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DOI: 10.1111/ijlh.12857
ORIGINAL ARTICLE
1
St Vincent’s Pathology (SydPath), St
Vincent’s Hospital Sydney, Darlinghurst, Abstract
NSW, Australia Introduction: Immunophenotyping by flow cytometry is routinely employed in dis-
2
St Vincent’s Clinical School, UNSW Sydney,
tinguishing between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma
Sydney, NSW, Australia
3 (MCL). Inclusion of CD200 has been reported to contribute to more reliable differen-
St Vincent’s Centre for Applied Medical
Research, Darlinghurst, NSW, Australia tiation between CLL and MCL. We investigated the value of CD200 in assessment of
4
NSW Health Pathology and ICPMR, atypical CLL cases.
Westmead, NSW, Australia
5
Methods: CD200 expression on mature B cell neoplasms was studied by eight-color
Haematology Department, St Vincent’s
Hospital Sydney, Darlinghurst, NSW, flow cytometry in combination with a conventional panel of flow cytometry markers.
Australia The study included 70 control samples, 63 samples with CLL or atypical CLL pheno-
6
Garvan Institute of Medical Research,
type, 6 MCL samples, and 40 samples of other mature B cell neoplasms.
Darlinghurst, NSW, Australia
Results: All CLL samples were positive for CD200, whereas MCL samples were dim
Correspondence
or negative for CD200. Of the CLL samples, 7 were atypical by conventional flow
William A. Sewell, Garvan Institute,
Darlinghurst, NSW, Australia. cytometry, with Matutes scores ≤3. These cases were tested for evidence of a
Email: w.sewell@garvan.org.au
t(11;14) translocation, characteristic of MCL, and all were negative, consistent with
Funding information their classification as atypical CLL. All these atypical CLL samples were strongly posi-
SydPath, St Vincent’s Hospital Sydney;
tive for CD200.
St. Vincent’s Clinical School, Faculty of
Medicine, UNSW Sydney Conclusion: CD200 proved to be a useful marker for differentiation between CLL
and MCL by flow cytometry. In particular, CD200 was useful in distinguishing CLL
samples with atypical immunophenotypes from MCL.
KEYWORDS
CD200, chronic lymphocytic leukemia, flow cytometry, immunophenotypic analysis,
lymphoma
1 | I NTRO D U C TI O N contrast to CLL, which can be indolent, MCL, a less common disor-
der, has an aggressive disease course that bears one of the worst
Chronic lymphocytic leukemia (CLL) is the most common form of prognoses among all the B cell lymphomas. Not only are the prog-
leukemia in adults in the Western world.1 The disease course is noses of MCL and CLL different, the treatment regimens for the two
highly variable, with some patients having a normal life span and disorders are not the same; therefore, the implications for misdiag-
others surviving only a few years postdiagnosis. With the advent of nosis are serious.5-7
sophisticated laboratory techniques such as flow cytometry and im- Flow cytometry, IHC, and anatomical pathology have particular
munohistochemistry (IHC), it is now usually possible to differentiate limitations in the diagnosis of CLL versus MCL due to overlapping
CLL from most mature B cell neoplasms. 2,3 Unfortunately, there may immunophenotypes and the fact that the wide range of histological
be a risk of misdiagnosis between CLL and mantle cell lymphoma patterns of MCL may resemble CLL.8 This problem may be addressed
(MCL), which share morphological features and CD5 positivity.4 In by performing cytogenetics or fluorescence in situ hybridization
Int J Lab Hem. 2018;1–7. © 2018 John Wiley & Sons Ltd | 1
wileyonlinelibrary.com/journal/ijlh
|
2 TING et al.
(FISH) to assess the t (11;14) translocation, the hallmark of MCL, that nonhematopoietic cells. CD200 and its ligand CD200R have an
causes overexpression of cyclin D1. FISH is particularly sensitive for important role in the inhibition of autoimmunity, inflammation,
the t (11;14) translocation, which can be detected in 97% of all MCL and adaptive immune responses via induction of regulatory T cells
cases.9 Unfortunately, FISH is expensive and not performed on all and effects on cytokine production.17 CD200 has been described
cases of mature B cell neoplasms. Alternatively, overexpression of as positive in CLL and negative in MCL, therefore offering an addi-
cyclin D1 can be directly evaluated by immunohistochemistry of tis- tional flow cytometry marker to improve the distinction between
sue sections, although this test may be falsely negative for MCL.10 CLL and MCL.18-20 The differential expression of CD200 in CLL
Furthermore, in many cases of CLL, the diagnosis is made on blood and MCL has been attributed partially to the different activation
samples only, by morphology and flow cytometry.11 Hence, there is of various intracellular signaling pathways in these two condi-
a critical need for effective flow cytometry markers to discriminate tions.19,20 The aim of this study was to evaluate the use of CD200
between CLL and MCL. in the differential diagnosis between CLL and MCL, in particular
Diagnosis of CLL and distinction from MCL and other lymphomas in cases where traditional flow cytometry markers demonstrate
by flow cytometry has been guided by the Matutes scoring system, atypical CLL.
