Skip to main content

Immunophenotypic Characterization and Ploidy Analysis of Neoplastic Plasma Cells by Multiparametric Flow Cytometry


Flow cytometric (FCM) immunophenotyping is an important tool for generating diagnostic and prognostic information in plasma cell dyscrasias. This study aimed to evaluate the immunophenotype and ploidy status of plasma cells (PCs) in patients of myeloma and its correlation with other laboratory parameters. Bone marrow of 70 newly diagnosed cases of myeloma were subjected to FCM using a panel of antibodies; CD138, CD38, CD19, CD45, CD28, CD81, CD56, CD200, and CD229. FxCycle Violet (FCV) dye was used for the ploidy analysis of clonal PCs. Median age was 60 years with M:F ratio of 3.2:1. A positive correlation was noted between the morphological and FCM-based PC enumeration (r = 0.4, p = 0.001). Aberrant expression of CD56, CD200, CD28, CD117, CD81 and CD19 and was observed in 88.5%, 77%, 29%, 37%, 23% and 17% cases respectively. Two aberrant antigens were noted in all cases. CD81 + cases had a relatively higher quantity of monoclonal-protein (> 1 g/dl, p < 0.05) and renal insufficiency (Cr > 2 mg/dl, p < 0.05) as compared to the CD81- cases. CD229 was expressed in all the cases, with a median MFI in PCs significantly higher than other hematopoietic elements. Hyperdiploid PCs (median DI-1.59, range, 1.16–2.6) were noted in 80% cases (n = 48), diploid/ near-hyperdiploid PCs in 8% (n = 5) cases and hypodiploidy in 3% (n = 1) cases. Bright CD56/CD200 and CD45- can identify abnormal PC in the majority of the cases. CD81 appears to correlate with disease burden and might be useful as a prognostic marker. CD229 is a reliable gating marker for plasma cells. Ploidy analysis may be incorporated in routine workup to guide in the identification of patients with poor prognosis.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3


  1. Paiva B, Almeida J, Pérez-Andrés M, Mateo G, López A, Rasillo A, Vídriales MB, López-Berges MC, Miguel JF, Orfao A (2010) Utility of flow cytometry immunophenotyping in multiple myeloma and other clonal plasma cell-related disorders. Cytometry B Clin Cytom 78(4):239–252.

    CAS  Article  PubMed  Google Scholar 

  2. Peceliunas V, Janiulioniene A, Matuzeviciene R, Griskevicius L (2011) Six color flow cytometry detects plasma cells expressing aberrant immunophenotype in bone marrow of healthy donors. Cytometry B Clin Cytom 80(5):318–323.

    CAS  Article  PubMed  Google Scholar 

  3. Robillard N, Wuillème S, Moreau P, Béné MC (2014) Immunophenotype of normal and myelomatous plasma-cell subsets. Front Immunol 31(5):137.

    CAS  Article  Google Scholar 

  4. Flores-Montero J, de Tute R, Paiva B, Perez JJ, Böttcher S, Wind H, Sanoja L, Puig N, Lecrevisse Q, Vidriales MB, van Dongen JJ, Orfao A (2016) Immunophenotype of normal vs. myeloma plasma cells: toward antibody panel specifications for MRD detection in multiple myeloma. Cytometry B Clin Cytom 90(1):61–72

    CAS  Article  Google Scholar 

  5. Lin P, Owens R, Tricot G, Wilson CS (2004) Flow cytometric immunophenotypic analysis of 306 cases of multiple myeloma. Am J Clin Pathol 121(4):482–488.

    Article  PubMed  Google Scholar 

  6. Mateo G, Montalbán MA, Vidriales MB, Lahuerta JJ, Mateos MV, Gutiérrez N, Rosiñol L, Montejano L, Bladé J, Martínez R, de la Rubia J, Diaz-Mediavilla J, Sureda A, Ribera JM, Ojanguren JM, de Arriba F, Palomera L, Terol MJ, Orfao A, San Miguel JF (2008) PETHEMA study group; GEM Study Group. Prognostic value of immunophenotyping in multiple myeloma: a study by the PETHEMA/GEM cooperative study groups on patients uniformly treated with high-dose therapy. J Clin Oncol 26(16):2737–2344

    Article  Google Scholar 

  7. Guo J, Su J, He Q, Li X, Zhao Y, Gu S, Fei C, Chang C (2016) The prognostic impact of multiparameter flow cytometry immunophenotyping and cytogenetic aberrancies in patients with multiple myeloma. Hematology 21(3):152–161.

