|

Received: 12 July 2017    Accepted: 11 April 2018

DOI: 10.1111/ijlh.12857

ORIGINAL ARTICLE

CD200 is a useful diagnostic marker for identifying atypical

chronic lymphocytic leukemia by flow cytometry

Y. S. Ting1,2 | S. A. B. C. Smith1 | D. A. Brown1,2,3,4 | A. J. Dodds1,2,5 | K. C. Fay1,5 | 

D. D. F. Ma1,2,3,5 | S. Milliken1,2,5 | J. J. Moore1,2,5 | W. A. Sewell1,2,6

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
Chronic lymphocytic leukemia (CLL) is the most common form of
leukemia in adults in the Western world.1 The disease course is
highly variable, with some patients having a normal life span and
others surviving only a few years postdiagnosis. With the advent of
sophisticated laboratory techniques such as flow cytometry and im-
munohistochemistry (IHC), it is now usually possible to differentiate
CLL from most mature B cell neoplasms. 2,3 Unfortunately, there may
be a risk of misdiagnosis between CLL and mantle cell lymphoma
(MCL), which share morphological features and CD5 positivity.4 In
contrast to CLL, which can be indolent, MCL, a less common disor-
der, has an aggressive disease course that bears one of the worst
prognoses among all the B cell lymphomas. Not only are the prog-
noses of MCL and CLL different, the treatment regimens for the two
disorders are not the same; therefore, the implications for misdiag-
nosis are serious.5-7
Flow cytometry, IHC, and anatomical pathology have particular
limitations in the diagnosis of CLL versus MCL due to overlapping
immunophenotypes and the fact that the wide range of histological
patterns of MCL may resemble CLL.8 This problem may be addressed
by performing cytogenetics or fluorescence in situ hybridization

Int J Lab Hem. 2018;1–7. © 2018 John Wiley & Sons Ltd |  1
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2       TING et al.

