Modern Pathology (2004) 17, 918927

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Flow cytometric analysis of normal and

reactive spleen
Adriana I Colovai1, Christina Giatzikis1, Eric K Ho1, Mushahid Farooqi1,
Nicole Suciu-Foca1, Giorgio Cattoretti1 and Attilio Orazi2
1

Department of Pathology, Columbia University, New York, NY, USA and 2Department of Pathology and
Laboratory Medicine, School of Medicine, Indiana University, Indianapolis, IN, USA

Spleen is surgically removed for both non-neoplastic and neoplastic pathologies. A significant proportion of
splenectomy specimens require distinguishing between reactive and neoplastic conditions (eg lymphoma). To
establish a normal reference range for the spleen lymphocyte subsets, fresh samples of benign, reactive
spleens obtained from adult patients (N 12) and samples of normal spleen obtained from cadaveric transplant
donors (N 14) were analyzed using three- and four-color flow cytometry. Study of pan-B, -T, and -NK marker
expression revealed that the frequency of T cells is higher and that of B cells is lower in reactive (nonneoplastic) compared to normal (cadaveric) spleen. Furthermore, our study established a frame of reference for
cell markers commonly used for immunophenotyping of lymphoma, and identified discrete lymphocyte
subsets, such as early plasma cells and T cells carrying the phenotype of the NK/T subset. These results will
facilitate an accurate interpretation of the flow cytometric analysis of human spleen lymphocytes.
Modern Pathology (2004) 17, 918927, advance online publication, 23 April 2004; doi:10.1038/modpathol.3800141
Keywords: spleen; flow cytometry; reference range

Flow cytometry is a sensitive technique currently

employed by pathologists for quantitative and
qualitative evaluation of hematopoietic cells.15 In
a clinical setting, the main purpose of flow cytometric analysis is to identify abnormal cells by
defining their immunophenotypic characteristics.
To accomplish this task, multiparameter analysis
represents a reliable approach that integrates the
information provided by forward scatter (FSC) and
side scatter (SSC) with multiple fluorescence parameters. Thus, the cell size and internal complexity
as well as the simultaneous expression of 34
different cell antigens are evaluated and used to
outline distinct cell populations. Importantly, the
multiparameter analysis permits the detection of
abnormal cells that occur at very low frequency in
the context of normal populations, such as in the
case of minimal residual disease.6,7
Three-color flow cytometry is currently used by
many laboratories for the analysis of leukemia and
lymphoma, although technology is moving rapidly
towards more complex types of analysis, employing
Correspondence: Dr AI Colovai, PhD, College of Physicians and
Surgeons of Columbia University, 630 W168 Street, P&S 14-402,
New York, NY 10032, USA.
E-mail: [email protected]
Received 22 October 2003; revised 24 February 2004; accepted 25
February 2004; published online 23 April 2004

six or more fluorescence parameters.8 In three-color

analysis, a widely used strategy for the gating of
lymphocytes is based on CD45 expression and SSC
characteristics.9,10 This approach permits the analysis of aged samples with a better lymphocyte
recovery compared to FCS vs SSC gating. Therefore,
gating by CD45/SSC represents the strategy of choice
for immunophenotyping blood lymphocyte subsets,
for example, monitoring CD4 counts in AIDS
patients.9 This strategy is also successfully applied
for the analysis of many lymphoid malignancies
detected in blood, bone marrow, and lymph
nodes.1,9
Specific immunophenotypic profiles have been
established for several lymphoid neoplasms.14 The
abnormal profiles are compared to the pattern of cell
marker expression normally seen in the organ/tissue
being examined, and a diagnostic interpretation is
made. While the normal reference ranges for
differentiation antigens expressed by lymphoid cells
from peripheral blood, bone marrow, and lymph
nodes are well established,1115 there is little
information concerning the immunophenotypic
profile of lymphocytes found in normal or reactive
spleen. The aim of our study was to provide a
detailed immunophenotypic characterization of
human spleen lymphocytes using three- and four-color
flow cytometry. Our analysis established reference
values for commonly used lymphoid markers and

Flow cytometric analysis of human spleen

AI Colovai et al
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defined discrete lymphocyte subsets in normal

(cadaveric) and reactive (non-neoplastic) spleen.

