ABNORMALITIES IN PATIENTS WITH CHRONIC FATIGUE SYNDROME
Umberto Tirelli M.D.°, Giuseppe Marotta
M.D.*, Salvatore Improta M.D.*, Antonio Pinto M.D.*
° CFS Unit, Division
of Medical Oncology and AIDS, Centro di Riferimento
(CRO), Aviano - Italy
* The Leukemia Unit, Division
of Medical Oncology, C.R.O., Aviano - Italy
|CFS: clinical and immunological study
|Prof. Umberto Tirelli
Division of Medical Oncology and AIDS
Centro di Riferimento Oncologico, IRCCS
33081 Aviano - Italy
phone: Italy - 434 - 659284
fax: Italy - 434 - 652997
||chronic fatigue syndrome, lymphocyte
subsets, NK cells, flow cytometry
Between January 1991 and January 1993,
265 patients who fulfilled the CDC criteria of the working case definition
of Chronic Fatigue Syndrome (CFS) have been observed at our Institution
and submitted to clinical and laboratory evaluation. One hundred and sixty-three
patients were females and 102 males, the median age was 35 years (range
4-55 years); all patients reported profound and prolonged fatigue, lasting
for a median of 3 years (range 6 months - 10 years), preceded or accompanied
at appearance by fever in 185 cases, and neuropsychologic problems including
inability to concentrate, difficulty in thinking, confusion, irritability,
forgetfulness, and depression. The fatigue was so severe to induce 102
patients to stop their working activities for a period of time ranging
from 3 months to 2 years (range 7 months). In 40 consecutive patients
a comprehensive immunologic testing by single and two-color flow cytometry
was performed and results compared with a group of 35 healthy, age- and
sex-matched controls. Whilst no significant differences were found in the
absolute numbers of circulating total T cells (CD3+) and of total helper/inducer
(CD4+) or suppressor/cytotoxic (CD8+) T cells, an evident reduction in
CD3-/CD16+ and CD57+/CD56+ NK lymphocytes along with an expansion of the
CD8+/CD56+ and CD16-/CD56+ NK subsets, were found in the CFS group.
In addition, CD56+ NK cells from CFS subjects were found to express an
increased amount of cell adhesion molecules (CD11b, CD11c, CD54) and activation
antigens (CD38). Both the percentage and absolute numbers of CD4+ T cells
bearing the CD45RA antigen appeared significantly reduced in CFS
patients, and CD4+ T lymphocytes from CFS subjects displayed an increased
expression of the intercellular adhesion molecule-1 (ICAM-1/CD54). Finally,
the total numbers of circulating (CD19+) B lymphocytes, were significantly
higher in CFS cases than in controls, and in 11 out of 30 CFS patients
the increase in circulating B cells was sustained by the expansion of the
CD5+/CD19+ subset of B lymphocytes. We conclude that CFS is a syndrome
not previously described in Italy, with the already known clinical characteristics
and appears associated with several immunologic abnormalities, including
those reported previously in cohort of patients from different countries.
We also show for the first time that CD56+ NK cell subsets from CFS patients
display an abnormally increased expression of cell adhesion molecules and
Chronic Fatigue Syndrome (CFS) is a recently
defined illness of unknown etiology, characterized by unexplained, disabling
fatigue lasting more than six months, chronic and recurrent low-grade fever,
adenopathy, pharyngitis, and neuropsychological symptoms such as
difficulties with concentration and depression . The Centers for
Disease Controls (CDC) have produced a working case definition for such
heterogeneous disease which relies on clinical and laboratory criteria
. A viral etiology for CFS has been suspected owing to the overlapping
clinical features with post-viral fatigue, and a number of different viruses
including Human herpes virus-6 (HHV-6) [3, 4], enteroviruses  and HTLV
II-related retroviruses , have been implicated in the development of
Results on immunological abnormalities
associated with CFS have been far more conflicting and include a decreased
number and function of NK cells [7-9], the presence of chronically activated
circulating T cells [10, 11], monocyte alterations , changes in B cells
subsets [8, 12], and abnormalities in cytokine serum levels or in vitro
response of lymphocytes to mitogenic stimulation [13-16]. More recently,
an altered distribution of CD4+ T cells subsets bearing CD45RA (naive T
cells) and CD45RO (memory T cells) antigens, along with the increased expression
of cell adhesion molecules on CD4+/CD45R0+ T lymphocytes have been described
in CFS patients by Straus and coworkers .
