Multiparameter flow cytometry is a useful tool for the diagnostic evaluation of mature B-cell neoplasms (MBN). Recently, it has been shown that CD200 may improve the distinction between chronic lymphocytic leukemia (CLL; CD200+) and mantle cell lymphoma (MCL; CD200−), but the role of CD200 expression in atypical CLL and other MBN remains to be established.
To address this issue, we investigated the expression of CD200 in 159 consecutive cases of MBN.
CD200 was strongly expressed in CLL and was revealed to be an excellent marker to distinguish CLL from MCL, even in cases of atypical CLL. However, lack of CD200 was not an exclusive finding of MCL, being also observed in other MBNs. Furthermore, CD200 was highly expressed in hairy cell leukemia, being useful in the differential diagnosis of lymphomas with villous lymphocytes. Herein, we propose an algorithm to classify CD5+ MBNs based on the expression of CD200, CD11c, heavy chain immunoglobulins, and Matutes score.
According to the World Health Organization (WHO) classification of tumors of the hematolymphoid system, the diagnosis of lymphoid neoplasms is based on a multifaceted approach, and many subtypes require a combination of clinical, morphologic, immunophenotypic and genetic features for a definitive diagnosis . Mature B-cell neoplasms (MBN), which represent more than 80% of all lymphoid tumors, are characterized by the presence of cells clonally derived from a transformed B-cell precursor at distinct stages of differentiation. The immunophenotypic resemblance to a given stage of B-cell differentiation is a relevant feature in the classification of these neoplasms, underscoring an essential role for multiparameter flow cytometry (MFC) in the diagnosis and classification of B-cell lymphomas .
In this context, the identification of CD5 and/or CD10 on the surface of tumor cells allows to restrict the diagnostic possibilities among the several subtypes of B-cell lymphomas, assisting in the selection of the subsequent diagnostic methods required for a definitive diagnosis [3-5]. The most frequent associations are CD5+/CD10−, whose main hypotheses are chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL); CD5−/CD10+, represented by follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma; and CD5−/CD10−, exemplified by marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma, and lymphomas with hairy/villous cells, such as splenic marginal zone lymphoma (SMZL) and hairy cell leukemia (HCL).
In the CD5+/CD10− subgroup, by far the most frequent when lymphoma cells are present in the peripheral blood (PB), cases with CD23-positive cells are usually classified as CLL, whereas most cases with CD23-negative cells are diagnosed as MCL. Because the immunophenotypic profile of CLL cells is quite homogeneous and easily recognized, MFC is the method of choice for diagnostic confirmation of CLL. However, the immunophenotypic profile of MCL cells is quite heterogeneous, and cases of CLL and MCL can frequently overlap to a significant degree . On those instances, the pattern of CD23 expression is not helpful for the diagnosis. CD23 expression is also not helpful in many cases of CLL whose cells do not show the classic phenotypic pattern—the so-called atypical CLL cases—as the resemblance to the more heterogeneous phenotype of MCL is even higher in those cases. In addition, other subtypes of B-cell lymphomas may show atypical expression of CD5, further complicating the differential diagnosis in the CD5+/CD10− subgroup [7-10].
Similarly, clinical and phenotypic overlapping also occurs among others B-cell lymphoma subgroups, as is the case in FL versus DLBCL  or HCL versus SMZL [12, 13]. In those instances, additional diagnostic methods are essential for the correct classification. Therefore, novel, informative, and nonredundant immunophenotypic markers could be helpful to improve the utility of MFC on the differential diagnosis of B-cell neoplasms.
Recently, it has been shown that the CD200 antigen (OX2) may improve the distinction between CLL (CD200 positive) and MCL (CD200 negative) cases [14-19]. Expression of high levels of this protein on the membrane of the cells has also been shown on HCL cases . Nevertheless, most studies so far have been performed mainly on typical CLL cases, and the role of CD200 expression in the distinction of atypical CLL from MCL remains to be established. Moreover, data on CD200 expression in other MBN are scarce, and further studies are necessary to investigate the precise role of CD200 on the differential diagnosis of B-cell lymphomas.
In this study, we have investigated the expression of CD200 in a series of 159 patients with MBN, evaluating the diagnostic utility of this biomarker alone and in association with other immunophenotypic markers using a conventional MFC approach.