Material and methods

Human spleen specimens were obtained from 11
males and 15 females, with an age range of 1779
years (mean age 42). The specimens included 14
normal spleens obtained from cadaveric organ
donors and 12 spleens obtained from patients with
non-neoplastic conditions, such as idiopathic
thrombocytopenic purpura (ITP) (six cases), splenic
infarct (three cases), benign vascular malformations
(two cases), and pancreatic fistula (one case). The
diagnosis of reactive spleen was established by two
experienced pathologists (AO and GC) based on
morphology findings. All spleen samples were
processed for flow cytometric analysis within 8 h
from harvesting, using a simple and rapid procedure. The tissue was first teased with forceps into
RPMI 1640 cell culture medium (MediaTech,
Herndon, VA, USA), allowed to settle, and large
debris was removed. The cell suspension was then
washed and resuspended in phosphate-buffered saline
(PBS) supplemented with 2% fetal calf serum
(FCS) and 0.1% sodium azide. Cell viability was
assessed using Trypan Blue. All specimens included
in the study showed lymphocyte viability higher
than 80%.

To compare the frequency of B-cell subsets

identified in the spleen and that found in peripheral
blood or lymph nodes, 11 samples of blood and 8
lymph nodes obtained from cadaveric organ donors
were included in this study. Lymph nodes were first
chopped into small pieces and the resulting cell
suspension was washed and resuspended in PBS
supplemented with 2% FCS and 0.1% sodium
azide. Lymph nodes with viability higher than
80% were included in the study.
Flow Cytometry

For three-color flow cytometric analysis, aliquots

of 5  105 cells/tube were stained with saturating
amounts of fluorescein isoththiocyanate (FITC)-,
phycoerythrin (PE)-, and peridinin chlorophyll
protein (PerCP)-conjugated antibodies, according
to the manufacturers instructions. As shown in
Table 1, the antibody panel included monoclonal
and polyclonal antibodies. CD45 antibody labeled
with the PerCP fluorochrome was added to each
tube for lymphocyte gating. To minimize the level of
nonspecific binding of antibodies used for the
detection of immunoglobulin (Ig) light and heavy
chains, cells were washed three times in PBS
supplemented with 10% FCS and 0.1% sodium
azide prior to staining.16 For intracellular staining,
cells were fixed and permeabilized using Fix and

Table 1 Antibody panel

CD marker (FITC/PE/PerCP)

Clone (FITC/PE/PerCP)

Source (FITC/PE/PerCP)

CD2/CD19/CD45
CD3/CD19/CD45
CD4/CD8/CD45
CD20/CD5/CD45
CD7/CD13/CD45
CD10/CD20/CD45
CD3/CD16+56/CD45
Kappa/Lambda/CD45
cy Kappa/cy Lambda/CD45
TCR alpha/beta/TCR gamma/delta/CD45
IgM/-/CD45
IgG/-/CD45
IgA/-/CD45
IgD/-/CD45
CD103/-/CD45
FMC7/-/CD45
cy TdT/-/CD45
-/CD11c/CD45
-/CD23/CD45
-/CD30/CD45
-/CD34/CD45
-/CD38/CD45
-/HLA-DR/CD45
-/cy CD79a/CD45

39C1.5/J4119/2D1
SK7/4G7/2D1
SK3/SK1/2D1
L27/L17F12/2D1
4H9/L138/2D1
W8E7/L27/2D1
SK7/B73.1/2D1
TB28-2/1-155-2/2D1
TB28-2/1-155-2/2D1
WT31/11F2/2D1
Polyclonal/-/2D1
Polyclonal/-/2D1
Polyclonal/-/2D1
Polyclonal/-/2D1
2G5/-/2D1
FMC7/-/2D1
Polyclonal/-/2D1
-/S-HCL-3/D1
-/EBVSC-5/2D1
-/Ber-H83/2D1
-/8G12/2D1
-/HB7/2D1
-/L243/2D1
-/HM47/2D1

Beckman Coultera/Beckman Coulter/BDb

BD/BD/BD
BD/BD/BD
BD/BD/BD
BD/BD/BD
BD/BD/BD
BD/BD/BD
BD/BD/BD
BD/BD/BD
BD/BD/BD
BioSource Internationalc/-/BD
BioSource International/-/BD
BioSource International/-/BD
BioSource International/-/BD
Beckman Coulter/-/BD
Beckman Coulter/-/BD
Beckman Coulter/-/BD
-/BD/BD
-/BD/BD
-/BD/BD
-/BD/BD
-/BD/BD
-/BD/BD
-/BD/BD

Beckman Coulter, Miami, FL, USA.