in which a point is given for each of the following five criteria: CD5+,
CD23+, FMC7-
, weak-
negative surface immunoglobulin and weak-
2 | M ATE R I A L S A N D M E TH O DS
negative CD22 or CD79b. According to this system, 87% of CLL scored
4 or 5 of 5, whereas only 0.3% of all other B cell lymphoproliferative
2.1 | Case selection
disorder cases achieved this score.12 However, this scoring system
is not completely reliable, and numerous CLL samples have atypical In this prospective study, blood and tissue samples were ob-
flow cytometry with scores of less than 4 of 5. Atypical CLL cases may tained in the routine course of patient management and sent
mimic MCL phenotype because of bright surface immunoglobulin, or to St Vincent’s Pathology for flow cytometry testing over a 7-
13
CD23 negativity, or FMC7 positivity. CD23 was proposed to differ- month period. Ethics approval for the study was obtained from
entiate MCL from CLL in IHC analysis, despite a lack of robust support- the Human Research Ethics Committee, St Vincent’s Hospital
ing data.4 A flow cytometry study found that MCL could be CD23+ Sydney. The tissue samples included bone marrow aspirates,
and reported that a diagnostic panel of the key markers CD5+, CD10− lymph node or tissue biopsies and pleural fluid. Control sam-
14
and CD23−, was only 76% sensitive. Further reports have demon- ples were obtained from healthy laboratory staff as well as
strated that CD23 can be positive in MCL by flow cytometry.15,16 patient samples submitted for assessment that had no abnor-
CD200, or OX-
2 , is an immunoglobulin superfamily mem- malities in all investigations. In the scenario where multiple
brane glycoprotein expressed on a variety of hematopoietic and samples were taken at different times from the same subject
F I G U R E 1 Representative plots of CD200 and CD5. Samples are as follows: (A) control; (B) typical CLL phenotype; (C) MCL; (D) atypical
CLL phenotype (case 4 in Table 2). For each sample, B cells (CD19+) are shown in the upper plot and other lymphocytes in the lower plot.
In the upper plots, blue cells are normal B cells, confirmed by polyclonal kappa and lambda light chain expression, and purple cells are
monoclonal cells. Cursors were placed according to CD3+ cell (red) and CD3-CD19-cell (green) internal controls shown in the lower plots
[Colour figure can be viewed at wileyonlinelibrary.com]
TING et al. |
3
at the same site, the sample with the largest number of B cells
2.3 | Case evaluation
was used.
Cursor placement was based on internal negative and positive controls
in the form of monocytes, T cells, and NK cells (Figure 1).21 Samples
2.2 | Procedure
were considered positive when >25% of monoclonal cells (or all B cells
All samples were acquired with an eight-color flow cytometer in the case of the control group) stained positive for the marker in
(FACSCanto II, Becton Dickinson, San Jose, CA) (BD) with acquisi- question. Monoclonal cells for gate placement were defined in CLL
tion target set at 50 000 leukocyte events. Plot figures shown in this samples as the CD5+ population, and in other B cell populations as the
report were prepared by FACSDiva software (BD). Data analysis was monoclonal population as defined by kappa and lambda antibodies in
conducted using FCS Express 4 Flow Research Edition (DeNovo, Los Tube 1. Blood samples with a monoclonal CLL phenotype B cell popu-
Angeles, CA). lation less than 100 x 109/L were excluded from the study.