CFS has been mainly described in the U.S.,
Australia, and Great Britain, but also in Canada, New Zealand, Israel,
Spain, and France  while it has never been described, to our knowledge,
in Italy with the exception of a preliminary report from our group .
We describe here the results of a prospective
clinical study on 265 patients with CFS observed in Italy, and of
a detailed immunophenotypic study of circulating lymphocytes from 40 consecutive
CFS subjects and 35 matched healthy controls.
Between January 1991 and January 1993, 265 patients who fulfilled the CDC
criteria of the working case definition of CFS  have been observed at
our Institution and submitted to clinical and laboratory evaluation .
This large number of patients is explained by the fact that our group was
the first in Italy to report cases of CFS and at least during 1991 we were
the only referral center. Moreover, large coverage by the media on this
apparently new syndrome in Italy was made.
All subjects had a longer than 6 months
history of a new onset of debilitating fatigue and fullfilled 6 or more
minor criteria (symptoms and physical findings), along with the exclusion
of any alternative diagnosis for their symptoms. In particular, chronic
psychiatric disease, such as endogenous depression, hysterical personality
disorder, anxiety neurosis, chronic use of antidepressive medications were
excluded as required by the case definition also by the use of the Minnesota
Multiphasic Personality Inventory (MMPI)  and structured interview.
In the same period of time, 1750 individuals were referred at our Institution
with suspicion of CFS, but they were actually diagnosed as affected by
other diseases. The diagnosis of depression was made in several patients
by psychiatrists and neurologists at other institutions, while patients
of this series were submitted to the MMPI . From September 1991 to
March 1992, 40 consecutive patients underwent extensive immunophenotypic
analysis of peripheral blood lymphocytes. The reason of the selection of
such limited group for extensive immunological analysis was mainly economic.
However these 40 patients reported a debilitating fatigue of more than
6 months duration along with the other symptoms fulfilling the CDC criteria
of the working case definition of CFS , therefore with characteristics
superimposable to the overall group of 265 patients. As a control group,
35 age- and sex- matched healthy individuals were selected among blood
donors and hospital staff. All control subjects underwent physical
examination and a routine laboratory workup to assess their healthy condition.
None of the control subjects had experienced a period of fatigue or unexplained
malaise lasting more than one week, in the six months preceding immunological
assessment. Both patients and control subjects were free from medications
(with a special regard to steroids and non-steroideal antiflammatory drugs)
for a minimum period of two months before blood sampling, as assessed by
the attending and referring physicians.
Cell separation and immunophenotyping.
