Manuscript Title:
Evaluation of a Method Allowing Preservation of Fresh Lymph Nodes for Flow Cytometric
Immunophenotyping.
Running Title:
Preservation of Fresh Lymph Nodes for FCI.
Authors:
Ms. Patricia Brincat
MSc. Haematology (University of the West of England)
Haematology Laboratory Pathology Department
Mater Dei Hospital
Malta
Dr. James Degaetano
Histopathology Laboratory
Pathology Department
Mater Dei Hospital
Malta
Dr. Craig Donaldson
Centre for Research in Biosciences
Department of Applied Sciences
University of the West of England, Bristol United Kingdom
Corresponding Author:
Patricia Brincat
Haematology Laboratory
Pathology Department
Block B, Level 1,
Mater Dei Hospital,
Tal-Qroqq, MSIDA
MALTA
MSD 2020
Tel: (+356) 25456350
Fax: (+356) 25456359
E-mail: patricia.farrugia@gov.mt
Key Words:
Flow Cytometric Immunophenotyping; Haematolymphoid Neoplasia; Lymph Nodes; Cryopreservation
Abstract
Background: Flow cytometric immunophenotyping (FCI) of lymph nodes (LN) requires fresh unfixed tissue,
with analysis being carried out within few hours post surgery. This study evaluated a novel method for fresh
LN preservation, in order to allow histomorphology-based FCI.
Methods: This study was carried out prospectively on 30 LN with suspected involvement by
Haematolymphoid Neoplasms (HLN). FCI was performed on each fresh and post cryopreserved LN cell
suspension. Percentage positivities (PP) and Mean Fluorescent Intensities (MFI) were calculated on both
preparations for a combination of T and B-lymphoid antigens together with viability.
Results: The cryopreservation method applied within this study did not affect significantly PP and had minor
impact on MFI of the mentioned antigens. Overall there was minimal decrease in PP and MFI on the
cryopreserved cells when compared to fresh cells, for most antigens with only a mild increase in apoptotic
cells. However these changes were not diagnostically significant, since both reactive processes and HLN
present within cryopreserved LN could be identified and differentiated. Viability was more than 75% for all
cryopreserved LN composed of haematolymphoid cells.
Conclusions: The method presented in this study confers the possibility of storing fresh LN biopsies for later
FCI, thus allowing a morphology-based immunophenotypic approach. This would allow a more sensitive,
specific and cost-effective management of LN specimens, whilst maintaining the important benefits
provided by FCI.
Key Terms: Flow Cytometric Immunophenotyping; Haematolymphoid Neoplasia; Lymph Nodes;
Cryopreservation.
Introduction
Immunophenotyping of Haemotolymphoid Neoplasms HLN presenting in solid tissues such as
lymph nodes (LN) is an important aspect in the identification and classification of these malignancies. The
major drawback of FCI on LN is the requirement of fresh, unfixed tissue1, with FCI being performed prior to
histomorphological evaluation. Lack of histomorphological guidance necessitates the use of a broad
monoclonal antibody (moab) panel with possible waste of reagents, instrument and technologist’s time
resulting in increased expenses to the patient and the health care system. Additionally, FCI may be carried
out unnecessarily since the panel selected may not be useful in diagnosing the patient’s medical
condition2,3. For FCI to be cost effective and efficient, an initial morphological evaluation of the tissue
should ideally precede FCI, providing specific indications on the need for FCI and also a morphology-based
panel selection. The Clinical and Laboratory Standards Institute (CLSI) guidelines (2007)4 state that any
chemically fixed tissue would be unsuitable for FCI. Such pre-requisite implies that tissue samples have to be
assayed within a few hours after surgery. It is not recommended to postpone FCI on fresh tissue until after
the histological examination is complete, since time delay adversely affects cell viability5. Histopathological
and immunohistochemical analysis of LN may be sufficient for the diagnosis of HLN. However FCI may still
be required particularly for those morphologically challenging neoplasms. Identification of clonal B-cell
populations can also prove difficult by immunohistochemistry due to nonspecific staining of kappa and
lambda light chains. The aim of this study is to develop a method to preserve cells from fresh LN tissue for
FCI, mainly for the detection of HLN, thus allowing a morphology based approach. This study will evaluate if
such a method impacts on antigen detection, both quantitative (percentage positivity), and also qualitative
(antigenic expression intensity), together with overall cell viability.