BD BioSciences, San Jose, CA, USA.
c
BioSource International, Inc., Camarillo, CA, USA.
cy, cytoplasmic.
b

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AI Colovai et al
920

Perm reagents (Caltag Laboratories, Burlingame, CA,

USA), and then incubated with specific antibodies,
as indicated in Table 1. Following staining, all
aliquots were subjected to red cell lysis using
FACSLyse solution (BD Biosciences, San Jose, CA,
USA). The presence of at least 90% CD45-positive
cells within all acquired events was considered
indicative of efficient red blood cell lysis.
To study the immunoprofile of splenic B cells,
four-color flow cytometry was performed. Splenocytes were stained with the following antibody
combinations: CD20 FITC/Kappa PE/CD45 PerCP/
CD19 APC (APC: allophycocyanine), CD20 FITC/
Lambda PE/CD45 PerCP/CD19 or isotype matched
control antibodies (BD Biosciences). To test the
expression of CD38 and CD138 markers, typically
expressed by plasma cells, splenocytes were stained
with CD20 FITC/CD38 PE/CD45 PerCP/CD19 APC
(BD Biosciences) and CD20 FITC (BD Biosciences)/
CD138 PE (Serotec Inc., Raleigh, NC, USA)/CD45
PerCP/CD19 APC (BD Biosciences).
To compare B-cell populations found in spleen,
whole blood and lymph nodes, splenocytes, whole
blood and lymphocytes isolated from lymph nodes
were stained with CD20 FITC/CD45 PerCP/CD19
APC or isotype matched control antibodies (BD
Biosciences). For this, 50 ml of whole blood or
5  105 lymphocytes isolated from lymph nodes
were incubated with each of the indicated antibody
mix, then lysed and washed.
Samples were run and analyzed on a FACSCalibur
instrument (BD Biosciences). The sensitivity of the
fluorescence detectors was set daily using Calibrite
beads (BD Biosciences). FSC and SSC measurements
were made using linear amplifiers, whereas fluores-

cence measurements were made with logarithmic

amplifiers. Flow cytometric two-parameter dot plots
and quadrant statistics were generated using Cellquest software (BD Biosciences).

Statistical Analysis

The mean and standard deviation (s.d.) values for

percent positive cells were calculated as described.11 Students t-test was used to compare the
frequency of lymphocyte subpopulations in normal
(cadaveric) vs reactive (non-neoplastic) spleens.
Matched t-test was used to assess the differences
between paired results obtained for B-cell subsets.
The correlation between age progression and the
frequency of lymphocyte subpopulations was determined by simple linear equation using BMPD
statistical software.17

Results
The immunophenotypic profile of human spleen
lymphocytes was studied using three- and fourcolor flow cytometry. The spleen specimens were
obtained from organ cadaveric donors (normal
group) and from patients with nonmalignant conditions that required splenectomy and showed minimal involvement of the spleen (reactive group). The
antibodies used in this study recognize pan-T, -B,
-natural killer (NK), and myeloid/monocytic antigens, covering markers that are essential for evaluation of lymphoma (Table 1).18 In addition, the
antibody panel included markers that allow a

Figure 1 Gating of human spleen lymphocytes. (a) Lymphocytes (L), monocytes (M), and granulocytes (G) from normal spleen were gated
using the display of CD45 (x-axis) vs SSC, (y-axis). (b) Spleen monocytes were identified based on the bright expression of CD45 and
CD14 markers.
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Table 2 Expression of surface and intracellular markers by

human spleen lymphocytes
Cell marker

Normal
(cadaveric)
spleen
N

CD2
14
CD3
CD4
CD5
CD7
CD8
CD4/CD8 ratio
CD10
CD11c
CD13
CD16/56+CD3
CD16/56+CD3+
CD19
CD20
CD20+CD5+
CD23
CD30
CD34
CD38
CD103
cy CD79a
HLA-DR
IgM
IgG
IgA
IgD
FMC7
Kappa
Lambda
Kappa/Lambda
cy Kappa
cy Lambda
cy TdT
TCR alpha/beta
TCR gamma/delta
a

