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Abstract
Background: Identification of plasma cells into abnormal (APC) and normal (NPC) compartments is of utmost importance in flow cytometric (FC) analysis of multiple myeloma (MM) and related plasma cell dyscrasias for diagnosis, prognosis, and follow-up. No single phenotypic marker is sufficient to distinguish NPC from APC. Materials and Methods: 43 newly diagnosed cases of MM and 13 controls were included in the study. Bone marrow (BM) samples from the 2nd pass were processed on the same day with antibodies against CD38, CD138, CD19, CD81, CD45, CD117, CD200, CD56, cytoKappa, and cytoLambda in a 4-color experiment with CD38 and CD138 as gating antibodies. Results: Mean APC% in cases was 96.5%. The expected Immunophenotype (IP) of APC which is CD19-/56+/45-/81-/117+/200+ was found in only 13/43 MM cases. In 30/43 cases, APC revealed deviation from expected IP either for single or a combination of markers. Sensitivity for APC detection was highest for CD19 (95.2%) followed by CD56 (90.4%) and CD81 (83.7%). Specificity was highest for CD19 (100%), CD56 (100%), and CD81 (100%) followed by CD117 (92.3%). Combination of markers with maximum sensitivity to detect APC (97.6%) was CD81- or CD19- and CD200+ or CD56+ (two markers); and for NPC (92.3%) was CD81+ and CD19+ and CD56- (three markers). Conclusion: Plasma cell IP can be highly variable with multiple minor subpopulations in both cases and normal controls. CD 19 and CD56 are highly informative markers for a 4-color experiment. Assessment of multiple markers in an 8–10 color experiment is more informative but the lack of advanced flow cytometers should not limit the use of FC in a 4-color approach. Our results emphasize that even basic equipment with limited fluorochrome can provide meaningful information if used appropriately.
Keywords: Abnormal plasma cells, CD marker, flow cytometry, normal plasma cells, plasma cells
How to cite this article: Introduction 
Multiple myeloma (MM) is the second most common hematologic malignancy and comprises approximately 1% of all tumors.[1] The proliferation of plasma cells (PC) with aberrant immunophenotype secreting a single type of light chain is a characteristic and leads to a plethora of clinical manifestations. The use of multiparametric flow cytometry (MFC) in the diagnosis of MM and other monoclonal gammopathies has significantly increased and may be considered mandatory in specific areas of routine clinical practice. MFC allows distinguishing the normal (NPC) versus abnormal clonal plasma cells (APC) with higher sensitivity and specificity.[1] It has been repeatedly concluded that no single marker can help in the differentiation of NPC from APC with 100% sensitivity and specificity.[2],[3],[4] Intratumoral heterogeneity plays an important role in MM pathogenesis. In the majority of myeloma cases, aberrant immunophenotypes are observed at diagnosis. Lack of CD19 has been described as a hallmark of malignant PC.[5] However, CD19+ and CD19-, including CD19-CD56+ PC have been reported in healthy BM.[6]
Identification of plasma cells into abnormal (APC) and normal (NPC) compartments is of utmost importance in flow cytometric (FC) analysis of MM and related plasma cell dyscrasias for diagnosis, prognosis, and follow-up. The current study investigates the immunophenotypic characterization of normal and abnormal plasma cells in BM samples of MM by 4-color MFC. We have determined the sensitivity and specificity of markers (CD19, CD45, CD117, CD56, CD200, and CD81). In addition, various combinations of markers were studied for the identification of NPC and APC to add to the existing knowledge on plasma cell immunophenotyping.
[TAG:2]Materials and Methods[/TAG:2]
Patients and sample
Diagnostic BM samples were analyzed from 43 patients with MM and 15 controls for one year, at the flow cytometry lab, Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow. A diagnosis of MM was based on the criteria of the International Myeloma Working Group (IMWG) 2014.[7] Cases that fulfilled the above criteria with >/=10% clonal plasma cells (based on a morphological assessment) or biopsy-proven plasmacytoma, together with at least one of the myeloma defining events were included in the study.
Controls
BM specimens from 13 control cases were collected and processed, which included 5 staging marrows from cases of Hodgkin lymphoma/solid tumors, 2 hypoplastic, 1 megaloblastic, 1 immune thrombocytopenia, 2 cases of chronic renal failure, and 2 cases of reactive BM.