Materials and Methods
Specimens: A prospective study was carried out on 30 fresh, consecutive LN from patients with suspicion of
HLN, arriving at the Histopathology Laboratory, Mater Dei Hospital (Malta). No specific selection criteria
were applied and all fresh LN were included in the study. Once surgically removed, LN reached the
laboratory within few hours post-surgery. Fresh LN were transported to the laboratory wrapped in sterile
saline-soaked gauze. These were cut transversely and excessive fat was trimmed from the periphery.
Tissue Processing: The tissue section for FCI was suspended in RPMI 1640 medium (Sigma-Aldrich). Cells
from the tissue were recovered in RPMI media by gentle dissociation using a scalpel. The homogenate was
filtered through a 100µm wire mesh; cells washed once in 2.0ml RPMI, centrifuged at 1800g for 3 minutes
and resuspended in fresh RPMI. Each suspension was divided into two tubes; one to be stained directly by
moab (cell count 10-12 x 109/L) and another to be cryopreserved (cell count 12-15 x 109/L).
Cryopreservation of single cell suspension: Cell suspension to be cryopreserved and a cryovial were placed
on an ice block and 200µL of RPMI were added to the cells. Then 200µL of cold Fetal Calf Serum (FCS, Gibco)
were added to the cells. Six hundred (600µL) of 20% of dimethyl sulfoxide (Sigma Aldrich) were added drop
wise to the cells swirling the tube continuously while keeping the cells cool. The cell suspension was then
transferred to the cryovial and frozen to -80°C using a Cryo 1°C freezing container (Nalgene) filled up with
isopropyl alcohol, which provided repeatable, -1°C/minute cooling rate. The frozen cells were stored at 80°C for a minimum of 10 days and a maximum of 20 days. Cells were not subjected to any lysing agent.
Thawing of single cell suspension: The cryovial containing the frozen cell suspension was placed in a 37°C
waterbath for rapid thawing. Once thawed, cells were transferred to a 15ml tube and suspended in thawing
medium (RPMI enriched with FCS). To approximately 2.0mL of cell suspension, 10ml of thawing medium
were added drop wise. The thawed cells were then washed once in RPMI, centrifuged at 1800g for 3
minutes and resuspended in cell wash (cell count 12-15x109/L). Cells were ready for moab staining and FCI
as described below.
Monoclonal antibody staining of single cell suspensions and FCI: Cell suspensions were stained with 3
colour fluorochrome-conjugated antibodies (Table 1). Each cell suspension was tested for viability utilising
7-Aminoactinomycin D (7-AAD) and an isotype control was run with each sample (IgG1FITC/IgG2aPE). All
antibodies, viability dye and isotype control were obtained from Becton Dickinson Biosciences (BD).
Tubes were analysed on a 4 colour, dual-laser FACSCalibur FC, (BD Immunocytometry Systems, San Jose,
CA). For each sample 10,000 events were acquired and analysed using Cell Quest Pro software (BD). The
flow cytometer was calibrated before each LN analysis using BD Calibrite 3 Beads, thus ensuring uniformity
in day-to-day analysis.
Percentage Positivity: Flow cytometric analysis of LN-derived cells was carried out for identification of the
phenotypic properties and viability of haematolymphoid cells. For each LN, FCI was performed on both fresh
and cryopreserved cells and results compared. Percentage positivity (PP) was obtained using low Side
Scatter (SSC)/CD45 gating for all antigens except kappa/lambda, where SSC/CD19 was used.
Mean Fluorescent Intensity: Quantification of antigen expression intensity was calculated using the Mean
Fluorescence Intensity (MFI) for all the antigens, on both fresh and cryopreserved cells. Antigen expression
intensity was calculated as the mean fluorescence, along a linear scale and represented as histogram
expressing MFI. Intensity values were calculated on low SSC/CD45 lymphocyte gate for all antigens except
kappa and lambda, which were calculated on low SSC/CD19 lymphocyte gate.
Statistical Analysis
Statistical Analysis was carried out using SPSS v11.01 software. The degree of linear correlation between
fresh and cryopreserved was carried out using Pearson’s Correlation Coefficient. The paired t-test was used
to evaluate the difference between results obtained on fresh and cryopreserved samples (p-value of <0.05
was taken as significant difference between the two methods). The Intra Class Correlation (ICC) was used as
a measure of agreement between the two methods (ICC value of >0.7 considered as an agreement).