    CAS  Article  PubMed  Google Scholar 

  8. Pojero F, Flores-Montero J, Sanoja L, Pérez JJ, Puig N, Paiva B, Bottcher S, van Dongen JJ, Orfao A (2016) EuroFlow group. Utility of CD54, CD229, and CD319 for the identification of plasma cells in patients with clonal plasma cell diseases. Cytometry B Clin Cytom 90(1):91–100

    CAS  Article  Google Scholar 

  9. Darzynkiewicz Z, Halicka HD, Zhao H (2010) Analysis of cellular DNA content by flow and laser scanning cytometry. Adv Exp Med Biol 676:137–147.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Tembhare P, Badrinath Y, Ghogale S et al (2016) A novel and easy FxCycleTM violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping. Cytometry A 89(3):281–291.

    CAS  Article  PubMed  Google Scholar 

  11. Morgan RJ Jr, Gonchoroff NJ, Katzmann JA, Witzig TE, Kyle RA, Greipp PR (1989) Detection of hypodiploidy using multi-parameter flow cytometric analysis: a prognostic indicator in multiple myeloma. Am J Hematol 30(4):195–200.

    Article  PubMed  Google Scholar 

  12. Van Wier S, Braggio E, Baker A, Ahmann G, Levy J, Carpten JD, Fonseca R (2013) Hypodiploid multiple myeloma is characterized by more aggressive molecular markers than non-hyperdiploid multiple myeloma. Haematologica 98(10):1586–1592.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Gupta R, Bhaskar A, Kumar L, Sharma A, Jain P (2009) Flow cytometric immunophenotyping and minimal residual disease analysis in multiple myeloma. Am J Clin Pathol 132(5):728–732.

    Article  PubMed  Google Scholar 

  14. Cho YU, Park CJ, Park SJ, Chi HS, Jang S, Park SH, Seo EJ, Yoon DH, Lee JH, Suh C (2013) Immunophenotypic characterization and quantification of neoplastic bone marrow plasma cells by multiparametric flow cytometry and its clinical significance in Korean myeloma patients. J Korean Med Sci 28(4):542–549.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Shim H, Ha JH, Lee H, Sohn JY, Kim HJ, Eom HS, Kong SY (2014) Expression of myeloid antigen in neoplastic plasma cells is related to adverse prognosis in patients with multiple myeloma. Biomed Res Int 2014:893243.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Salama ME, Du S, Efimova O et al (2015) Neoplastic plasma cell aberrant antigen expression patterns and their association with genetic abnormalities. Leuk Lymphoma 56(2):426–433.

    CAS  Article  PubMed  Google Scholar 

  17. Gupta S, Karandikar NJ, Ginader T, Bellizzi AM, Holman CJ (2018) Flow cytometric aberrancies in plasma cell myeloma and MGUS - correlation with laboratory parameters. Cytometry B Clin Cytom 94(3):500–508.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Chen F, Hu Y, Wang X, Fu S, Liu Z, Zhang J (2018) Expression of CD81 and CD117 in plasma cell myeloma and the relationship to prognosis. Cancer Med 7(12):5920–5927.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Draxler DF, Wutzlhofer LM, Slavka G, Hübl W, Ludwig H, Schreder M, Reynolds J, Willheim M (2019) Flow Cytometric Evaluation of Traditional and Novel Surface Markers for the Diagnosis of Plasma Cell Dyscrasias. Indian J Hematol Blood Transfus 35(4):673–682.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Alapat D, Coviello-Malle J, Owens R, Qu P, Barlogie B, Shaughnessy JD, Lorsbach RB (2012) Diagnostic usefulness and prognostic impact of CD200 expression in lymphoid malignancies and plasma cell myeloma. Am J Clin Pathol 137(1):93–100.

