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Utility of CD200 expression by flow cytometry in lymphoproliferative disorders and plasma cell dyscrasias

1 Department of Pathology, Nizams Institute of Medical Sciences, Hyderabad, Telangana, India
2 Department of Medical Oncology, Nizams Institute of Medical Sciences, Hyderabad, Telangana, India
3 Department of Hematology, Nizams Institute of Medical Sciences, Hyderabad, Telangana, India

Date of Submission19-May-2021
Date of Decision06-Jul-2021
Date of Acceptance27-Feb-2022

Correspondence Address:
Navatha Vangala,
Department of Pathology, Nizams Institute of Medical Sciences, Hyderabad, Telangana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjdrdypu.mjdrdypu_373_21


Background: The cluster of differentiation 200 (CD200) is a recently introduced marker, used to differentiate various lymphoproliferative disorders (LPDs) and is a potential target for chemotherapy. Objective: The objective is to study the utility of CD200 expression by flow cytometry (FC) in various LPDs and plasma cell disorders. Materials and Methods: This is a retrospective study done over a period of 2 years. The study group included 52 cases with a clinical suspicion of LPD (n = 40) or plasma cell disorder (n = 12). Clinical data, morphological data on peripheral blood, and/or bone marrow examination were analyzed and correlated with the final results on FC. Results: Out of 40 LPDs, chronic lymphocytic leukemia (CLL) accounted for a majority of the cases accounting for 57.5% (23 cases). Plasma cell myelomas (PCM) were the most common plasma cell disorders accounting for 75% (nine cases). All cases of CLL showed CD200 expression and the two cases of mantle cell lymphoma (MCL) were CD200 negative. Splenic marginal zone lymphomas (MZL) involving marrow showed dim CD200 expression. Bright CD200 expression was also observed in all cases of hairy cell leukemia (HCL) and 67% of cases diagnosed as PCM. Conclusion: CD200 is a very useful marker in the diagnosis of various LPDs especially CLL, HCL, and PCMs. It can be used as an additional marker particularly in distinguishing CLL/small lymphocytic lymphoma (SLL) from MCL and atypical CLL from other CD5+ B-cell neoplasms and extranodal MZL.

Keywords: CD200, lymphoproliferative disorders, plasma cell disorders

How to cite this URL:
Divya P, Vangala N, Uppin MS, Uppin SG, Gundeti S, Bacchu S, Radhika K K, Parvathi A, Paul TR. Utility of CD200 expression by flow cytometry in lymphoproliferative disorders and plasma cell dyscrasias. Med J DY Patil Vidyapeeth [Epub ahead of print] [cited 2022 Dec 6]. Available from: https://www.mjdrdypv.org/preprintarticle.asp?id=343839

  Introduction Top

The cluster of differentiation 200 (CD200) (OX-2 membrane glycoprotein) is a newly discovered antigen expressed in B lymphocytes and a subset of T cells. It is a recently introduced marker, used to differentiate various lymphoproliferative disorders (LPDs) and is a potential target for chemotherapy. Low-grade lymphoid neoplasms such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are difficult to distinguish especially if they show partial CD5 expression.[1],[2] Bright CD200 expression is seen in CLL, unlike MCL. The present study aims to assess the utility of CD200 expression by flow cytometry (FC) in various LPDs and plasma cell disorders.

  Materials and Methods Top

This is a retrospective study done from January 2019 to December 2020. The study was approved by the institutional ethics committee. The study was approved by the institutional ethics committee with approval number of EC/NIMS/2713/2021, 53rd ESGS No: 1115/2021 dated 24.3.2021. All the data was retrieved from the departmental archives and patient identity was never disclosed in the entire study. CD200 was recently introduced in our lab and has been used in flow cytometric immunophenotyping for over 2 years. The samples subjected to FC were freshly collected peripheral blood or bone marrow aspirates. Informed consent was taken from all patients before the collection of samples. The study group included 52 cases with a clinical suspicion of lymphoproliferative or plasma cell disorder. FC was done on peripheral blood in 35 cases and on bone marrow aspirate in 17 cases. Heparin or ethylenediaminetetraacetic acid (EDTA) anti-coagulated peripheral blood or bone marrow sample (100 μl) was washed with PBS. To this various cocktails of titrated fluorochrome, tagged antibodies were added. These included the primary and secondary panels of monoclonal antibodies, which were used, based on clinical findings and morphology. The primary panel included CD45, CD3, CD19, CD20, CD5, CD23, CD38, kappa, lambda, CD10, CD200, and the secondary panel for B-cells included immunoglobulin M (IgM), CD103, CD180, CD20, CD79b, CD11c, CD25, CD123, and CD43. In addition, CD138 and CD56 were included in the secondary panel for plasma cells. The cell suspensions were then incubated in dark for 15 min followed by the addition of 500 μl of OptiLyse C (Beckman Coulter company) to lyse the red blood cells. To this, 4 ml of isotone solution was added and centrifuged at 1500 rpm for 5 min. This is followed by decanting supernatant and further addition of 500 μl of isotone solution followed by analysis on Navios Ex (Beckman Coulter company) 3 lasers, 10 colors analyzer. Gating of lymphoid cells was done based on CD45 versus side scatter analysis. Plasma cells were gated based on CD138 versus side scatter analysis. On FC, a CD marker was considered positive if it was expressed in more than 20% of cells. CD200 expression was evaluated semi-quantitatively by comparison with negative control and the results were designated as dim or 1+ (<1 log shift in mean fluorescence intensity [MFI] compared with negative control), moderate or 2+ (1-2 log shift in MFI), and bright or 3+ (>2 log shift in MFI). Clinical data, morphological findings on peripheral blood, and/or bone marrow examination were analyzed and correlated with the final results on FC.