Results
Out of the 30 LN anaylsed fresh and post cryopreservation, 28 had a population of more than 75% of the
total cells expressing strong CD45 and low SSC, with variable presence of CD45 negative cells, with variable
presence of CD45 negative cells including epithelial, non haematological cells. The other 2 LN had a
population with low CD45 and high SSC, compatible with non-haemopoietic cells. The latter were diagnosed
exclusively by histomorphology and IHC, since the study was not intended to investigate antigenic
expression on non-HLN, the data obtained from such LN was excluded from any statistical analysis carried
out in relation to T and B-lymphoid antigens. Antigenic expressions for the LN analysed can be seen in Table
4.
CD45 Antigenic Expression
Correlation analysis of CD45 PP showed linear correlation between fresh and cryopreserved cells (r: 0.849,
p: <0.001). CD45 showed a statistically significant difference between fresh and cryopreserved cells (p:
0.003), with -7.03% mean difference (Figure 1). However measure of agreement analysis, revealed an ICC
value of 0.7543, indicating constant agreement in expression of this antigen between the two preparations.
MFI for CD45 on fresh cell suspensions correlated very well with that obtained from cryopreserved cells (r:
0.766, p: <0.001). No statistically significant difference was seen in the MFI of CD45 between the fresh and
cryopreserved analysis (p-value: 0.621). Additionally, measurements of agreement showed that CD45 MFI
on fresh cells consistently agreed with MFI on cryopreserved cells (ICC: 0.7653), on both reactive and
neoplastic LN.
B and T-Lymphoid Antigenic Expressions on Fresh and Cryopreserved LN
Surface antigens CD4/CD8; CD3/CD5; CD19/CD10; CD20/CD23 were quantified as PP out of the total CD45
positive/low SSC cells. Kappa/lambda were quantified as PP out of the total CD19 positive/low SSC cells.
Correlation analysis showed a linear relationship between PP for the antigens tested on both fresh and
cryopreserved LN (CPL) (Table 2). Measure of agreement analysis on PP of the above antigens showed
strong agreement between the two methods (Table 2).The MFI of all antigens was not markedly affected by
cryopreservation since correlation analysis and measure of agreement analysis showed that MFI values
obtained on fresh cell suspensions correlated very well with those obtained on cryopreserved cells (Table
3).
Viability
Viability on the fresh cells was between 75 and 96% (mean 88.2%) on all cases except one involved with
metastatic tumour. Viability post thawing was between 70 and 94% (mean 83.5). Correlation analysis
carried out to assess the viability of cells within fresh and CPL suspensions showed that there was
remarkable correlation between the two preparations (r: 0.882 and p: <0.001). Statistical analysis carried
out to assess significant difference between the viability on fresh and cryopreserved cells showed a
statistically significant difference (p: 0.004). However measure of agreement analysis, gave an ICC value of
0.8166, clearly demonstrating strong agreement between the two methods.
Discussion
Lymphoma diagnosis requires a multi disciplinary approach and FCI has proved to play a critical role in
antigen identification and lineage assignment together with providing diagnostic and prognostic
information. The fact that FCI of LN has to be carried out on fresh, unfixed tissue entails that analysis is
performed within a few hours post surgery. The possibility of storing fresh LN biopsies while maintaining
intact the phenotypic properties of its constituents has important advantages. Primarily it allows a
morphology-based immunophenotypic approach, leading to a more sensitive, specific and cost-effective
management. This study evaluated a method by which fresh LN can be preserved for the time necessary for
histological examination to be ready, so that a more directed immunophenotypic approach can be taken.