Patients, Materials, And Methods
A total of 102 PB, 48 bone marrow (BM), 7 lymph nodes (LN), and 2 cerebrospinal fluid (CSF) samples obtained for diagnostic purpose from 159 patients diagnosed with mature B-cell neoplasm (MBN) were studied. The study was approved by the Institution Review Board and was in line with the guidelines of the local ethics committee and the Helsinki Declaration.
The diagnosis of mature B-cell neoplasm was established according to the WHO classification based on clinical data, and on morphologic, immunophenotypic, and genetic criteria. The revised Matutes score system, based on the immunophenotypic analysis of five membrane markers (CD5+, CD23+, FMC7−, and negative/weak expression of surface immunoglobulin and CD79b), was employed to classify CLL cases . Typical CLL cases were defined by a score ≥ 4; and atypical cases were identified by a score < 4 or lack of CD23.
Patients were distributed according immunophenotypic classification  as follows (Table 1): CD5+: 81 cases (typical CLL, 45; atypical CLL, 11; MCL, 14; CD5+ non-Hodgkin lymphoma non-CLL, non-MCL–NHL, 11); CD10+: 16 cases (FL, 11; DLBCL, 5); CD5−/CD10− lymphomas with hairy/villous lymphocytes, 20 [HCL, 13; SMZL, 6; unclassifiable lymphoma-splenic diffuse red pulp small B-cell lymphoma (SRPBL), 1]; prolymphocytic leukemia (PLL), 7; lymphoplasmacytic lymphoma (LPL), 4; and unclassified CD5−/CD10− lymphomas: 31 cases. This group was composed by cases with no LN tissues for examination and whose PB/BM morphologic, immunophenotypic, and cytogenetic data most probably corresponded to MZL or CD10− DLBCL cases.
Table 1. Distribution and Phenotypic Characteristics of CD200 on 159 Cases of MBN
No. of cases
MFI median (range)
Pattern of CD200 expression
CD5+ NHL: CD5+ non-Hodgkin lymphoma non-CLL, non-MCL; CLL: chronic lymphocytic leukemia; DLBCL: diffuse large B cell lymphoma; FL: follicular lymphoma; HCL: hairy cell leukemia; LPL: lymphoplasmacytic lymphoma; MCL: mantle cell lymphoma; MFI: mean fluorescence intensity; PLL: prolymphocytic leukemia; SD: standard deviation; SMZL: splenic marginal zone lymphoma; SRPBL, splenic diffuse red pulp small B-cell lymphoma.
Moderate to strong
Moderate to strong
Dim to strong
1 Case: dim; 1 case: strong
Dim to moderate
Dim to moderate
Dim to moderate
Dim to strong
Diagnosis of MCL was confirmed by immunohistochemical detection of cyclin D1 in tissue biopsies or detection of t(11;14)(q13;q32) by FISH. In addition, absence of IGH/CCND1 rearrangement and CLL related cytogenetic abnormalities were demonstrated in all cases of CD5+ NHL non-CLL, non-MCL by FISH. Diagnosis of PLL was considered in cases with high lymphocyte counts associated to > 55% of PB prolymphocytes, absence of lymphadenopathy, and no previous history of MBN. No cyclin D1 expression and/or t(11;14) were detected in the CD5+ PLL cases. In the CD5+ NHL non-CLL, non-MCL, non-PLL, and unclassified CD5−/CD10− subgroups, cases were considered MBN not otherwise specified in the final report, and a note recommending further tests was included.
Multiparameter Flow Cytometric Immunophenotyping
All samples were collected in tubes containing K3 EDTA as anticoagulant. Cells in suspension (2 × 106 cells in 50–100 μL per tube) from the PB, BM, CSF, and LN samples were stained with monoclonal antibodies (MAb) directed against cell surface markers using a stain-lyse-and-then-wash, direct immunofluorescence technique, as previously described . The following panel of 4-color combinations of MAbs—fluorescein isothiocyanate (FITC)/phycoerythrin (PE)/peridinin chlorophyll protein (PerCP-Cy5.5)/allophycocyanin (APC)—was used in all cases: (sIg)λ/(sIg)κ/CD20/CD5, FMC7/CD10/CD19/CD38, CD23/CD19/CD3/CD79b, CD103/CD123/CD19/CD22, −/CD25/CD19/−, sIgM/CD19/−/CD11c, sIgG/CD19/−/−, sIgA/CD19/−/−, and sIgD/CD19/−/CD200. A tube containing Ig isotype controls for FITC/PE/PerCP-Cy5.5/APC was performed in all cases. The source of MAbs was as follows: Ig isotype controls, CD3, CD5, CD10, CD11c, CD19, CD20, CD22, CD25, CD38, CD79b, CD103, CD123, and FMC7 were from Becton Dickinson Biosciences (BDB), San José, CA, USA; and CD23, λ, κ, IgM, IgG, IgA, and IgD were from Dako (Glostrup, Denmark). CD200 identification was performed using the clone OX104 (eBioscience, San Diego, CA).