Reactive
(non-malignant)
spleen

Mean7s.d.
(%)

Mean7s.d.
(%)

38710
3179
1778
3278
37711
1475
1.273
171
28710
171
1577
572
55711
4979
876
34713
171
272
79713
271
52712
71711
42714
575
977
36712
38713
2875
2175
1.370.2
3176
2275
o1
2979
272

12

50714
43714
2377
42713
44712
1977
1.270.2
171
25710
171
1275
574
45714
42712
1177
35712
171
271
83712
271
44710
71712
40717
372
672
35712
30710
2578
1776
1.470.2
2978
1676
o1
37712
571

P-value

0.015
0.009
NS
0.028
NS
0.028
NS
NS
NS
NS
NS
NS
0.04
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS

NS, not significant (P40.05).

detailed analysis of abnormal cells, beyond the

initial evaluation of the case.
As indicated in Figure 1, spleen lymphocytes
were gated based on bright expression of the CD45
marker and low SSC. Among the nucleated cells,
lymphocytes accounted in average for 82% of the
cells. Monocytes were identified in each sample
using the CD14 antibody, and were found to
represent about 2% of all nucleated cells. The mean
percentage and s.d. values for each marker or
combination of markers analyzed within the lymphocyte gate are indicated in Table 2.
The comparison between normal and reactive
spleen using the Students t-test of significance
indicated that, for several T- and B-cell markers,
the differences between cell frequencies found in
the two groups of specimens were statistically
significant. Thus, the frequency of lymphocytes
expressing CD2, CD3, CD5, or CD8 was significantly
higher in reactive compared to normal spleen, while

the frequency of CD19-positive cells was lower in

reactive vs normal spleen (Table 2). The mean
percentages of cells expressing other T-cell markers,
such as CD4 or T-cell receptor (TCR), were also
higher in reactive vs reactive spleen, although the
differences did not reach statistical significance.
Likewise, the mean percentages of lymphocytes
expressing pan-B-cell markers, such as CD20,
FMC7, CD79a, heavy and light Ig chains, were lower
in reactive compared to normal spleen. The frequency of NK cells appeared only slightly increased
in normal vs reactive spleen.

B-cell Profile

A typical immunoprofile of spleen B lymphocytes

obtained by three-color analysis is indicated in
Figure 2. The lymphocytes were gated using the
CD45/SSC display (Figure 1) and analyzed for the
expression of the following markers: CD19, CD20,
FMC7, cytoplasmic CD79a, surface and cytoplasmic
Kappa and Lambda Ig chains, surface IgA, IgD, IgG
and IgM chains, HLA-DR, CD10, and CD23. As
shown in Table 2, the frequency of CD19 and
cytoplasmic CD79a-positive cells had mean values
of 55 and 52%, respectively, in normal spleens, and
45 and 44%, respectively, in reactive spleen.
Students t-test of significance indicated that there
were no statistical differences between the frequency of CD19 and cytoplasmic CD79a-positive
cells within each group (P40.05). However, a
statistically significant difference was found when
the frequency of cells expressing CD19 was compared with the frequency of CD20 or FMC7 expressing cells in paired samples of normal (cadaveric)
spleen. Thus, CD20 was found on 49% (P 0.01),
while FMC7 was present on 38% (Po0.001) of the
lymphocytes in normal spleen. In reactive spleen,
the difference between the frequencies of cells
expressing CD19 and CD20 did not reach statistical
significance, yet the difference between CD19 and
FMC7 expressing cells was significant (Po0.001).
The percentage of the lymphocytes expressing
Kappa and Lambda Ig light chains was 49% in
normal spleen, being significantly lower than the
percentage of CD19 positive cells (Po0.01) and
matching the frequency of CD20-positive cells
(P40.05). In reactive spleen, the frequency of the
lymphocytes expressing Ig light chains was also
lower than that of the cells expressing the CD19
marker, although the difference was not statistically
significant. Notably, the frequency of lymphocytes
displaying both surface and cytoplasmic expression
of Kappa and Lambda chains matched closely the
percentage of CD19-positive cells in normal (53%)
and reactive (45%) spleen, respectively (P40.05).
To further define the phenotypic profile of splenic
B cells, four-color flow cytometric analysis was
performed. As shown in Figure 3, nearly all
CD19 CD20 cells were positive for either surface
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Figure 2 Immunophenotypic profile of splenic B cells. Human spleen lymphocytes were gated as indicated in Figure 1 and studied for
expression of pan-B-cell markers by three-color flow cytometry. The oval highlights the population of CD20-positive B cells coexpressing
the CD5 marker.