Lymphocyte Antigenic Expression
CD45 expression showed good correlation between fresh and cryopreserved cells, with minimal increase in
percentage positivity between CPL and fresh as expected, since the CPL cell suspension had a cell count
higher (12-15 x 109/L) than the fresh LN cell suspension (10-12 x 109/L). Additionally cryopreservation did
not significantly compromise the expression intensity of CD45. Such findings are of utmost importance since
CD45 is considered to have a central role in FCI investigating HLN, due to its ability to differentiate between
cells of haempoietic origin from non haemopoietic ones and also indicates the stage of maturation of the
cells. Studies have also outlined the important role of CD45 MFI in differentiating between diagnostically
challenging mature lymphoid neoplasms, such as CLL and MCL. Although these two B-cell neoplasms have
distinct phenotypic characteristics, some cases may present with phenotypic shifts making differential
diagnosis problematic. The typical phenotype of MCL is the co-expression of CD5/CD19/CD20 and lack
CD23, however atypical CLL may also present with such phenotype. Therefore CD45 MFI could be used to
differentiate between these two neoplasms (Carulli et al 2008) 6. Within this study results demonstrate that
cryopreservation (1) had no substantially adverse effects on CD45 expression and intensity and (2) had
minimal impact on cell loss. The intact expression of this marker confers the possibility that this antigen is
used as a gating marker together with differentiating cellular ontogeny and identifying neoplastic
phenotypes of cells. Within this study CD45 was used as a gating marker together with Side Scatter (SSC)
(Figure 3). The cryopreservation method applied within this study had no major effects on the light scatter
parameters, both forward scatter and side scatter.
B-cell and T-cell Antigens
FCI of cryopreserved cell suspensions showed patterns very similar to FCI carried out directly on fresh tissue.
Although highly correlated CD10 PP showed a significant difference between the fresh and cryopreserved
results (p: 0.07). This could be attributed to the fact that most LN examined had very low percentage of
CD10 positive cells (0%-34%, mean: 9.6%). This difference did not impact on diagnosis of HLN on CPL (Figure
3). In fact the LN involved with Follicular Lymphoma had a CD10 positivity of 34% on fresh and 27% on CPL.
Alternatively, PP for all B-cell antigens including CD10 showed strong agreement between both methods.
Excellent correlation in PP between fresh and cryopreserved cells was seen for all T-lymphocyte antigens.
Mean Fluorescent Intensity
MFI for the different antigens analysed in this study were evaluated in order to assess whether
cryopreservation affected significantly cellular antigen quantification. Correlation analysis showed that
there is linear relationship between MFI obtained by the two methods. Difference analysis for MFI between
fresh and cryopreserved cells showed no significant difference for all B-lymphoid antigens, except for CD23
(p: 0.041). These results illustrate that MFI are higher on the fresh cells and the cryopreservation/thawing
process tends to lower MFI of CD23, as was also reported by Deneys et.al., (7) post cryopreservation of
peripheral blood. Although a discrepancy was observed on CD23 MFI between the two preparations, such
difference did not impact in any way on the final interpretation of both reactive and neoplastic LN.
Measurement of agreement in MFI for all B-lymphoid antigens showed reproducibility between fresh and
cryopreserved antigenic expression, however a lack of agreement was observed for CD19 and kappa
analysis. Such a disagreement in CD19 MFI may have a diagnostic effect, as reported by several publications
which outlined the importance of CD19 intensity expression for differentiating HLN8,9,10. Light chain
analysis on both fresh and CPL gave very distinct patterns and were interpretation was very clear. The
difference in MFI seen for kappa did not interfere in any way in the final interpretation and clonality could
be established in both reactive process and also when there was light chain restriction in cases of HLN.
T-lymphoid antigens, CD4 and CD8 also seem to be down regulated by cryopreservation, and this may
impact on the diagnosis of T-cell HLN. However PP of the latter were unaffected by the method and
therefore the impact on diagnosis and differentiation of T-cell neoplasia may be minimal.
Viability
Good correlation and strong agreement in cell viability was obtained between fresh and CPL suspensions.
The significant difference seen between the two preparations (p: 0.004) is probably due to some inevitable
loss in cell viability, induced by the cryopreservation process. Guidelines issued by the CLSI (4) recommend
cell viability of more than 75% for reliable immunophenotypic analysis. The results obtained from this study
show that the cryopreservation method applied maintains haemopoietic cell viability, a crucial feature for
reliable FCI of HLN.