Data acquisition was performed immediately after completion of sample staining, using a FACSCalibur flow cytometer and the CellQUEST software (BDB). For each sample, data from at least 5 × 104 events per tube was acquired. The Infinicyt software (Cytognos, SL, Salamanca, Spain) was used for the analysis of flow cytometry data. Daily instrument quality control was performed using Calibrite beads (BDB) to ensure consistent determination of fluorescence intensity during the study.
Merge of CD200 Flow Cytometry Data Files
Merge of data files corresponding to aliquots of each individual sample was performed as previously described by Costa et al. , after gating on CD19+ lymphoma cells. Briefly, CD19+ B-cells were selected in the CD200 data file using conventional gating strategies (forward and sideward light scatter and the pattern of CD19 expression). The B-cell population was composed almost exclusively by neoplastic B-cells (median: 99.7%, range 86–100%). Whenever identified, residual normal B-cells—detected by different expression of CD19, IgD, and/or light scatter chararacteristics—were excluded from analysis. Data from gated B-cells were saved in a new file, and then merged into a single virtual data file, using the Infinicyt software, to create a reference pool of CD200 expression by lymphoma cells from all 159 patients investigated in this study. The mean fluorescence intensity values (MFI; relative linear units scaled from 0 to 104) of CD200 from each leukemic sample was represented by only one dot (square), enabling the comparison of its expression among different lymphoma groups and distinct subtypes (Fig. 1). Intensity of expression evaluated by MFI was categorized as dim, moderate, strong and very strong. Dim staining was defined as slightly increased when compared to the negative control (isotype control). Moderate staining was defined between first and second log decades; strong staining, between second and third log decades; and very strong staining, as MFI above the third log decade. Isotype controls and internal reference populations were used to circumvent possible autofluorescence interference in cases presenting large size B-cells.
The two-sided Student T-test and the Mann-Whitney U test were used to calculate the statistical significance of differences observed between groups. P values < 0.05 were considered significant. All statistical analyses were performed with the SPSS 13.0 package (SPSS, Chicago, IL).
Patient Characteristics and Expression of CD200 on Isotype Controls
Mean (SD) age of the patients—82 males and 77 females—was 65  years (median: 65 years; range: 32–99 years). The patients' hemoglobin levels ranged from 7.2 to 16.1 g/dL (median 12.7 g/dL), the white blood cell counts ranged from 2.6 to 282 × 109 cells/L (median 23.7 × 109 cells/L) and the platelet counts ranged from 21 to 310 × 109 cells/L (median 156 × 109 cells/L). Neoplastic B-cells percentage ranged from 2.5 to 91% (median 53%) and residual normal B cells from 0 to 3.1% (median 0.16%). In PB samples, the absolute number of neoplastic B-cells ranged from 0.4 to 297 × 109 cells/L (median 16.7 × 109 cells/L). The CD200 median MFI expression on isotype control was 5.75 (range = 2–13) and on residual normal B-cells was 87 (range = 32–273). In PB samples, the CD19neg lymphocytes (e.g., NK and T-cells) presented negative to dim expression of CD200 (MFI: median = 13.6, range = 5.6–36).
CD200 Expression on CD5+ MBN
Neoplastic B-cells from all 45 cases of typical CLL presented homogeneous and strong expression of CD200 (MFI: median = 271), whereas all 14 cases of MCL were negative for CD200 expression (P < 0.0001) (Figs. 1B and 2A). Four MCL cases showed partial expression of the CD23 antigen. In addition, all MCL cases were negative for CD11c and expressed both IgM and IgD with high intensity. All 11 cases of atypical CLL also expressed CD200 (MFI: median = 131), but with lower intensity than that observed in typical CLL (P = 0.002) (Fig. 1B). Of note, CD23 was absent or dimly expressed in six cases of atypical CLL and the following antigens were expressed with moderate/strong intensity: FMC-7, 8 cases; CD79b, 5 cases; and sIg, 4 cases of atypical CLL.