Written informed consent was taken from the patients and controls. The study was approved by the authors' Institutional Ethical Committee. Any case which did not meet the diagnostic criteria for MM as per IMWG, 2014 were excluded from the study. A detailed clinical history and records related to the diagnostic workup of MM were collected from the patient's hospital records.
Sample collection and processing
Second pass BM aspirate was collected (0.3 to 0.5 ml) in EDTA anticoagulant and processed within the same working day. A panel of antibodies against CD38, CD138, CD19, CD81, CD45, CD117, CD200, CD56, Kappa, and Lambda light chains was used [Table S1]. The panel was standardized after titration of antibodies (BD Biosciences, San Jose, CA). The specimen was processed as per the protocol using the whole blood lysis technique.
In brief, for assessing surface antigens, 1 × 106 cells were labeled with fluorescently conjugated antibodies followed by lysis using BD FACSLyse (1x) (Cat no. 349202). For cytoplasmic kappa and lambda light chain expression, lysis was performed by BD Pharmylse (1x) (BD Biosciences, USA, Cat no. 555899) followed by washing and surface staining with CD38 and CD138. The cells were then fixed and permeabilized by Cytofix/cytoperm Kit (BD, Biosciences, Cat no- 554714) followed by the addition of Kappa and Lambda antibodies (BD Biosciences, USA, cat no. 349516). After the final washing in phosphate-buffered saline, cells were suspended in 1% paraformaldehyde. Sample acquisition was done on BD FACS Calibur flow cytometer (BD Biosciences, USA) equipped with 2 lasers for 4-color immunophenotyping using Cell Quest Pro software. At least 105 events or more were acquired in each tube. Negative limits were set by autofluorescence. Expression of any marker by more than 20% of cells was considered positive. Data analysis was performed on FCS Express 6 (De Novo Software, CA, USA).
Definition of APC and NPC
Plasma cells were identified by gating the cells on CD 138 positivity and bright CD38 expression. In addition, CD45 expression and light scatter properties were utilized to exclude lymphocytes and debris. Following criteria were used for the definition of APC: at least 2 aberrant antigens or monoclonal intracellular light chain expression (K: L >3:1 or <0.5:1) or both.[8],[9] Plasma cells in control samples were deemed to be NPC. In MM cases, NPC were identified based on polyclonal intracellular light chain expression and/or a typical nonneoplastic/reactive plasma cell immunophenotype (CD19+/CD45+/CD81+/CD200-/CD117-/CD56-).
Statistical analysis
GraphPad Software Inc. (version 8.0) was used for data analysis. Data was presented in median (Q1-Q3) and numbers (%). The Mann–Whitney U and Pearson correlation tests were used to estimate the statistical significance of differences between groups. One-way ANOVA was applied to test the significance between the three groups. Receiver operating curve (ROC) analysis was performed to check the diagnostic. 2 × 2 tables were built categorizing a combination of CD19 and CD56 test as positive or negative based on individual cut-offs obtained from ROC curves. A value of ≤0.05 was considered statistically significant in all statistical analyses.
Results The study included 43 cases (27 male and 16 female) and 13 controls (09 male and 04 female). Characteristics of cases and controls concerning age, sex, plasma cell percentage on BM aspiration morphology (Morphology: BM PC%), and flow cytometry (FCM: BM PC%) are given in [Table S2]. The median age of cases and control groups were 42 and 57 years, respectively. Morphological analysis of plasma cells in BM aspirate showed a median (Q1-Q3) count of 32% (18–60) in cases and 5% (2–7) in controls. In flowcytometry median (Q1-Q3) plasma cell count was 5.4% (1.7–13) in cases and 0.48% (0.19–1.55) in controls. In MM cases, APC was seen in 24 (56%) cases while both APC and NPC were seen in 19 (44%) cases [Table S2]. Identification of NPC in MM cases was facilitated by a small cluster of plasma cells with the typical immunophenotype of NPC other than the major population of APC or by expression of polyclonal light chains or both [Figure 1]a, [Figure 1]b.