Such findings provide evidence that cryopreservation is a reliable method, suitable for fresh LN cell
suspensions and has minimal impact on both T and B-lymphoid lineages, light chain expression detection
and CD45 analysis. MFI values can be successfully evaluated and utilized on the cryopreserved cells for most
antigens. Viability is maintained up to the critical standards required for reliable diagnostic FCI. No
differences were observed in the expression of the different markers between reactive and neoplastic
processes. Since the number of HLN neoplasia included in this study is relatively small, the method could be
applied to investigate if it has any effect on antigenic expression exclusively on HLN. Although the current
study investigated fresh unfixed lymph node tissue, when the method was applied to fresh tonsils, antigenic
preservation was also maintained and both PP and MFI correlated very well. Such data was not included
because the sample size for other lymphoid tissue biopsies is too low.
The method investigated in this study eliminates the use of a broad panel of antibodies. Having
histomorphological and immunohistochemical guidance on the presence and the possible type of HLN
enhances the sensitivity and specificity of the technique, limiting costs and time employed to reach a final
diagnosis. Another major advantage of a morphology-based FCI approach is that such an analysis is carried
out only when indicated.
In conclusion, this study showed that cryopreservation of cell suspensions obtained from fresh LN is a
reliable method which does not compromise the relative percentages of B and T-lymphoid markers and the
total number of cells. Antigenic expression intensities were also maintained for most markers tested and
can eventually be identified and quantified by multicolor FCI. The results obtained within this study clearly
indicate that the two methods are interchangeable, thus conferring the possibility of storing fresh LN
specimens, allowing a histomorphology based immunophenotypic approach.
Acknowledgments
The authors acknowledge Dr. Paul White and Dr. Neville Calleja for their statistical assistance. Ms. Sharon
Falzon for her assistance and advice. The management of the Pathology department and all the staff at the
Haematology and Histopathology departments within Mater Dei Hospital, Malta for their support.
References
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Table 1: Table summarises the different monoclonal antibody combinations, together with their respective
fluorochromes and gating markers.
FITC
PE
PerCP (Gating marker)
Tube 1
CD4
CD8
CD45
Tube 2
CD5
CD3
CD45
Tube 3
CD19
CD10
CD45
Tube 4
CD20
CD23
CD45
Tube 5
Kappa
Lambda
CD19
FITC; fluorescein isothiocyanate, PE; phycoerythrin and PerCP; peridinin chlorophyll protein.
Table 2: Summary of correlation, statistical differences and measurement of agreement obtained for
percentage positivities on the different antigens tested.
Antigen
Pearson’s
Correlation
Coefficient
r-value
Paired t-test
p-value
Significance
2-tailed
(p-value)
Mean
Difference
Intraclass
Correlation
Coefficient
95% CI
Lower
Upper
95%
Confidence
Interval
Lower
Upper
CD4
0.966
<0.001
0.945
0.07
-2.03
2.17
0.9661
0.9280
0.9842
CD8
0.883
<0.001
1.000
0.00
-0.94
0.94
0.8854
0.7672
0.9453
CD5
0.890
<0.001
0.894
0.25
-3.55
4.05
0.8926
0.7812
0.9489
CD3
0.958
<0.001
0.761
0.39
-2.23
3.01
0.9587
0.9128
0.9806
CD20
0.932
<0.001
0.874
0.25
-2.96
3.46
0.9344
0.8633
0.9691
CD23
1.000
<0.001
0.118
1.50
-0.40
3.40
0.9653
0.9259
0.9839
CD10
0.986
<0.001
0.007
1.15
0.34
1.95
0.9696
0.9126
0.9876
CD19
1.000
<0.001
0.629
-0.61
-3.15
1.94
0.9519
0.8992
0.9774
Kappa
0.936
<0.001
1.000
0.00
-2.42
2.42
0.9366
0.8677
0.9702
Lambda
0.972
<0.001
0.193
-1.18
-2.99
0.63
0.9673
0.9310
0.9847
CD45
0.849
<0.001
0.003
-7.03
0.7543
Table 3: Summary of correlation, statistical differences and measurement of agreement obtained for MFI on
the different antigens tested.