Mononuclear cells were isolated by standard Ficoll-Hypaque centrifugation
of citrate-dextrose anticoagulated peripheral blood. Specimens were processed
within two hours from sampling. In general, two patient samples and
at least one control specimen were tested toghether on the same day. For
direct immunofluorescence analysis, cells (5.0 x 105) were first preincubated
(30 min at 4°C) with Hank's balanced saline solution containing 10%
rabbit serum and 0.01% sodium azide to prevent Fc receptor binding, and
subsequently incubated (30 min at 4°C) with saturating concentrations
of fluorescein- (FITC) and phycoerythrin- (PE) labeled monoclonal antibodies
as described [20, 21]. Two-color direct imunofluorescence was performed
as described [20, 21]. Monoclonal antibodies used along with their
recognized cluster of differentiation (CD) and source were as follows:
Leu 4/CD3-PE, Leu 3/CD4-PE, Leu 2/CD8-PE and -FITC, Leu 11a/CD16-FITC,
Leu 12/CD19-FITC, anti-HLA-DR-FITC, Leu 19/CD56-PE, Leu 7/CD57-FITC (Becton-Dickinson,
Mountain View, CA); MO1/CD11b-FITC, 4B4/CD29-FITC, 2H4/CD45RA-FITC (Coulter
Immunology, Hialeah, FL); 84H10/CD54-FITC, UCHL.1/CD45RO-FITC, T16/CD38-FITC
(Immunotech, Marseille, France); KB90/CD11c-FITC (Dakopatts A/S, Glostrup,
Denmark). In 30 CFS patients and 23 healthy controls CD5+ B cells
were also evaluated with a preconstituted CD5-PE/CD19-FITC antibody combination
(Immunotech). The percentage of CD5+ B cells was expressed as a percentage
of the total lymphocyte population in the analysis gate. Non
specific binding of monoclonal antibodies was assessed by labeling cells
with phycoerythrinated and fluoresceinated isotype-matched control mouse
Igs (Becton-Dickinson). Viable, antibody-labeled lymphocytes were identified
according to their forward and right angle scattering, electronically gated
and analyzed for surface fluorescence on a FACScan flow cytometer (Becton
Dickinson). Cells identified by the lymphocyte gating were checked with
a anti-CD45-FITC+anti-CD14-PE antibody combination (LeucoGATE, Becton-Dickinson).
Less than 1% of CD14+ monocytes were identified in the lymphocyte gate.
Fluorescence data were collected and analyzed by a Lysis II software (Becton-Dickinson).
Laser output was mantained constant and the fluorescence gain settings
were adjusted daily to obtain the same fluorescence signals from calibrating
microspheres (Polyscience Inc., Warrington, PA) to allow fluorescence intensity
comparisons among different experiments. In 10 patient samples and 9 control
specimens immunophenotypic characterization was performed on the same day
with a whole blood lysis method , obtaining superimposable results,
as also supported by previous studies .
The statistical significance of mean antibody
reactivities and of absolute numbers of antigen positive lymphocytes comparisons
among CFS and control groups were analyzed by the Mann-Whitney U test for
non-gaussian distribution .
Clinical findings. One hundred
and sixty-three patients were females and 102 males, the median age was
35 years (range 4-55 years); all patients reported profound and prolonged
fatigue, lasting for a median of 3 years (range 6 months - 10 years), preceded
or accompanied at appearance by fever in 185 cases, and neuropsychologic
problems including inability to concentrate, difficulty in thinking, confusion,
irritability, forgetfulness, and depression (see Table 1). The other symptoms
detected were, among the others, in agreement with the CDC definition,
muscle weakness in all patients, low grade fewer in 185 patients, sore
throat in 190, painful laterocervical or axillary lymphnodes in 140, myalgia
in 260, sleep disturbances, either insomnia or hypersomnia in 261, headaches
in 260, migratory arthralgia in 245, photophobia and transient visual scotomata
in 235 patients. Depression, diagnosed mainly at other institutions, was
reported to appear several months after the onset of fatigue in 95 patients,
being therefore rather considered reactive to a debilitating and unpredicatable
disease. The fatigue was so severe to induce 102 patients to stop their
working activities for a period of time ranging from 3 months to 2 years
(range 7 months). There were three pairs of brothers, one mother and daughter
and three children (11, 11 and 4 years old) in this case series. Laboratory
examinations, in particular complete blood count, erythrocyte sedimentation
rates, standard serum chemistry test, testing for collagen vascular diseases
and thyroid function test, were normal. Antibodies to HIV were negative
in the patients tested. On clinical examination, non essudative pharyngitis
was observed in 155 patients, while in 65 patients lymph nodes were increased
in size without any suspicious characteristics for malignancies.