(FISH) to assess the t (11;14) translocation, the hallmark of MCL, that
causes overexpression of cyclin D1. FISH is particularly sensitive for
the t (11;14) translocation, which can be detected in 97% of all MCL
cases.9 Unfortunately, FISH is expensive and not performed on all
cases of mature B cell neoplasms. Alternatively, overexpression of
cyclin D1 can be directly evaluated by immunohistochemistry of tis-
sue sections, although this test may be falsely negative for MCL.10
Furthermore, in many cases of CLL, the diagnosis is made on blood
samples only, by morphology and flow cytometry.11 Hence, there is
a critical need for effective flow cytometry markers to discriminate
between CLL and MCL.
Diagnosis of CLL and distinction from MCL and other l­ymphomas
by flow cytometry has been guided by the Matutes scoring ­system,
in which a point is given for each of the following five criteria: CD5+,
CD23+, FMC7-­
, weak-­
negative surface immunoglobulin and weak-­
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
disorder cases achieved this score.12 However, this scoring system
is not completely reliable, and numerous CLL samples have atypical
flow cytometry with scores of less than 4 of 5. Atypical CLL cases may
mimic MCL phenotype because of bright surface immunoglobulin, or
13
CD23 negativity, or FMC7 positivity. CD23 was proposed to differ-
entiate MCL from CLL in IHC analysis, despite a lack of robust support-
ing data.4 A flow cytometry study found that MCL could be CD23+
and reported that a diagnostic panel of the key markers CD5+, CD10−
14
and CD23−, was only 76% sensitive. Further reports have demon-
strated that CD23 can be positive in MCL by flow cytometry.15,16
CD200, or OX-­
2 , is an immunoglobulin superfamily mem-
brane glycoprotein expressed on a variety of hematopoietic and
nonhematopoietic cells. CD200 and its ligand CD200R have an
important role in the inhibition of autoimmunity, inflammation,
and adaptive immune responses via induction of regulatory T cells
and effects on cytokine production.17 CD200 has been described
as positive in CLL and negative in MCL, therefore offering an addi-
tional flow cytometry marker to improve the distinction between
CLL and MCL.18-20 The differential expression of CD200 in CLL
and MCL has been attributed partially to the different activation
of various intracellular signaling pathways in these two condi-
tions.19,20 The aim of this study was to evaluate the use of CD200
in the differential diagnosis between CLL and MCL, in particular
in cases where traditional flow cytometry markers demonstrate
atypical CLL.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Case selection
In this prospective study, blood and tissue samples were ob-
tained in the routine course of patient management and sent
to St Vincent’s Pathology for flow cytometry testing over a 7-­
month period. Ethics approval for the study was obtained from
the Human Research Ethics Committee, St Vincent’s Hospital
Sydney. The tissue samples included bone marrow aspirates,
lymph node or tissue biopsies and pleural fluid. Control sam-
ples were obtained from healthy laboratory staff as well as
patient samples submitted for assessment that had no abnor-
malities in all investigations. In the scenario where multiple
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.
at the same site, the sample with the largest number of B cells
was used.
2.2 | Procedure
All samples were acquired with an eight-­color flow cytometer
(FACSCanto II, Becton Dickinson, San Jose, CA) (BD) with acquisi-
tion target set at 50 000 leukocyte events. Plot figures shown in this
report were prepared by FACSDiva software (BD). Data analysis was
conducted using FCS Express 4 Flow Research Edition (DeNovo, Los
Angeles, CA).
All samples were stored at 4°C and tested within 24-­36 hours
of collection. Any solid tissue samples were disaggregated into
cell suspensions by pushing through a cell strainer (BD). 0.5 mL
of cell suspensions or bone marrow aspirates were washed twice
in phosphate-­
buffered saline (PBS), centrifuged for 5 minutes at
800 g and resuspended in 0.5 mL 1% (w/v) bovine serum albumin
in PBS. Peripheral bloods were not washed, except for kappa and
lambda analysis. 50 μL of sample was then added to each FACS tube.
Antibodies were cocktailed with the amount based on routine lab-
oratory protocols, added to the assay tubes, and then incubated for
10 minutes at room temperature. Red cells were lysed using FACS
Lyse (BD) (2 mL), washed in PBS (2 mL), and resuspended in fixative
solution.
All tubes contained backbone markers CD19-­APC-­H7, CD20-­
Per-­CP, and CD45-­V500. The other markers were as follows: Tube
1, CD3-­Pacific Blue (PacB), kappa-­FITC, lambda-­PE, CD10-­PE-­Cy7,
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
CD200-­APC and CD54-­PacB (BioLegend, San Diego) and CD160-­PE
(Beckman Coulter, Marseille). A CD200-­PacB antibody (BD) was
also tested and gave similar results to the CD200-­APC antibody, on
which the data in this manuscript are based.
TA B L E   1   Source of samples
Controls (n = 70) CLL (n = 63)
Age (Mean ± SD) 55 ± 17 70 ± 11
Male/Female 36/34 44/19
Peripheral blood 56 49
Bone marrow 8 12
Lymph node 2 0
Fine needle aspirate 2 1
Tissue biopsy 1 0
Pleural fluid 1 1
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).
|
      3
2.3 | Case evaluation
Cursor placement was based on internal negative and positive controls
in the form of monocytes, T cells, and NK cells (Figure 1).21 Samples
were considered positive when >25% of monoclonal cells (or all B cells
in the case of the control group) stained positive for the marker in
question. Monoclonal cells for gate placement were defined in CLL
samples as the CD5+ population, and in other B cell populations as the
monoclonal population as defined by kappa and lambda antibodies in
Tube 1. Blood samples with a monoclonal CLL phenotype B cell popu-
lation less than 100 x 109/L were excluded from the study.
2.4 | Statistics
Statistical analysis was conducted using Microsoft Excel and IBM
SPSS Statistics 20 Program. Samples were assessed for normality
using the Shapiro-­Wilk test. As the groups with larger number of
subjects (control and CLL groups) were not found to be normally
distributed, and the remaining groups (MCL group, subgroups of
mature B cell neoplasms) had low sample numbers, the Kolmogorov-­
Smirnov test was used to assess differences between any of the
sample groups with a 5% overall significance level. Subsequently, the
Mann-­Whitney U test was employed to test statistical significance
between individual groups (CLL vs MCL; atypical CLL vs MCL; typi-
cal CLL vs atypical CLL). To ensure the overall significance level, a
Bonferroni correction was used so a P value of less than .017 (.05/3)
was used to judge the significance of pair wise comparisons.
3 | R E S U LT S
The B cells in 179 samples were classified by flow cytometry with-
out reference to the CD200 results. Samples were scored accord-
ing to the Matutes system, with one point for each of the following
Other monoclonal B cell
MCL (n = 6) populations (n = 40)
68 ± 11 68 ± 14
6/0 21/19
3 23
1 6
1 2
1 6
0 2
0 1
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4       TING et al.