Antigen
Pearson’s
Correlation
Coefficient
r-value
p-value
Paired t-test
Significance
2-tailed
(p-value)
Mean
Difference
Intraclass
Correlation
Coefficient
95% Confidence
Interval
95% CI
Lower
Upper
Lower
Upper
CD4
0.932
<0.001
0.016
22.04
4.51
39.56
0.9181
0.8048
0.9638
CD8
0.959
<0.001
<0.001
481.14
283.53
678.75
0.9252
0.5832
0.9759
CD5
0.939
<0.001
0.172
26.39
-12.17
64.95
0.9367
0.8686
0.9702
CD3
0.901
<0.001
0.418
65.46
-97.76
228.68
0.8988
0.7950
0.9517
CD20
0.727
<0.001
0.369
116.82
-145.43
379.08
0.7230
0.4874
0.8611
CD23
0.898
<0.001
0.041
136.54
5.77
267.30
0.8309
0.6534
0.9198
CD10
0.962
<0.001
0.332
11.32
-12.19
34.83
0.9532
0.9024
0.9779
CD19
0.582
0.002
0.121
-105.54
-240.95
29.88
0.5576
0.2493
0.7656
Kappa
0.713
<0.001
0.493
-50.96
-201.58
99.65
0.5759
0.2646
0.7785
Lambda
0.753
<0.001
0.816
43.25
-334.49
420.99
0.7390
0.5083
0.8704
CD45
0.766
<0.001
0.621
0.7653
Table 4: Table representing immunophenotype of the LN specimens analysed.
Haematolymphoid
Neoplasia
CLL
DLBCL
FL
MCL
T-NHL
Hodgkin’s Lymphoma
Reactive Hyperplasia
Immunophenotype
No. of Cases
Pos: CD5, CD20, CD19, CD23, Kappa
Neg: CD10, Lambda, CD3, CD4, CD8
Pos: CD20, CD19, Lambda
Neg: CD10, CD23, Kappa, CD5, CD3, CD4, CD8
Pos: CD20, CD19, CD10, Kappa
Neg: CD23, Lambda, CD5, CD3, CD4, CD8
Pos: CD5, CD20, CD10, Lambda
Neg: CD23, Kappa, CD3, CD4, CD8
Pos: CD4, CD5, CD3
Neg: CD8, CD19, CD10, CD20, CD23, Kappa,
Lambda
T-cell predominance with some B-cell markers
present and no light chain restriction
Variable expression of all T-cell and B-cell
markers with no light chain restriction
1
1
1
1
1
2
21
Figure 1: Bar chart representing a comparison between fresh and cryopreserved mean percentage
Haematolymphoid Neoplasia
Immunophenotype
No. of Cases
positivities, for all the tested markers.
120.0
Percent Positivity
100.0
80.0
60.0
Fres h Lymph Nodes
Cryopres erved Lymph Nodes
40.0
20.0
45
AA
D
CD
a
La
m
bd
a
19
Ka
pp
10
CD
23
CD
CD
3
20
CD
5
CD
8
CD
CD
CD
4
0.0
Axis Title
Figure 2: Bar chart representing a comparison between fresh and cryopreserved mean fluorescent
intensities, for all tested antigens.
7000
Mean Fluorescent Intensity
6000
5000
4000
Fresh Lymph Nodes
3000
Cryopreserved Lymph
Nodes
2000
1000
0
CD4
CD8
CD5
CD3
CD20
CD23
CD10
CD19
Kappa Lambda
CLL
Pos: CD5, CD20, CD19, CD23, Kappa
Neg: CD10, Lambda, CD3, CD4, CD8
1
DLBCL
Pos: CD20, CD19, Lambda
Neg: CD10, CD23, Kappa, CD5, CD3, CD4, CD8
1
FL
Pos: CD20, CD19, CD10, Kappa
Neg: CD23, Lambda, CD5, CD3, CD4, CD8
1
MCL
Pos: CD5, CD20, CD10, Lambda
Neg: CD23, Kappa, CD3, CD4, CD8
1
T-NHL
Pos: CD4, CD5, CD3
Neg: CD8, CD19, CD10, CD20, CD23, Kappa, Lambda
1
Hodgkin’s Lymphoma
T-cell predominance with some B-cell markers present and no
light chain restriction
2
Reactive Hyperplasia
Variable expression of all T-cell and B-cell markers with no light
chain restriction
21
Table 4: Table representing immunophenotype of the LN specimens analysed.
A
B
Figure 3: A case of MCL analysed on fresh (A) and cryopreserved cells (B), with minimal variation in phenotypic pattern
between the two. Such concordance is also seen for the other LN analysed. FCI gave a phenotype suggestive of MCL
involvement within the LN, with the typical antigenic expression pattern of CD5+/CD20+/CD23-/CD10- and light chain
restriction.