During the 2 year period of the study,
8 patients experienced the complete disappearance of symptoms, and 22 patients
referred a substantial decrease of symptoms, while in the other 235 cases
symptoms persisted although with spontaneous remissions and relapses. No
patient died for any reason.
Immunophenotyping of peripheral lymphocytes.
Immunological studies were performed in 40 consecutive patients and results
compared with those obtained from 35 age- and sex- matched healthy controls.
The absolute numbers of circulating lymphocytes were comparable in the
two groups (CFS, 1804 ± 354 x 106/l; normal controls 1927±
401 x 106/l). No statistically significant differences were found
in the absolute numbers of circulating total T cells (CD3+) and of total
helper/inducer (CD4+) or suppressor/cytotoxic (CD8+) T cells (Table 2).
CD4/CD8 ratios in CFS patients were also superimposable to values obtained
in the control group (data not shown). Circulating B cells, as evaluated
by an anti-CD19 antibody appeared to be significantly higher in the CSF
patients as compared to healthy subjects (210 ± 71 vs 116
± 36 x 106/l; p<0.05) (Table 2). As shown in table 3, two-color
immunofluorescence disclosed a significant decrease in the numbers of
circulating CD3-/CD16+ NK lymphocytes in the CFS group (80 ±
14 vs 136 ± 27 x 106/l; p<0.05) (Table 3). Subset analysis
of NK cells showed that whilst CD56+/CD57+ cells appeared to be decreased
in the CFS group (123 ± 35 x 106/l) as opposed to normal controls
(186 ± 50 x 106/l) (p < 0.05), the CD8+/CD56+ subpopulation of
NK cells appeared significantly increased in CFS cases (165
± 55 vs 126 ± 37 x 106/l) (p < 0.05).
In addition, the CD16-/CD56+ subset of NK lymphocytes appeared also significantly
expanded in CFS cases (138 ± 48 x 106/l) as compared to control
subjects (86 ± 23 x 106/l) (p<0.05). Interestingly, most of CD56+
cells in CFS patients displayed an increased expression of adhesion molecules
CD11b, CD11c, CD54 and of the activation antigen CD38 as shown by two-color
flow cytometry (Fig. 1). The percentages of CD56+ NK cells expressing such
membrane molecules appeared higher than in healthy controls, even though
a statistical significance was reached only when differences in CD56+/CD38+
cells were analyzed (Fig. 1). Subset analysis of CD4+ lymphocytes with
antibodies recognizing different isoforms of the CD45 antigen, evidenced
a significant reduction of both absolute numbers and relative percentages
of CD4+/CD45RA+ T cells in CFS patients as opposed to healthy subjects
(Table 3 and Fig 2). No significant changes were conversely detected when
subpopulations of CD4+ cells bearing CD45RO and CD29 molecules were analyzed
in both patients and controls (Table 3 and Fig 1).
Circulating T cells of CFS patients
were found to display an "activated" phenotype. The total number of CD3+
T lymphocytes expressing HLA-DR antigens (Table 3) was significantly increased
(138.6 ± 61 x 106/l) as compared to normal controls (81.4 ±
39 x 106/l) (p < 0.05). In addition, the percentage of CD3+ T cells
expressing the activation antigens CD11b, CD11c and CD54 appeared
also increased in the CSF group (Fig 3A), but such difference
did not reach a statistical significance. Conversely, the subset analysis
of the intercellular adhesion molecule-1 (ICAM-1/CD54) expression revealed
a significant increase of both relative percentages (Fig 3B) and
absolute numbers (Table 3) of CD4+/CD54+ T cells in CFS patients
(228.8 ± 142 x 106/l) as compared to controls (71.4 ± 31
x 106/l) (p < 0.05). No differences were conversely found in the levels
of CD8+/CD54+ T cells among CFS cases and normal subjects (Fig 3B
and Table 3). Representative dot plots showing the phenotypic profile
(CD45RA, CD29 and CD54) of circulating CD4+ lymphocytes from a CFS
patient and from a normal control subject are presented in Fig. 4.