Among the seven PLL cases investigated, three expressed CD5 (two cases with partial expression and one case with strong expression); and two expressed CD200 (dimly by one of them and brightly by the other). Among the CD5+ PLL cases, two were IgM+/IgD+, one was IgA+, and all three showed CD11c expression with moderate/strong intensity. The remaining four CD5-negative PLL cases were also negative for CD200: three expressed IgM/IgD; one, IgG; and only one, CD11c.
Among the CD5+ B-cell lymphomas (non-CLL, non-MCL, and non-PLL), nine out of 11 cases expressed CD200 (MFI: median = 83; Fig. 1B). In this group, all cases presented dim expression of CD5; eight cases expressed IgM/IgD; three, IgG; and four, CD11c.
CD200 Expression on CD5-Negative MBN
CD10+ B-cell lymphomas
There was no difference in the pattern or intensity of CD200 expression among CD10+ lymphomas. CD200 was negative in 4 out of 5 cases of CD10+ DLBCL; the positive case showed weak expression of this antigen (MFI: 40). Among the 11 FL cases, CD200 expression was negative in three cases; dim in six; and moderated in two (MFI: median = 29.4; Figs. 1B and 2C).
CD10-negative B-cell lymphomas
The expression of CD200 was negative in 10/31 cases of CD5-/CD10- lymphomas, and highly heterogeneous in 21/31 positive cases (MFI: median = 52.7). In the LPL group, one case had dim and another had moderate expression of CD200 (MFI: median = 47); two LPL cases were negative for CD200 (Figs. 1B and 2D).
Lymphomas with hairy/villous lymphocytes
All HCL cases were CD200 positive and this group presented the highest intensity of fluorescence (MFI: median = 442; Figs. 1B and 2B). SMZL had lower expression of CD200 than HCL (MFI: median = 73.8, P < 0.0001), with dim to moderate intensity, and this pattern was also observed in the single case of SRPBL (MFI = 61.6).
CD200 is a type-1 membrane glycoprotein belonging to the immunoglobulin superfamily, expressed by dendritic cells, neurons, subsets of T-cells , B-cell precursors and mature B-cells [16, 25]. This glycoprotein is not expressed by normal plasma cells and NK cells . CD200 can be expressed by distinct hematologic malignancies, including acute leukemia, multiple myeloma [26, 27] and T- and B-cell lymphomas [14-17, 20, 24, 25]. Gene expression profile studies suggested that CD200 positivity is associated with unfavorable outcomes in MM  and AML .
In this study, we investigated the expression of CD200 in a representative series of patients, and evaluated if the inclusion of this marker in a conventional immunophenotypic panel could be useful to improve the diagnostic algorithm of B-cell lymphomas by flow cytometry [23, 25, 30-33].
Different groups have emphasized the strong expression of CD200 on HCL cells [15, 20] and its potential usefulness in the differential diagnosis of CD5+ B-cell lymphomas, especially CLL and MCL [14, 15, 17]. Nevertheless, some critical issues need further clarification, as most studies to date included predominantly cases of MCL and typical CLL, but little is known about the pattern of CD200 expression in atypical CLL, an entity that often presents with features of both CLL and MCL, and whose diagnosis can be very challenging in some cases, underscoring a potential role for CD200 in this specific setting. In addition, little is known about the pattern of CD200 expression in other B-cell lymphoma subtypes that less frequently, but not rarely, can also present with CD5+ cells (PLL, CD5+ NHL, CD5+ lymphomas with hairy/villous lymphocytes) and thus need to be included in the differential diagnosis in some cases.
In our series, CD200 was constantly expressed in typical CLL cases and was an excellent marker for the differential diagnosis with MCL, as previously shown by others [14-19]. Clonal B-cells from typical CLL cases frequently have high expression of CD23, but cases of atypical CLL can present with weak expression or even no expression of this antigen, as observed in several cases from our series. However, MCL cells, whose lack of CD23 is one of their main features, can exhibit CD23 expression in about 20–30% of cases [34-36], further complicating the differential diagnosis with atypical CLL. In our series, CD200 was expressed by all atypical CLL cases, therefore being helpful for the distinction with MCL, particularly on those cases with absent or dim CD23.