TA B L E   2   Features of atypical CLL samples

Sample
no., age, Sample Light Matutes PB clone
gender site CD5 CD22 CD23 FMC7 chain score Other investigations size x 109/L

1, 59, F Peripheral + Dim + + Bright 3 Immunohistochemistry for cyclin D1 0.97c

blood (on previous bone marrow):
negative.
Cytogenetics: Previous BMA showed
no abnormality
FISH: Previous BMA showed no
evidence of t(11;14); no other
probes tested.
2, 47, F Bone + Dim Dim + subset Dim 3 Immunohistochemistry for cyclin D1: 33.4
marrow negative.
Cytogenetics: No abnormality
detected.
FISH: 13q loss. Normal copy number
of 11q, 12 and 17p.
3, 66, M Peripheral + Dim + + Dim 3 Immunohistochemistry for cyclin D1 6.9
blood (on previous bone marrow):
negative.
Cytogenetics: Previous BMA showed
no abnormality detected.
FISH: not performed.
4a, 89, M Peripheral + Dim + subset + Bright 2 Cytogenetics: Too few metaphases 0.51c
blood to interpret.
FISH: No evidence of t(11;14); no
other probes tested.
5, 81, F Peripheral + Dim −­ + subset Moderate 2 Cytogenetics: not performed. 9.8
blood FISH: No evidence of t(11;14) or
copy number changes for 11q, 12,
13q or 17p.
6b,69, M Bone + Dim + + Moderate 3 Immunohistochemistry for cyclin D1: 0.24c
marrow negative.
Cytogenetics: No abnormality
detected.
FISH: not performed—refer to this
patient’s peripheral blood (sample 7).
7b, 69, M Peripheral + Dim + + Moderate 3 Cytogenetics: No abnormality 0.24c
blood detected.
FISH: Peripheral blood: No evidence
of t (11;14) or copy number changes
for 11q, 12, 13q or 17p.
a
Dot plots of case 4 are shown in Figure 1D.
b
Samples 6 and 7 are bone marrow and peripheral blood, respectively, from the same patient.
c
Of the cases with clone size <5 × 109/L, case 1 had been previously treated for CLL. Cases 4 and 6 had not been previously treated and were considered
MBL.