Figure 1: (a) Bone marrow from a control patient (staging marrow) with plasma cells shown in green: CD38+, CD138+, CD19+, CD56-, CD45+, CD117-, CD81+, CD200-. CD19/CD56; dot plot shows three populations of plasma cells CD19 + CD56-(major, blue arrow); CD19+CD56+ (red arrow) and CD19-CD56+ (black arrow) both minor populations. (b) Bone marrow from a case of multiple myeloma with plasma cells shown in green. Both abnormal plasma cells (red arrow) and normal plasma cells (blue arrow) are shown. APC are kappa restricted while a cluster showing Lambda positivity is NPCs
Antigen expression in MM cases for identification of plasma cells
CD 45 and CD117 expression could be evaluated in 42 cases. CD45 was expressed as a homogenous single cluster (dim or moderate) in 5 (12%), absent in 15 (36%), and heterogeneous in 22 (52%) cases. The homogenous expression of CD56 was most frequent and was present in 38 (90.5%) cases followed by CD200 in 34 (79%), CD117 in 22 (52%), CD81 in 07 (16%), and CD19 in 2 (4.76%) cases [Table 1].
Table 1: Antigen expression pattern and sensitivity and specificity of various antigens in multiple myelomaAntigen expression in control for identification of plasma cells
Controls showed a typical immunophenotype of NPC in 8/13 (61%). Among the remaining 5 controls, the following aberrancies were revealed: dim expression of CD200 in 5/5, dim CD117 expression in 1/5, and negative to a dim heterogeneous expression of CD45 in 2/5. CD45 was expressed in 11/13 (85%) controls.
Sensitivity and specificity of markers (CD19, CD45, CD117, CD56, CD200, and CD81) and their combinations for identification of abnormal plasma cells
Sensitivity and specificity of various markers in detection of APC are depicted in [Table 1]. Sensitivity of CD19 was highest (95.24%) followed by CD 56 (90.48%), CD 81 (83.7%), CD 200 (79.07%), CD 117 (52.38) and specificity was 100.00%, 100.00%, 100.00%, 53.85%, and 92.31%, respectively.
Analysis with the combination of CD19 and CD56
Compared with individual sensitivity and specificity of various markers, combined diagnostics using one or more test positivity as a positive test, and all two tests negative as negative interpretation, the combination of CD19 and CD56 was assessed: various subpopulations of CD19+56-, CD19-56+, CD19+56+, and CD19-CD56- were identified. The combination of CD19-CD56+ and CD19+ CD56- showed significant difference in cases compared to controls (85.14 ± 27.30% vs. 6.28 ± 4.49%, P = <0.0001 and 11.66 ± 14.32% vs. 76.00 ± 22.01%, P = <0.0001), respectively, as depicted in [Table 2]. ROC curves were drawn for distinguishing cases from controls. Area under curve (AUC) for the combination of CD19-56+ and CD19+56- were 0.973 and 0.987, respectively. The combination CD19-56 + showed a sensitivity and specificity of 97.62% and 100.0% followed by the combination of CD19+56- (83.33% and 100.00%) with a diagnostic accuracy 97.96% and 92.00%, respectively [Table 3].
Table 2: Markers expression for combination of CD19 and CD56 in cases and controls
Table 3: Diagnostic value of different combinations of CD19 and CD56 using ROC curve analysisSensitivity of combination of markers for detection of abnormal plasma cell
We analyzed various combinations of 2, 3, 4, 5, and 6 markers (total 18 combinations studied) and found three different types of combinations including two, three, and four marker combinations as giving sensitivity of >85% for detection of APC and is depicted in [Table 4]. The combination of 2 markers (CD81- or CD19- and CD200 + or CD56+) showed highest sensitivity of 97.61%. The combination of 3 markers (CD45- or CD81- or CD19- [any 1 negative] and CD200+ or CD56+ or CD117+ [any 2 positive]) resulted in a sensitivity of 90.47% followed by the combination of 4 markers: (CD45- or CD81- or CD19- [any 2 negative] and CD200+ or CD56+ or CD117+ [any 2 positive]) with a sensitivity of 85.71%. For the other combinations, the sensitivity ranged from 04.76 to 80.9% (data not shown).
