant plasma cells in the S phase had inferior relapse-free and overall survival. 30[48]

Laboratory Aspects of Flow Cytometric Ploidy Assessment
A successful FCM DNA analysis warrants fulfilling of the prerequisites discussed in the following sections.

Sample-Related Prerequisites
FCM DNA analysis pertinent to hematological malignancies is commonly performed on fresh, unfixed, fluid-state samples like peripheral blood, bone marrow, body fluids, and fine needle aspirates. 18In indications requiring sample fixation (like transported samples), precipitating fixatives (acetone and alcohol) are preferred over cross-linking fixatives (formaldehyde and glutaraldehyde). 4This is because the nucleic acid cross-linking resulting from these fixatives will interfere with the desired stoichiometric biding of the dyes. 4In the era of univariate and bivariate FCM DNA analysis, it was recommended to perform the assay in samples with a minimum of 15 to 20% malignant cells of interest. 49With the advent of multiparametric FCM DNA analysis, the assay could be successfully performed even in samples with less than 1% malignant cells. 8f the assay has to be performed on archived paraffin blocks, the following factors have to be taken care of.A cell suspension must be prepared from a section of the paraffin block that contains both the tumor cells and normal resident cells (selected by light microscopic evaluation).The thickness of the section is determined by the size of the tumor cell's nucleus (a 50-nm-thick section would be optimal). 5The quality of DNA binding dye staining is relatively inferior as the tissue is already exposed to formaldehyde during Indian Journal of Medical and Paediatric Oncology Vol.44 No. 5/2023 © 2023.The Author(s).
Flow Cytometric DNA Analysis Bommannan 529 fixation; hence, stoichiometric staining might not be optimal. 3,4In this scenario, 4',6-diamidino-2-phenylindole (DAPI) is the preferred nucleic acid binding dye as it is least affected by formaldehyde-induced cross-linking. 4 Since the nucleus of cells is cut through during sectioning, it is common to get a sub-G0/G1 population during analysis.This population must not be interpreted as a hypodiploid clone of cells.In such situations, curve-fitting algorithms/software

Assay-Related Prerequisites
For any FCM DNA assay, it is mandatory to validate the linearity, degree of resolution, and robustness of sample processing in a machine and panel-specific setting.Commercially Table 3 Common issues encountered during flow cytometric DNA analysis and the possible reasoning and solutions to consider

Issue
Reasons Solution

Doublets and cell aggregates Higher relative centrifugal force (RCF)
The ideal RCF has to be calculated to achieve an optimal centrifugation force of 500-540 g 52 Aged sample.Autolysis of cells cause release of sticky nuclear content, causing cell-to-cell adhesions Sample should be processed as fresh as possible, ideally within 24-48 h from collection 52 Sample acquired at high acquisition rate The sample must be acquired at a low event rate, ideally 100-200 events/s 52 Harsh handling of samples during processing Mixing steps involved during processing must be gentle, preferably using a Pasteur pipette 52 Nonstoichiometric staining (ideally >90% cells in a sample must be stained with the dye) 52 Low concentration of dye used for staining Re-standardize the assay with gradients of dye concentration to identify optimal dye concentration to be used.Note: the cell concentration and incubation time have to be kept constant

Excess of cells in the cell suspension being stained
Re-standardize the assay with gradients of cell concentrations to identify optimal cell concentration to be used.Note: the dye concentration and incubation time have to be kept constant