All samples were stored at 4°C and tested within 24-36 hours
of collection. Any solid tissue samples were disaggregated into
2.4 | Statistics
cell suspensions by pushing through a cell strainer (BD). 0.5 mL
of cell suspensions or bone marrow aspirates were washed twice Statistical analysis was conducted using Microsoft Excel and IBM
in phosphate-
buffered saline (PBS), centrifuged for 5 minutes at SPSS Statistics 20 Program. Samples were assessed for normality
800 g and resuspended in 0.5 mL 1% (w/v) bovine serum albumin using the Shapiro-Wilk test. As the groups with larger number of
in PBS. Peripheral bloods were not washed, except for kappa and subjects (control and CLL groups) were not found to be normally
lambda analysis. 50 μL of sample was then added to each FACS tube. distributed, and the remaining groups (MCL group, subgroups of
Antibodies were cocktailed with the amount based on routine lab- mature B cell neoplasms) had low sample numbers, the Kolmogorov-
oratory protocols, added to the assay tubes, and then incubated for Smirnov test was used to assess differences between any of the
10 minutes at room temperature. Red cells were lysed using FACS sample groups with a 5% overall significance level. Subsequently, the
Lyse (BD) (2 mL), washed in PBS (2 mL), and resuspended in fixative Mann-Whitney U test was employed to test statistical significance
solution. between individual groups (CLL vs MCL; atypical CLL vs MCL; typi-
All tubes contained backbone markers CD19-APC-H7, CD20- cal CLL vs atypical CLL). To ensure the overall significance level, a
Per-CP, and CD45-V500. The other markers were as follows: Tube Bonferroni correction was used so a P value of less than .017 (.05/3)
1, CD3-Pacific Blue (PacB), kappa-FITC, lambda-PE, CD10-PE-Cy7, was used to judge the significance of pair wise comparisons.
CD22-APC; Tube 2, CD38-PacB, FMC7-FITC, CD11c-PE, CD5-
PE-Cy7, CD23-APC; Tube 3, CD54-PacB, IgM-FITC, CD160-PE,
CD5-PE-Cy7, CD200-APC. All antibodies were from BD except 3 | R E S U LT S
CD200-APC and CD54-PacB (BioLegend, San Diego) and CD160-PE
(Beckman Coulter, Marseille). A CD200-PacB antibody (BD) was The B cells in 179 samples were classified by flow cytometry with-
also tested and gave similar results to the CD200-APC antibody, on out reference to the CD200 results. Samples were scored accord-
which the data in this manuscript are based. ing to the Matutes system, with one point for each of the following
TA B L E 1 Source of samples
For each site, the study includes only one sample per patient. Some patients are represented at more than one site; 70 samples were collected from 66
controls, 63 samples with typical or atypical CLL phenotype from 56 patients (of which 13 were monoclonal B cell lymphocytosis (MBL)), 6 samples
from 3 MCL patients and 40 samples from 38 patients with other monoclonal B cell populations, (of which 22 were diagnosed lymphomas with subtype,
and 18 were unclassified, of which 10 were diagnosed lymphomas without subtype, and 8 were MBL).
|
4 TING et al.
Sample
no., age, Sample Light Matutes PB clone
gender site CD5 CD22 CD23 FMC7 chain score Other investigations size x 109/L
flow cytometric criteria: CD5 positive, CD22 dim/negative, CD23 translocation. If there was no evidence of such a translocation by
positive, light chain dim/negative, FMC7 negative.12 Typical CLL cytogenetics or FISH, or if cyclin D1 was negative by IHC, the case
cases were bright positive for CD23. If CD23 was dim, or if there was classified as atypical CLL. The 179 samples consisted of 70
was a CD23-negative subset, the case did not receive a point for controls (polyclonal B cells only), 63 samples with CLL phenotype,
the CD23 criterion. Cases scoring 4 or 5 points of 5 were classi- of which 56 were typical and 7 were atypical, 6 MCL samples and
fied as CLL. In all these cases, this diagnosis was consistent with 40 other monoclonal B cell populations (Table 1). Details of the
other pathology testing including microscopy, and where available, atypical CLL cases are presented in Table 2. Of the atypical CLL
IHC, cytogenetics, and FISH. In cases with a CD5+ monoclonal B samples, 3 had dim or negative CD23 expression, all had partial or
cell population, which scored one point for CD5+, if the total score total FMC7 expression, and 5 had moderate-bright surface light
was less than 4, samples were evaluated for evidence of a t(11;14) chain.
TING et al. |
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4 | D I S CU S S I O N
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How to cite this article: Ting YS, Smith SABC, Brown DA,
kemia and mantle cell lymphoma immunologically very close: flow
cytometric distinction by the use of CD20 and CD54 expression.
et al. CD200 is a useful diagnostic marker for identifying
Leukemia. 2001;15:1458‐1465. atypical chronic lymphocytic leukemia by flow cytometry. Int
23. Farren TW, Giustiniani J, Liu FT, et al. Differential and tumor- J Lab Hem. 2018;00:1–7. https://doi.org/10.1111/ijlh.12857
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