CD200 expression can also be useful to distinguish MCL from most cases of CD5+ NHL, since CD200 was expressed with moderate intensity in more than 80% of cases. However, in the CD5+ NHL group, lack of CD200 was not an exclusive finding of MCL, being also observed in two cases of CD5+ NHL and in most cases of PLL.
Other antigens had been evaluated in an attempt to distinct atypical CLL from MCL; generally, they present discrepancy in a significant number of cases. Kraus et al.  demonstrated that CD11c is constantly negative or dimly expressed in MCL cases, whereas it was expressed in one third of CLL cases, being useful to distinguish both disorders only in a subset of patients. Besides, CD11c is frequently expressed by PLL  and MZL subtypes [3, 39], and this marker could be valuable to separate MCL from CD5+ MZL and PLL cases.
PLL is a rare and aggressive MBN, characterized by splenomegaly, marked lymphocytosis, and >55% prolymphocytes on PB. Approximately one third of cases express CD5 , and the differential diagnosis of CD5+ PLL includes CLL cases that develop an increased number of prolymphocytes , as well as the nucleolated variant of MCL [42, 43]. CD200 was negative or dimly expressed on most PLL cases, suggesting that it is a good marker to differentiate from CLL, but not from MCL cases with increased number of prolymphocytes. Nevertheless, PLL cases reported in our series frequently presented CD11c expression, and some cases had surface heavy chain immunoglobulins other than IgM and IgD.
It is postulated that the normal counterparts of MCL are originated from pregerminative B cells before the switch class of immunoglobulin, even though some cases presented mutation of IgVH , and virtually all MCL cases presented strong expression of surface IgM and IgD. Since most cases of CD5+ NHL are originated from cells from the germinal center or postgerminal center , the use of heavy chain immunoglobulins profile could be useful in the distinction of MCL from CD5+ NHL in a subset of patients. In line with this, one third of CD5+ NHL cases presented expression of IgG.
Based on the combined expression patterns of CD200, CD11c, heavy chain immunoglobulins, and the Matures score, in addition to the cytomorphological analysis, a diagnostic algorithm can be proposed to classify correctly the distinct subtypes of CD5+ MBNs (Fig. 3). In cases with Matutes score ≤ 3, the CD200 expression is pivotal to MBN classification. Whenever CD200 is positive, the differential diagnosis among atypical CLL and CD5+ NHL should be considered, even though one PLL patient presented high expression of CD200 in our series. In this situation, the Matutes score associated with the quantification of prolymphocytes by morphology will guide the classification. Cases lacking CD200 and CD11c but showing high expression of IgM and IgD are highly suggestive of MCL. Conversely, the presence of CD11c, IgA, or IgG are unlikely features of MCL, and the diagnosis of CD5+ NHL or PLL should be taken in account. Nevertheless, caution should be taken when using immunophenotypic data as an exclusive basis for the classification of B-cell lymphomas, and searching for the t(11;14) by FISH and/or the presence of cyclin D1 by immunohistochemistry are essential steps in the multifaceted approach for the final diagnosis.
Several studies have demonstrated that CD200 is highly expressed in HCL by either immunohistochemistry or MFC [14, 15, 20]. Likewise, HCL cells from our study presented the brightest expression of CD200 among MBNs. In turn, SMZL cases and SRPBL presented dim to moderate expression of CD200, and this marker could be helpful in the differential diagnosis of MBN with hairy/villous lymphocytes. Nevertheless, two of these previous reports demonstrated that CD200 was not expressed in 14 SMZL cases [14, 15]. This could be explained by the use of different methodology or distinct CD200 MAb clones. Unfortunately, no case of HCL variant was evaluated in our study, and the role of CD200 expression in the distinction of classical HCL and variant HCL remains to be established.
In the CD10+ MBN group, CD200 was negative or dimly expressed, being not useful to distinguish FL from DLBCL. Likewise, CD200 was not useful for the diagnosis of the CD5−/CD10− MBN subgroup other than lymphomas with hairy/villous cells, since the expression of CD200 was heterogeneous in these cases. These results were comparable to the data reported by the Euroflow Consortium that demonstrated a varied pattern of CD200 expression in cases of CD10− DLBCL and MZL . In conclusion, our results expand the understanding of the CD200 expression in MBNs, giving further support for the inclusion of this marker in the flow cytometric panels for the differential diagnosis of MBNs.