    Article  PubMed  Google Scholar 

  21. Olteanu H, Harrington AM, Hari P, Kroft SH (2011) CD200 expression in plasma cell myeloma. Br J Haematol 153(3):408–411.

    Article  PubMed  Google Scholar 

  22. Nair JR, Carlson LM, Koorella C, Rozanski CH, Byrne GE, Bergsagel PL, Shaughnessy JP Jr, Boise LH, Chanan-Khan A, Lee KP (2011) CD28 expressed on malignant plasma cells induces a prosurvival and immunosuppressive microenvironment. J Immunol 187(3):1243–1253.

    CAS  Article  PubMed  Google Scholar 

  23. Hundemer M, Klein U, Hose D, Raab MS, Cremer FW, Jauch A, Benner A, Heiss C, Moos M, Ho AD, Goldschmidt H (2007) Lack of CD56 expression on myeloma cells is not a marker for poor prognosis in patients treated by high-dose chemotherapy and is associated with translocation t(11;14). Bone Marrow Trans 40(11):1033–1037.

    CAS  Article  Google Scholar 

  24. Kraj M, Sokołowska U, Kopeć-Szlezak J, Pogłód R, Kruk B, Woźniak J, Szpila T (2008) Clinicopathological correlates of plasma cell CD56 (NCAM) expression in multiple myeloma. Leuk Lymphoma 49(2):298–305.

    CAS  Article  PubMed  Google Scholar 

  25. Sahara N, Takeshita A, Shigeno K, Fujisawa S, Takeshita K, Naito K, Ihara M, Ono T, Tamashima S, Nara K, Ohnishi K, Ohno R (2002) Clinicopathological and prognostic characteristics of CD56-negative multiple myeloma. Br J Haematol 117(4):882–885.

    Article  PubMed  Google Scholar 

  26. Tembhare PR, Ghogale S, Tauro W, Badrinath Y, Deshpande N, Kedia S, Cherian K, Patkar NV, Chatterjee G, Gujral S, Subramanian PG (2018) Evaluation of CD229 as a new alternative plasma cell gating marker in the flow cytometric immunophenotyping of monoclonal gammopathies. Cytometry B Clin Cytom 94(3):509–519.

    CAS  Article  PubMed  Google Scholar 

  27. Yousef S, Kovacsovics-Bankowski M, Salama ME, Bhardwaj N, Steinbach M, Langemo A, Kovacsovics T, Marvin J, Binder M, Panse J, Kröger N, Luetkens T, Atanackovic D (2015) CD229 is expressed on the surface of plasma cells carrying an aberrant phenotype and chemotherapy-resistant precursor cells in multiple myeloma. Hum Vaccin Immunother 11(7):1606–1611.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Almeida J, Orfao A, Ocqueteau M, Mateo G, Corral M, Caballero MD, Blade J, Moro MJ, Hernandez J, San Miguel JF (1999) High-sensitive immunophenotyping and DNA ploidy studies for the investigation of minimal residual disease in multiple myeloma. Br J Haematol 107(1):121–131.

    CAS  Article  PubMed  Google Scholar 

Download references


The work was sponsored as an intramural project by Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, U.P. India.

Author information

Authors and Affiliations



RG and KR were involved in the conceptualization of the idea and the preparation of the manuscript. RG had acquired the funds for the project. PA and SB were involved in the data acquisition. MKS was involved in carrying out the lab experiment. MKS and DC have assisted in the formal analysis of data. AG and SN were involved in the management of the patients and assisted in clinical data.

Corresponding author

Correspondence to K. Rahman.

Ethics declarations

Conflict of interest

The authors declare that they do not have any conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (TIF 163 kb)

Supplementary file2 (DOCX 14 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gupta, R., Gupta, P., Rahman, K. et al. Immunophenotypic Characterization and Ploidy Analysis of Neoplastic Plasma Cells by Multiparametric Flow Cytometry. Indian J Hematol Blood Transfus 38, 473–480 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Plasma cell dyscrasia
  • Immunophenotype
  • FxCycle Violet
  • Ploidy
  • CD229