Kappa or Lambda light chain. However, a distinct

population carrying the CD19 CD20 phenotype
and accounting for 6 and 3% of all CD19-positive
cells in normal and reactive spleen, respectively,
showed very dim or no expression of Kappa and
Lambda Ig light chains. To determine whether these
B cells represent plasma cells, expression of CD38
and CD138 was analyzed by four-color flow cytometry. As illustrated in Figure 4a, CD19 CD20 B
cells showed expression of high levels of CD38 yet
were negative for CD138. These results are consistent with the phenotype of early plasma cells.1921
To compare the frequency of CD19 CD20 B
cells from human spleen and that from normal
peripheral blood or lymph nodes, 11 samples of
blood and 8 lymph nodes were analyzed by threecolor flow cytometry for expression of CD45, CD20,
and CD19. The frequency of CD19 CD20 B cells
was lower than 1% in peripheral blood and reached
a mean of 1% in lymph nodes (Figure 4b). Thus,
CD19 CD20 B cells appear to be more abundant
in spleen as compared to peripheral blood or lymph
nodes.
Modern Pathology (2004) 17, 918927

A significant subset of splenic B cells, representing 8 and 11% of the lymphocytes (16 and 26% of
CD20-positive cells) in normal and reactive spleen,
respectively, was found to coexpress the CD20 and
CD5 markers (Figure 2, Table 2). As illustrated in
Figure 2, the level of CD5 expression on B cells was
typically one log less compared to the level seen on
T cells (CD5 CD20) in any of the spleen samples.
CD5 B cells have been previously identified as the
B-1a subset and shown to represent a major subset
during fetal life and childhood.22 To determine
whether age-related changes in the frequency of
CD5 CD20 cells occur in the spleen, the distribution of cell frequency values vs donors age was
studied using simple linear equation. As shown in
Figure 5, there was a statistically significant
decrease in the percent of CD5 CD20 B cells
with age (Po0.02). The relationship between the
frequency of other lymphocyte subsets, as defined
by the expression of CD3, CD19, CD20, CD16/56, or
CD23 marker, and donors age was also studied. No
significant correlation was found for any of these
cell subsets (data not shown).

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Figure 3 Expression of Kappa and Lambda Ig light chains by splenic B-cell subsets. Spleen lymphocytes were gated as indicated in
Figure 1 and analyzed by four-color flow cytometry using CD20/Kappa/CD45/CD19 and CD20/Lambda/CD45/CD19 antibody cocktails.
CD19 CD20 (gate R2) and CD19 CD20 (gate R3) B-cell subsets were studied for expression of Kappa and Lambda Ig light chains, as
illustrated by the histograms. Dotted lines indicate staining with isotype matched antibody used as negative control.

CD10 (CALLA), a characteristic marker of germinal center B cells and a hallmark of follicular
lymphoma,5,23 was expressed on a small number of
cells from normal (mean 1%) and reactive (mean
2%) spleens. CD103, a useful marker for the
diagnosis of hairy cell leukemia,7 was expressed at
similarly low frequency. Markers that are frequently
expressed by immature cells, such as CD34 or TdT,
were found on 2% or less of the spleen lymphocytes
(Table 2).

markers CD16/CD56 were expressed in the absence

of CD3 on 15 and 12% of the lymphocytes from
normal and reactive spleen, respectively, and coexpressed with CD3 on 5% of lymphocytes in
both groups (Figure 6, Table 2). CD30, a useful
marker for the diagnosis of anaplastic large cell
lymphoma,24 was virtually absent on normal splenic
lymphocytes.