  Results Top

The study group included 52 cases with 40 cases suspicious for LPDs and 12 cases suspicious for plasma cell disorders. There was a wide age range of 21 to 78 years with a median of 60 years for LPDs and 49 to 76 years with a median age of 61 years for plasma cell disorders. The male to female ratio was 1.6:1 for LPDs and 2.6:1 for plasma cell disorders.

Out of 40 LPDs, CLL accounted for a majority of the cases accounting for 57.5% (23 cases). Plasma cell myelomas (PCM) were the most common plasma cell dyscrasias accounting for 75% (nine cases). The final diagnosis and CD200 positivity in the rest of the suspected LPDs and plasma cell disorders is depicted in [Table 1]. The expression pattern in cases positive for CD200 is depicted in [Figure 1].
Table 1: CD200 expression in cases suspected for LPDs and plasma cell disorders

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Figure 1: Histogram showing the expression pattern in cases positive for CD200

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Cases diagnosed as CLL presented with marked leukocytosis ranging from 27,600/mm3 to 2, 25,700/mm3 with lymphocytosis ranging from 75% to 97%. FC revealed positivity for CD19, CD200, and CD20 (dim), CD5 and CD23 in all cases [Figure 2]. Matutes score was 4 or 5 in 18 cases and score 3 in 5 cases. One of the cases was a 58-year-old male who was a diagnosed case of CLL and is on treatment for 4 years. He presented with mild leukocytosis of 13,700/mm3 and the differentials showed 33% lymphocytes. FC was done and the CD19 gated population was positive for CD200, CD5, CD23, lambda, and ROR1. This was diagnosed as a case of minimal residual disease (MRD) in a diagnosed and treated case of CLL based on positivity for ROR1 in CD19 gated cells.
Figure 2: Chronic lymphocytic leukemia. (a and b) Peripheral smear showed lymphocytosis with smudge cells (Giemsa, ×100, ×400); (c) Bone marrow aspirate showed focal aggregates of small lymphoid cells (Giemsa, ×400); (d) Trephine biopsy showed interstitial lymphoid aggregates (H&E, ×400); (e-h.) Flow cytometry of peripheral blood lymphocytes showed expression of CD19, CD20, CD5, CD23, CD 200, kappa

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Two cases of MCL were diagnosed which expressed CD19, CD5, CD38, CD20, surface Ig, lambda, CD43, and were negative for CD200, CD23, and CD10. Immunohistochemistry with Cyclin D1 done on the trephine sections was positive.

There was one rare case of adult B-acute lymphoblastic leukemia (ALL) diagnosed in a 65-year-old male which on FC showed positivity for CD19+, CDcy79a+, CD34+, and was negative for CD200, TdT, and CD117. Among suspected LPDs, two cases were reported as reactive T lymphocytosis as both showed an immunophenotype of CD3+, CD4+, CD8+, CD19-, CD20-, CD23-, CD200- on FC.

Two cases of hairy cell leukemias (HCL) were diagnosed which expressed CD45, CD19, CD20, CD79b, CD103, CD11c, CD25, CD 180, and kappa with bright expression of CD200 [Figure 3]. CD23 and CD123 were expressed only in one of these cases. Two cases of HCL-variant were diagnosed of which only one case showed dim CD200 expression. There were two cases of marginal zone lymphomas (MZL) involving marrow with splenomegaly, and classified as splenic MZL. Both cases had an immunophenotype of CD45+, CD20+, CD19+, CD79b+, CD 200+, CD38+, kappa+, CD5-, CD23- and also negative for HCL markers on FC.
Figure 3: Hairy cell leukemia. (a) Peripheral smear showed lymphocytes with hairy cytoplasmic projections (Giemsa, ×400) (inset × 1000); (b and c) Trephine biopsy- (b) Showed diffuse sheets of lymphoid cells with fried egg appearance (H&E, ×400); (c) Increased reticulin condensation (Reticulin, ×400); (d-f) Flow cytometry of peripheral blood lymphocytes showed expression of CD20, CD11c, CD103, CD 180, CD200, kappa