Table 4: Sensitivity of combination of markers for abnormal plasma cells and normal plasma cellsSensitivity of combination of markers for detection of normal plasma cell
The combination of the three markers (CD81+ and CD19+ and CD56-) resulted in a sensitivity of 100.0%, followed by the combination of 4 markers (CD81+ and CD19+ and CD56- and CD117-) showed 92.30% sensitivity for detection of NPC [Table 4].
Discussion This study demonstrates the diagnostic utility of markers and their combinations, in a 4-color experiment, for the characterization and quantification of plasma cells by MFC in newly diagnosed MM patients. The results can be extrapolated to other plasma cell dyscrasias as aberrant immunophenotypes of plasma cells are shared by most entities in this group. Morphologic analysis and immunohistochemical analysis of PCs remain the gold standard and an important diagnostic measure for MM in routine practice. Quantification of PCs using MFC has now become a standard practice in monitoring treatment response in MM. It is well-accepted that the percentage of PCs is usually underrepresented by MFC as compared to other cytologic methods.[10] Contributing factors include, but are not limited to, second pull BM for MFC which is hemodiluted as compared to first pull, uneven distribution of plasma cells associated with lipid-enriched BM spicules in the morphology slides, as opposed to lipid-depleted liquid BM analyzed by MFC, and time delay in sample procurement and processing.[11],[12],[13]
We came across a similar discrepancy in our results with PC% on smear morphology and by MFC as 32% vs. 5.4% and 5% vs. 0.57% for cases and controls. It is well established that compared to a conventional morphologic and immunohistochemical method, MFC has high sensitivity in the identification of PCs.[14]
It has been suggested by leading researchers that combined assessment of CD138 and CD38, together with CD45, CD19, CD56, CD27, CD81, and CD117 would be ideally suited for MRD monitoring in virtually every MM patient.[15] In keeping with this, we aimed to include as many markers as feasible in a 4-color experiment. Accordingly, we have included CD138 and CD38 as gating markers with CD45 in one tube to aid in PC identification as recommended by European Myeloma Network, 2008. Additionally CD19, CD56, CD200, CD81, and CD117 were assessed.
In MM cases, only APCs were detected in 56% cases, whereas 44% cases revealed a mixed bag of APCs and NPCs with the latter being in small fractions. We were able to identify PCs with CD138 and CD38 gating in all cases with additional confirmation by CD45 and scatter properties. However, because of no or heterogeneous expression of CD45 on PCs, it cannot be relied completely. Downregulation of CD138 is seen in aged samples and therefore sample needs to be processed rapidly to avoid counting falsely low numbers.[9],[15],[16],[17] It is important to analyze all PCs population for the complete immunophenotypic profiling and functional analysis of subpopulations of interest.[18]
Markers considered essential for detecting aberrant phenotypes, e.g. CD19, are not strictly aberrant on their own. CD19 has recurrently emerged as the most informative marker for flow-MRD in MM because it is absent in tumor PCs from virtually every MM case; there are a significant fraction of all normal/reactive BM PCs (around 30%) that are also negative for CD19.[3],[19] The demonstration of restricted immunoglobulin coupled with an abnormal immunophenotype can be used to distinguish between reactive and neoplastic conditions.[12] We have found cytoplasmic light chain expression to be most useful for assessing the clonal nature of PCs.
CD45 was heterogeneously expressed in 52% cases and absent in 36% cases. In controls, CD45 was expressed in 85% and was negative to dim in a heterogeneous manner in 15%. Liu et al.[19] evaluated the characteristics of PC and found the heterogeneous expression of CD45 with a stronger expression of CD28, CD56, or CD117. Similar findings have been reported by others.[20] Therefore, it can be concluded that CD45 individually does not provide critical information about the nature of PCs but in 8–10 color panels for MRD evaluation, it can assist in gating and exclusion of non-PCs.
CD19 is highly sensitive for the detection of APC ranging from 95–100% in various studies and we found a similar value of 95.2%.[2],[8] But we also found a high specificity (100%) as CD19 was always expressed in controls, in contrast to other studies, which have reported that CD19 may be absent in a small population of NPCs as well.[2] We found very frequent (90.5%) positivity for CD56 in newly diagnosed MM cases similar to previous reports.[21] CD56 has long been claimed to be a myeloma-specific PC marker; however, a small subset of normal/reactive BM PCs show unequivocal CD56 expression at variable levels. Raswstron et al.[22] reported the expression of CD56 throughout the disease and CD56 value above 15% is considered abnormal on PCs. We did not find CD56 expression in control subjects probably because of high threshold levels used to define positive or negative.