Insufficient incubation time
Re-standardize the assay with gradients of incubation time to identify optimal incubation time.Note: the dye and cell concentration have to be kept constant Samples from patients on DNA binding anticancer drugs Repeat the assay in a treatment naïve sample (if available) Undesired RNA binding 4 The degree of undesired RNA binding depends on the dye's RNA binding propensity • While using dyes with higher RNA binding propensity (e.g., propidium iodide), the sample has to be treated with RNAse before incubation with the dye 4 • A dye without RNA binding capability (e.g., FxCycle violet) can be used 9,52 Sub-G0/G1 population 4 (the cells that have DNA content less than that of a normal diploid control) Apoptosis: apoptosis results in DNA fragmentation of specific lengths.During sample processing, apoptotic cells leak out their intracellular contents (including smaller DNA fragments).Hence, apoptotic cells have a lower total DNA content than normal diploid control and manifest as a distinct sub-G0/G1 peak • If a sub-G0/G1 peak is also present in diploid controls, then the sub-G0/G1 population present in the malignant cells can be considered apoptotic and ignored; however, this approach is subjective and risks exclusion of true hypodiploid malignant cells 9 • Concurrent staining with annexin V will aid objective identification of apoptotic cells 9 Necrosis: necrosis involves random-sized DNA fragmentation, resulting in a sub-G0/ G1 "smearing pattern" against a perfect peak generated by apoptosis/hypodiploid cells Samples should be fresh and processed within 24-48 h of collection 52 available chicken or trout (they inherently have a mixture of single, double, triple, and quadruple nucleated red blood cells [RBCs]) erythrocytes are used for this purpose. 4,10ccording to the 1993 DNA cytometry consensus conference's recommendations, indigenous normal cells (stromal cells in the case of paraffin-embedded tissues) present in a tumor-rich sample should be used as internal diploid controls.In scenarios where such internal controls are not available/sparsely present within the sample, normal diploid cells sourced from healthy controls can be spiked to the tumor sample before processing.
In the context of FCM DNA analysis pertinent to hematological malignancies, lymphocytes are used as diploid controls.Neutrophils and monocytes are not ideal controls as the former are highly fragile and the latter have increased stainability (especially for propidium iodide) due to their unique chromatin conformation. 4

Sample Processing-Related Prerequisites
The protocols pertinent to sample processing toward FCM DNA analysis depend on the nature of the sample, the cell-permeant ability of the dye, and its extent of RNA binding capability (refer to ►Supplementary Table S1, available in the online version).Detailed discussion regarding the specific modifications required for each dye is beyond the scope of this review but is readily available in the literature. 2,3,50,51The vital features are that the samples must be processed within 24 to 48 hours of collection.A stable dye binding equilibrium is achieved only if the concentration of dye used for staining is nearly 100 times above the concentration of the cells to be stained.Hence, the optimal concentrations of cells and the dye used must be standardized to achieve stoichiometric staining. 52The processed samples must be acquired immediately and the acquisition must be at a low event rate, around 100 to 200 events per second. 52A minimum of 200 diploid control cells must be acquired. 52

Analysis-Related Prerequisites
For optimal FCM DNA evaluation, a minimum of 90% of cells must be stained with the DNA binding dye. 52The quality of an FCM DNA assay is evaluated based on the coefficient of variation (CV) of the G0/G1 phase of diploid control cells present in the processed sample.This CV is the result of sample quality, sample processing errors, heterogeneity in individual cells' DNA content, and the degree of chromatin compaction.For optimal FCM DNA analysis, the CV of the G0/ G1 phase of diploid control must be less than 6%, with CV less than 3% yielding the best results. 4,18,52he common pitfalls encountered during FCM DNA analysis are depicted in ►Table 3. 3,4,52

Conclusion
FCM DNA assessment is an objective, reproducible, and sensitive assay to evaluate ploidy and SPF of any cells of interest.FCM-determined ploidy correlates with CG-determined ploidy and patient survival.The FCM DNA assay can be used even in scenarios where the load of tumor cells is low or conventional CG assessment is not feasible or fails.

G1•
Time gap between a recently concluded mitosis and the onset of the next DNA synthesis process • Cells undergo synthesis of RNA and ribosomal protein machinery needed for DNA synthesis S • Synthesis and replication of nuclear DNA content before division • Indicates proliferative intent of a cell G2 • The interval between completion of DNA synthesis and onset of mitosis • Opportunity to repair any DNA damage that might have occurred in preceding cell cycles • Reorganization of DNA structure, enabling equal division of DNA between ensuing daughter cells Mitosis M • The actual process of cell division • Equal distribution of DNA, RNA, protein, and cellular organelles between the daughter cells

Fig. 1
Fig. 1 Flowcytometric DNA assay depicted as a multimodal histogram (shaded curves).The green, blue, and red colored lines represent the theoretical G0/G1, S, and G2/M phases, respectively.Y-axis: number of cells.X-axis: intensity of staining with FxCycle violet, a DNA-specific fluorescent dye.

Fig. 2
Fig. 2 DNA index assessment in B-lymphoblastic acute lymphoblastic leukemia using FxCycle violet, a DNA-specific dye.(a-c) From a sample with diploid blasts.(d-f) From a sample with high hyperdiploid blasts.Overlay histograms (c,f) show the G0-/G1-phase cells of both blasts and lymphocytes.The fluorescence intensity of FxCycle violet is depicted as Geometric mean (GMean) of expression.