Discussion
T and NK Cell Profile

A typical T-cell profile of spleen lymphocytes is

depicted in Figure 6. Pan-T cell markers CD2 and
CD3 were expressed on 38 and 31%, respectively, of
normal spleen lymphocytes, and on 47 and 40%,
respectively, of reactive spleen cells. Staining with
antibodies specific for TCR a/b and g/d chains
indicated that the a/b heterodimer was expressed
on 29% of lymphocytes from normal spleen and
34% of lymphocytes isolated from reactive spleen,
while the g/d chains were present on only 2% of
lymphocytes from normal spleen and on 4% of
lymphocytes from reactive spleen. The NK-specific

The spleen is the largest lymphoid tissue of the

human body, accounting for approximately 25% of
the total number of lymphocytes. To establish the
immunophenotypic profile of lymphocyte subsets
found in human spleen, we performed three- and
four-color flow cytometric analysis of the cells
isolated from fresh spleen tissue obtained from
cadaveric donors and patients with nonmalignant
conditions. The extensive antibody panel used in
this study allowed a detailed characterization of the
lymphocyte subsets found in normal and reactive
spleen. The two groups of specimens showed
significantly different frequencies of CD2, CD3,
CD5, CD8, and CD19 expressing cells. These results
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Figure 4 (a) Expression of plasma cell markers on spleen lymphocytes. Spleen lymphocytes were analyzed by four-color flow cytometry
using CD20 FITC/CD38 PE/CD45 PerCP/CD19 APC and CD20 FITC/CD138 PE /CD45 PerCP/CD19 APC antibodies. B cells were gated
based on CD45 (as indicated in Figure 1) and CD19 positivity and analyzed for expression of CD20, CD38 and CD138. (b) Expression of Bcell markers by lymphocytes from peripheral blood and lymph nodes. A representative example of normal peripheral blood and lymph
node is shown. Lymphocytes were gated using the CD45/SSC dot plot and studied for expression of CD19 and CD20 markers.

Figure 5 Distribution of individual percent values for CD5 CD20 cells vs age. Simple linear equation was applied to determine P and
r2 values.

strongly suggest that reactive spleen displays a

higher frequency of T cells and a lower frequency
of B cells as compared to normal spleen.
Modern Pathology (2004) 17, 918927

Previous studies of normal B cells present in

human peripheral blood and lymph nodes showed
that pan-B cell markers CD20 and FMC7 were

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AI Colovai et al
925

Figure 6 Immunophenotypic profile of splenic T and NK cells. Spleen lymphocytes were gated as indicated in Figure 1 and analyzed for
expression of pan-T and NK markers by three-color flow cytometry. The oval highlights the NK/T cell subset (CD3 CD16/56 ).

coexpressed by nearly all CD19-positive B cells.11,15

In contrast, we found that the expression of CD20
and FMC7 did not parallel that of CD19 and CD79a
on splenic B cells. To exclude the possibility that the
differences between the results of our study and
those of previous reports are due to the use of
different flow cytometry protocols, lymphocytes
from normal peripheral blood and lymph nodes
were analyzed using the same method and reagents
as used for spleen cells. Our data indicate that the
frequency of CD19 CD20- B cells is, indeed, higher
in spleen as compared to peripheral blood or lymph
nodes.
The study of Ig light chain expression on splenic
B lymphocytes showed that surface Kappa and
Lambda chains were expressed by CD19 CD20
B cells yet were absent or expressed at very low
levels by CD19 CD20 B cells. However, cytoplasmic expression of Kappa and Lambda was found on
virtually all CD19-positive cells. Furthermore,
CD19 CD20 cells expressed high levels of CD38,
and were negative for CD138. These results are
consistent with the presence in the spleen of a
sizable population of early plasma cells,1921 carrying the CD45 CD19 CD38 bright, Kappa dim/
negative, Lambda dim/negative, CD20-CD138- phenotype, and accounting for about 6 and 3% of B cells in
normal and reactive spleen, respectively.
FMC7 antibody has been shown to recognize a
CD20-related epitope, presumably derived from a
multimeric CD20 complex.25,26 However, in some
neoplastic lymphoproliferative disorders, the levels
of FMC7 and CD20 expression show no correlation
and, in fact, the pattern of FMC7 and CD23
coexpression has been found to be valuable for the