Expression of CD19 and CD56 markers are heterogeneous on bone marrow PCs of healthy individuals and in MM cases.[6] In 2011, Peceliunas et al.[6] in a 6-color approach characterized four different subsets of PCs among normal PC, based on the expression or absence of CD19 and CD56: “normal” PC CD19+/CD56-, “aberrant” CD19-/CD56+, “double positive” CD19+/CD56+ and “double negative” CD19-/CD56-. In a total of 11 normal BM samples, all subsets were observed in 5 subjects, 5 were lacking “double positive” PC, and 1 was lacking a significant amount of “aberrant” PC. The median proportions were 60.3%, 9.6%, 3%, and 29.9%, respectively. On similar lines, Robillard et al.[23] in 2014, discriminated bone marrow PCs in four groups based on CD19 and CD56/CD28 in normal BM samples. The authors reported median proportions of 53.7% for CD19+ (CD56/CD28)-, 12% CD19-(CD56/CD28)+, 7% dual positive, and 23% dual negative. Similar analyses on MM cases revealed median proportions of different populations, respectively, as 0.3, 96.5, 0.5, and 1.2% of all PC for the four subsets. We have found all four subgroups of PCs based on CD19/CD56 in both cases and controls. Mean values for CD19-CD56+ and CD19+ CD56- PCs showed a highly significant difference in cases compared to controls (P < 0.0001). In an attempt to derive the diagnostic cut-off of this combination in a 4-color experiment, AUC for a combination of CD19-56+ and CD19+ 56- was calculated, which revealed 0.973 and 0.987, respectively. The combination of CD19-56+ at a cutoff value of ≥24.0 showed 100.0% specificity and 97.62% sensitivity with 100.0% positive predictive value. The combination of CD19+ 56- at a cutoff point of ≤12 showed 100.0% specificity and 100.0% PPV; however, the sensitivity was 83.33%, suggesting this combination to be extremely useful for diagnostic immunophenotyping where several fluorescent channels are limited.
CD117 expression, which has a potential for targeted therapy, was present in 22 cases resulting in a sensitivity of 52.38%. Other researchers have reported sensitivity in the range of 30–50.5%.[8] However, it revealed high specificity of 92.31% as only one control revealed CD117 positive PCs (30% cells with dim expression). No CD117+ PCs have been reported in Normal/reactive BM making it a highly useful marker to detect NPCs.[14]
Studies have demonstrated CD81 expression on PCs to have diagnostic and prognostic efficacy. In half of the myeloma cases, dim to negative expression has been reported and CD81 positivity identified as an independent prognostic factor. In a study by Tembhare et al.,[2] the addition of CD81 in plasma cell dyscrasias panel improved the precision of APC enumeration and found strong expression of CD81 in NPCs, on the other hand, 95% of APCs showed negative or weak expression. In detection of APC, CD81 showed 100.0% specificity and 95.00% sensitivity. Our study is concordant, revealing 100% specificity and 83.70% sensitivity for APC. We have also found 100.0% sensitivity for the combination of CD81+/CD19+/CD56- in the identification of NPC.
CD200 is a transmembrane glycoprotein and acts as a therapeutic target due to its role in immune regulation and tolerance.[24] In PCM patients, CD200 expression has been associated with poor prognosis.[25]
Limitations of our study are relatively small study groups and the utilization of a 4-color platform, which is slowly becoming obsolete now especially in the developed world. However, a significant part of our world has either no access to a flow cytometer or relies on basic flow cytometers with a 2-laser configuration. Our results emphasize that even basic equipment with limited fluorochromes can provide meaningful information if used appropriately. We were also able to demonstrate different plasma cell subsets in the CD19/CD56 tube and this combination turned out to be highly informative. The present study reports the diagnostic value of different antigens in individual or combination for discrimination of abnormal plasma cells in MM cases.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
The study was funded by the Intramural research grant from Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow.
Conflicts of interest
There are no conflicts of interest.
References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_505_21
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