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A known case of diffuse large B-cell lymphoma (DLBCL), on treatment for 2 years presented with moderate leukocytosis (32,200/mm3). A diagnosis of DLBCL (spill over) was made and FC was done. The cells expressed CD19, CD20, CD23, CD38, CD5, and lambda and were negative for CD200 [Figure 4]. A case each of lymphoplasmacytic lymphoma, Burkitt lymphoma, B-cell ALL, and T-LPD were diagnosed, which were negative for CD200.
Figure 4: Diffuse large B-cell lymphoma spill over. (a) Peripheral smear showed large lymphoid cells with high N/C ratio, round to indented nuclei, prominent nucleoli, and fine nuclear chromatin (Giemsa × 400); (c) Bone marrow imprint smears showed focal aggregates of large lymphoid cells (Giemsa × 100); (d) Trephine biopsy showed diffuse sheets of large lymphoid cells with vescicular nuclei (H&E, ×100); (d-f.) Flow cytometry of peripheral blood lymphocytes showed expression of CD19, CD20, CD5, CD38, and negative for CD200, kappa

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PCM expressed CD200 in 67% cases. One of the cases was a 70-year-old male who is a known case of myelodysplastic syndrome (MDS) and presented with pancytopenia with a serum beta-2 microglobulin of 4930 μg/mL. Serum protein electrophoresis revealed a monoclonal spike and immunofixation studies showed monoclonal IgG kappa. Bone marrow examination was done and aspirates were particulate with increased cellularity for age. There was erythroid prominence with a myeloid to erythroid ratio of 1:2. Plasmacytoid cells accounted for 50% of marrow nucleated cells and there was dyspoiesis in the erythroid and myeloid series. FC revealed an immunophenotype of CD45+, CD19+, CD38+, CD138+, CD200+, kappa positive. In view of dyspoiesis, FISH studies were done suspecting MDS which showed deletion of 20q in the neoplastic cells. The case was finally diagnosed as smoldering myeloma with MDS.

A case of PCM with hairy cell morphology was diagnosed in a 68-year-old male who is a known diabetic with acute kidney injury on dialysis. He presented with marked leukocytosis (1, 16, 900/mm3) and peripheral smear showed 74% atypical plasmacytoid cells with hairy cytoplasmic projections (hairy cell-like). These cells had an immunophenotype of CD19+, CD56+, CD38+, CD138+, CD200+, and kappa+ and was negative for HCL markers (CD11c, CD22, CD103) on FC [Figure 5].
Figure 5: Plasma cell myeloma with hairy cell morphology. (a and b.) Peripheral smear showed plasma cells with eccentric nucleus and hairy cytoplasmic projections (Giemsa ×100, ×400); (c-f.) Flow cytometry of peripheral blood lymphocytes showed expression of CD19, CD38, CD138, CD56, CD200, kappa

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Reactive plasmacytosis was diagnosed in two cases. Marrow showed 15–18% plasma cells which were positive for CD38, kappa, lambda, and negative for CD200.

  Discussion Top

FC has a well-established role in the immunophenotypic categorization of lymphoid neoplasms. Several CD markers have been identified to date associated with lineage specificity. Antigens expressed by the neoplastic cells give them a characteristic immunophenotype identified on FC, thus aiding in diagnosis, sub-classification, staging as well as monitoring the MRD. Low-grade lymphoid neoplasms such as CLL and MCL are difficult to discriminate especially if the cells show partial CD5 expression.[1],[2] Differentiation between B-cell LPDs is important as MCL needs aggressive treatment whereas CLL has an indolent course.[3]

CD200 is a fairly recently introduced marker, useful for flow cytometric immunophenotyping of such low-grade neoplasms, particularly those of B-cell lineage. CD200 or OX-2 membrane glycoprotein is a human protein encoded by the CD200 gene mapped to chromosome 3q13.2.[4] CD200 is expressed by various cell types, such as follicular dendritic cells, thymocytes, B-cells, a subset of T cells, neurons, and endothelial cells.[5]

CD200 belongs to the immunoglobulin super-family and its receptor (CD200R1) is expressed specifically on granulocytes, monocytes, and a subset of T cells. CD200 generates an immunosuppressive signal by binding to its receptor and thus suppresses anti-tumor immunity.[6],[7],[8] CD200 positive neoplasms evade the host immune response and this is the basis for the anti-CD200 targeted therapy.