accurate and reproducible classification of B-cell

lymphomas.27 The results reported here regarding
the expression of FMC7 on normal splenocytes
provide a useful reference for the interpretation of
an abnormal B-cell immunophenotype. The frequency of splenic FMC7-positive lymphocytes was
found to be significantly lower than the frequency of
CD19 cells, and also lower than that of CD20positive cells. Therefore, the absence of FMC7 on
subpopulations of splenic B cells should not be
interpreted as an aberrant phenotype in the absence
of other abnormal phenotypic features.
A significant population of CD20-positive B cells
from the spleen was found to express CD5, namely 8
and 11% of the lymphocytes in normal and reactive
spleen, respectively. Previous studies have identified the B-cell population coexpressing CD5 and
CD20 antigens as the B-1a subset, and found that 2
3% of the lymphocytes from normal adult tonsils
and lymph nodes display this phenotype.28 During
fetal life and childhood, however, CD5 B cells
were shown to represent a major B-cell subset,
accounting for 4060% of fetal splenic B cells.29,30
Although the ontogeny and function of these cells
are not well understood, it is assumed that they
provide an innate immune defense early in life by
producing antibodies against ubiquitous bacteria.31
Our present study indicates that the frequency of
splenic CD20 CD5 cells decreases with age. This
finding is in accordance with previous data showing
the same type of correlation between the frequency
of CD5 B cells from peripheral blood and age.32
The level of expression of the CD5 marker
observed on normal splenic B cells is lower than
that seen on T cells. Thus, in most cases flow
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cytometry permits the distinction between normal B

cells, which show dim expression of CD5, and
leukemic B cells (eg chronic lymphocytic leukemia
(CLL) and mantle cell lymphoma),4,15 which typically appear as a well-defined cluster of CD20/CD5
double-positive cells, expressing CD5 at levels close
to those found on T cells.
Similar to the CD5 marker, CD23 is a critical
marker for the diagnosis of B-cell disorders, allowing the distinction between CLL and mantle cell
lymphoma.4 In contrast to CD5 pattern of expression, CD23 shows increased expression with age,
from 35% in cord blood to over 60% of B cells in
adult blood.15 We found that a relatively large cell
population expressed CD23, representing 34 and
43% of the lymphocytes from normal and reactive
spleen, respectively. However, no significant correlation between expression of the CD23 marker and
age could be established for spleen lymphocytes.
The study of T-cell populations present in the
human spleen revealed that the CD4/CD8 ratio is on
the average 1.2:1, similar to the ratio found in lymph
nodes and bone marrow yet lower than in peripheral
blood, where it usually exceeds 2:1.11 The frequency
of g/d TCR-positive cells was 6% and 11% of all CD3
positive T cells in normal and reactive spleen,
respectively. These values may appear much lower
than those previously reported by studies which
showed that g/d TCR-positive cells represent about
17% of all CD3 positive cells in the red pulp of the
spleen.3335 However, the same studies reported that,
in periarteriolar sheaths, g/d TCR expressing cells
were rare. Thus, the apparent discrepancy between
our results and previously reported data is caused
by the different approaches used to analyze and
report the frequency of g/d TCR-positive cells. While
previous studies used immunohistochemistry and
reported a relatively high frequency of g/d TCRpositive cells within the red pulp area of the spleen,
our study used flow cytometry and reported the
frequency of this subset within whole spleen.
A sizable proportion of splenic T cells exhibit
dual expression of T and NK cell markers, a
phenotypic signature of NK/T cells.36 This T-cell
subset has been intensively studied as a possible
bridge between innate and adaptive immunity. The
proposed role of NK/T cells in immune responses
ranges from suppression of autoimmunity to tumor
rejection.36,37 While in peripheral blood NK/T cells
account for less than 6% of CD3-positive lymphocytes, our study shows that about 16% of the CD3positive cells present in the spleen express CD16/56
antigen(s). A relatively high frequency of these cells
has been also reported in the liver, where up to 55%
(mean 27%) of CD3-positive lymphocytes express
NK markers.37 These findings support the concept
that spleen and liver lymphocytes have important
immunoregulatory function.
Using a clear description of biological and
technical aspects of sample analysis by flow cytometry, our study provides a frame of reference for B,
Modern Pathology (2004) 17, 918927

T, and NK subsets present in normal and reactive

spleen. These results will be useful for flow
cytometry assessment of spleen specimens obtained
following fine-needle aspiration or splenectomy,
facilitating an accurate diagnosis of lymphoid
malignancies of the spleen.

Acknowledgement
This work was supported by NIH grant RO1AI25210-16.

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