flow cytometric criteria: CD5 positive, CD22 dim/negative, CD23
positive, light chain dim/negative, FMC7 negative.12 Typical CLL
cases were bright positive for CD23. If CD23 was dim, or if there
was a CD23-­negative subset, the case did not receive a point for
the CD23 criterion. Cases scoring 4 or 5 points of 5 were classi-
fied as CLL. In all these cases, this diagnosis was consistent with
other pathology testing including microscopy, and where available,
IHC, cytogenetics, and FISH. In cases with a CD5+ monoclonal B
cell population, which scored one point for CD5+, if the total score
was less than 4, samples were evaluated for evidence of a t(11;14)
translocation. If there was no evidence of such a translocation by
cytogenetics or FISH, or if cyclin D1 was negative by IHC, the case
was classified as atypical CLL. The 179 samples consisted of 70
controls (polyclonal B cells only), 63 samples with CLL phenotype,
of which 56 were typical and 7 were atypical, 6 MCL samples and
40 other monoclonal B cell populations (Table 1). Details of the
atypical CLL cases are presented in Table 2. Of the atypical CLL
samples, 3 had dim or negative CD23 expression, all had partial or
total FMC7 expression, and 5 had moderate-­bright surface light
chain.
TING et al.
F I G U R E   2   CD200-­APC MFI according to lymphoma type.
For the control samples, the figure shows data for the polyclonal
B cells. For the other samples, the data for the monoclonal B
cells only are shown. Abbreviations: DLBCL, diffuse large B cell
lymphoma; LPL, lymphoplasmacytic lymphoma. See Table 1 Legend
for details of the “Unclassified” samples
F I G U R E   3   CD200-­APC MFI in samples with CLL phenotype
according to diagnosis or source. Samples with CLL phenotype
were divided into cases with a diagnosis of CLL or a diagnosis of
MBL. Samples with CLL phenotype were also divided into those
from peripheral blood or bone marrow
Representative examples of control, CLL, MCL, and atypical CLL
CD200 staining are shown in Figure 1. Of the 70 control samples, all
except one exhibited clear CD200 expression on polyclonal B cells
(Figure 2). There was CD200 staining on 40%-­100% of B cells, except
for the single negative sample, which had CD200 on 3.7% of B cells.
CD200 was strongly expressed on monoclonal cells of all typical CLL
samples (on 70%-­100% of cells) and on all 7 atypical CLL samples (on
87%-­100% of monoclonal cells). The CD200 MFI and percent of pos-
itive cells in the atypical CLL samples were similar to the typical CLL
cases (P = .55 and .83, respectively). By contrast, MCL samples were
|
      5
CD200 dim or negative (on 0.3%-­18.3% of monoclonal cells). CD200
MFI and % positive cells were significantly lower on MCL compared
with all CLL samples (P < .001 for MFI and % positive cells; Mann-­
Whitney U test) and compared with the atypical CLLs (P < .002 for
MFI and % positive cells) (Figure 2).
There was more spread of the CD200 MFI in control samples
compared to CLL samples, and the CLL samples were brighter than
the controls (median MFI 1482 and 821, respectively) (Figure 2).
Among the other samples of mature B cell neoplasms, apart from
CLL and MCL, the two hairy cell leukemia samples had brighter MFIs
for CD200 than all the other monoclonal samples. CD200 MFI was
variable on other types of mature B cell neoplasms (Figure 2).
When the CLL phenotype samples were divided into cases with
a diagnosis of CLL and cases with a diagnosis of MBL, the CD200
MFIs of the two groups were not significantly different (P = .78)
(Figure 3). As illustrated in Figure 3, CD200 MFIs of CLL samples in
bone m
­ arrow and peripheral blood were similar (P = .76). There was
no significant relationship between age and CD200 expression in
controls and CLL samples (data not shown).
CD54 and CD160 were assessed because they have been pro-
posed to distinguish between CLL and MCL. 22-24 However, CD54 did
not differ between CLL and MCL. While CD160 was brighter on CLL
than that on MCL, in the current study many CLL cells were dim or
negative for CD160 and a satisfactory distinction between CLL and
MCL was not obtained (data not shown).
4 | D I S CU S S I O N
A striking difference in CD200 expression on CLL versus MCL cells
was found, with all 56 typical CLL samples positive for CD200 and all
of the 6 MCL samples expressing little or no CD200, consistent with
previous reports.18-20,24 The median % of cells positive for CD200 in
our typical CLL cohort (99.5%) is very similar to the 94%,19 96%, 20
and 100%18 expression found in previous studies.
In the present study, all 7 atypical CLL samples were positive
for CD200. CD200 expression in the atypical CLL cases was clearly
much higher than that in the MCL cases, but was not significantly dif-