Sample for FC can be collected from peripheral blood, marrow, or the involved organ. However, CD200 expression was observed to be higher in peripheral blood and marrow samples when compared to archived tissues as the antigens are well preserved in fresh samples when compared with formalin-fixed tissues.[3] All samples in our study were either peripheral blood or marrow aspirates.

CLL is the most common leukemia in adults in western countries accounting for 25–30% of all leukemias,[9] while it accounts for 1.7–8.8% in the Indian subcontinent.[10] CLL and MCL are the two low-grade B-cell neoplasms expressing CD5.[11],[12],[13],[14] However, typical cases of CLL usually express CD23 with a dim expression of CD20 and surface Ig whereas MCLs are negative for CD23 with a bright expression of CD20 and surface Ig. This classic immunophenotype of CLL may not be seen in all cases making its distinction from MCL difficult.[15],[16],[17],[18] Several studies have observed that CD200 is efficacious in such cases in the differentiation of low-grade B-cell neoplasms where CLL usually showed bright positivity and MCL were CD200 negative.[1],[19],[20],[21],[22],[23] We observed that in all (n = 23) our cases of CLL, CD200 expression was bright to moderate and the two cases of MCL were CD200 negative. ROR1 is a useful marker to discriminate normal B-cells from neoplastic B-cells and is a marker for MRD.[24] In one case diagnosed with MRD in CLL, ROR1 was positive in CD19 gated cells.

MZLs have a variable CD200 expression based on the primary site of diagnosis. Similar studies showed that nodal MZLs show bright CD200 expression while extranodal MZLs showed a spectrum of expression with dim or absent expression in splenic MZLs.[3],[23],[25] We observed that the two cases of splenic MZLs involving marrow showed dim CD200 expression. Since CD200 expression is usually bright in CLL and dim to absent in MZL, it can be used to distinguish atypical CLLs from MZL involving peripheral blood or marrow. A subset of MZLs may show partial expression of CD5. Such cases of CD200+/CD5+ MZL are very rare and could be potentially difficult to distinguish from CLL.[26]

Bright CD200 expression was also observed in all cases of HCL similar to other studies,[18],[20],[23],[27],[28] with a dim expression noted in one case of HCL-variant. CD200 expression was seen in 67% of cases diagnosed as PCM. Alapat et al.[19] in their study reported that normal plasma cells are usually negative for CD200 and 71% of PCMs were CD200 positive.[20],[29],[30] Moreaux et al.[29] and Osman et al.[31] observed that PCM with CD200 expression was associated with a poor prognosis. CD200 was negative in the two cases of reactive plasmacytosis, included in this study.

One case of B-ALL diagnosed in our study was positive for CD45, CDCy79a, CD19, CD34, CD38, and negative for CD10, CD20, and CD200. Alapat et al.[19] reported that the highest level of CD200 was expressed in the most immature B-cell progenitors which comprise a minor subset of CD34+/CD19+/CD10 +/CD20-hematogones. T-lineage ALLs are usually uniformly negative for CD200 with variable positivity observed in B-ALLs.

Several ongoing clinical trials on the blockade of the immune checkpoint by anti-CD200 targeted therapy, especially in CLL and PCM, are under study.[3]

Anti-CD200 targeted therapy is the current research area of interest and several ongoing clinical trials have been proposed in CD200 + neoplasms including CLL/small lymphocytic lymphoma (SLL) and PCM.[6],[32],[33] The mechanism of action proposed was blockage of CD200 receptor–ligand interaction which enables the immune-mediated killing of tumor cells. Mahadevan et al.[34] investigated the therapeutic use of samalizumab as a CD200 immune checkpoint inhibitor in CLL and PCM and they observed that Samalizumab had a good safety profile and the tumor burden was reduced in a majority of cases with advanced CLL. These studies establish the role of anti-CD200 targeted therapy in other CD200+ neoplasms such as HCL, B-ALL, and classical Hodgkin lymphoma in near future.[35],[36],[37]

  Conclusion Top

CD200 is a very useful marker in the diagnosis of various LPDs especially CLL, HCL, and PCM. It can be used as an additional marker particularly in distinguishing CLL/SLL from MCL and atypical CLL from other CD5+ B-cell neoplasms and extranodal MZL. Differentiation between CD5 positive B-cell LPDs is important as CLL has an indolent course and MCL has to be treated aggressively. Lastly, CD200 positive neoplasms can be a potential target for anti-CD200 targeted therapy. Hence, it is recommended that CD200 be incorporated in the immunophenotypic panel for LPDs and plasma cell disorders.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/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


Conflicts of interest

There are no conflicts of interest.

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