ferent from the typical CLL cases. Two other studies reported posi-
tive CD200 expression on all atypical CLL cases; one of these found
CD200 on all 3 cases with a Matutes score of 3, and the other found
CD200 on all 11 atypical CLL cases with a Matutes score <4. 24,25 A
recent report on cases classified according to a similar 5-­point scale
found that 13 of 15 cases with a score of 3 were CD200 positive.
However, this report excluded MCL cases; therefore, strict compari-
son with the current report is difficult. 26 There was no MCL with the
aberrant immunophenotype of CD23+ in our series, so it was not
possible to evaluate the CD200 expression on CD23 +  MCL.
In our series, replacing CD23 with CD200 would have improved
the Matutes scoring system for better discrimination of CD5+
lymphoproliferative disorders with aberrant immunophenotypes.
Four of our 7 atypical CLL samples had ambiguous CD23 expres-
sion (dim or negative subset), but all were CD200+. The sensitivity
 |
6       TING et al.
and specificity of CD200 in the identification of CLL and MCL, in
terms of % expression, were both found to be 100%, overall per-
forming better than CD23, for which sensitivity and specificity were
93.6% and 100%, respectively. These findings carry significant clin-
ical relevance because atypical CLL as defined by the Matutes score
is quite common—the original paper proposing the score12 found
13% (52/400) of CLLs to be atypical, scoring ≤3, and another report
from this group found such cases to represent 11% of CLL cases, 27
­comparable to the figure of 7/63 (11%) in the present report. The
present study suggests that CD200 is very useful in classifying CD5+
monoclonal B cell populations with atypical phenotypes. However, a
recent study with a large number of mantle cell lymphomas indicated
that 3 of 61 of the MCL cases were positive for CD200, indicating
that the CD200 result is not completely reliable in distinguishing be-
tween CLL and MCL. 28
All the atypical CLL cases in the present study were CD5+. While
the vast majority of CD5+ B cell neoplasms are either CLL or MCL,
a few CD5+ cases are other types. For example, 5% of lymphop-
lasmacytic lymphomas and 5% of marginal zone lymphomas have
been reported to express CD5. 2 Diffuse large B cell lymphoma is
also occasionally CD5+, 2 but morphological examination is likely to
recognize such cells. While CLL is normally diagnosed by immuno-
phenotype,3 FISH analysis can be informative. FISH demonstrated
loss of 13q, a characteristic feature of CLL, 29 in one of our atypical
CLL cases. However in 2 cases, FISH analysis for the CLL probe set of
11q, 12, 13q and 17p demonstrated no copy number abnormality; in
2 patients, the CLL probe set was not used (the only probe used was
t (11;14) to exclude MCL); and in one case, FISH was not performed.
Although we have demonstrated that the atypical CLL cases are not
MCL, the possibility that one or more of these cases may not be
CLL cannot be excluded. However, marginal zone lymphoma seems
unlikely, as a report on CD200 expression in mature B cell neoplasms
found 4 cases of CD5+ marginal zone lymphoma all of which were
negative for CD200. 24
In our study, expression of CD200 on cells with CLL phenotype
was not significantly different between peripheral blood and bone
marrow cells. This conclusion contrasts with a study in which bone
marrow cells with CLL phenotype were significantly brighter for
CD200 than peripheral blood cells. 28 However, there is substantial
overlap between the two groups in both studies.
In conclusion, CD200 is a sensitive and specific marker for dis-
criminating between CLL and MCL in flow cytometry immunopheno-
typic analysis, and is particularly useful in recognizing atypical CLL.
AC K N OW L E D G E M E N T S
This project was supported by SydPath, St Vincent’s Hospital Sydney
and by St. Vincent’s Clinical School, Faculty of Medicine, UNSW Sydney.
The authors thank Steven Le and Liza Flores for technical assistance.
C O N FL I C T O F I N T E R E S T S
The authors have no competing interests.
AU T H O R C O N T R I B U T I O N
YST preformed the acquisition, analysis, and interpretation of data;
SABC designed the antibody panels and oversaw the acquisition and
analysis of data; DAB, AJD, KCF, DDFM, SM, and JJM contributed to
the research design; and WAS initiated and supervised the study. All
authors contributed to the drafting of the manuscript and approved
the submitted version.
ORCID
W. A. Sewell  http://orcid.org/0000-0002-3586-8761
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How to cite this article: Ting YS, Smith SABC, Brown DA,
et al. CD200 is a useful diagnostic marker for identifying
atypical chronic lymphocytic leukemia by flow cytometry. Int
J Lab Hem. 2018;00:1–7. https://doi.org/10.1111/ijlh.12857