In 11 out of 30 CSF patients analyzed
for B lymphocyte subpopulations, CD19+ B cells were found to coexpress
the CD5 antigen. The percentage of circulating CD19+/CD5+ B cells
in CSF patients appeared variable but usually superior to 15% (Fig
5). CD19+/CD5+ B cells ranged from 0.9 to 5.3% of total lymphocytes in
25 subjects of control group analyzed for such subpopulation of B cells
(data not shown). A representative flow cytometry dot plot showing the
coexpression of CD5 antigen on CD19+ B cells from CFS patients is shown
in Fig. 6.
We have observed a significant number
of cases of CFS at a single institution, suggesting that CFS, so far never
reported in Italy in a case series, is present in this country. This high
number of cases of CFS at a single institution can be explained by the
fact that our center was the only referral center for CFS in Italy in 1991.
The clinical characteristics of our patients
are similar to those reported in other countries [1-2]. We were able to
detect 3 groups of patients with distinct natural history. While in 8 patients
symptoms of CFS disappeared during the 2 year follow-up of this study,
and in 22 other patients there was a significant decrease of symptoms,
in other 235 patients symptoms of CFS persisted although with spontaneous
remissions and relapses. It must be stressed that the fatigue was so severe
to induce 102 subjects to stop their working activities for a relatively
long period of time, therefore affecting significantly the social life
of these patients. A debate exists in the literature as whether the increased
prevalence of psychiatric diseases seen in the CFS patients is the primary
cause of the syndrome or secondary to the debilitation of having a chronic
disease. Buchwald et al  reported abnormal magnetic resonance imaging
of the brain in 78% of patients with CFS tested and this was significantly
increased in comparison with healthy controls.
A detailed immunophenotypic study of circulating
lymphocytes was performed in 40 consecutive, strictly CDC-defined CFS patients
and 35 healthy matched controls. In our cohort of patients with CFS a number
of immunologic abnormalities involving specific subpopulations of NK cells,
T lymphocytes and B cells were detected. First, the absolute number of
CD3-/CD16+ and CD57+/CD56+ NK cells were reduced along with a concomitant
significant increase of CD56+/CD8+ and CD16-/CD56+ subpopulations. CD56+
NK cells also expressed an increased amount of CD11b, CD11c, and CD54 antigens.
Second, CD4+ T cells from CFS patients were found to show an increased
expression of the ICAM-1 (CD54) antigen as opposed to CD8+ T cells. Third,
the absolute numbers and relative percentage of CD4+/CD45RA+ "naive" T
cells were reduced in CFS patients, whilst CD4+/CD45RO+ and CD4+/CD29+
T cell subsets did not show appreciable variations in patients as compared
to healthy controls. Fourth, CD19+ B cells were overall increased in patients
and in 11 out of 30 patients the B cell expansion was sustained by an increase
of the CD5+/CD19+ subset of B lymphocytes.
The immunological results observed in
this study, although in agreement with previously data showing a
decrease in circulating NK cells [7-9], did not confirm the decreased proportions
of CD4+ and CD8+ T cells reported by Lloyd et al.  and by Behan et
al.  in CFS. In addition, we have shown that the NK cell decrease in
CFS appears to be subset-specific in that the numbers of CD8+/CD56+
cells in our patients were increased. Interestingly,
the CD16-/CD56+ subset of NK cells was
also expanded in our patients. This latter appears the most immature subpopulation
of NK cells and is thought to represent an "early precursor" subset
giving rise to mature CD16+/CD56+ NK lymphocytes [24, 25]. In addition,
CD16-/CD56+ cells were found to express the highest levels of CD11c,
but unable to secrete appropriate amounts of g-interferon (IFN) and
turned to be less efficient effectors of non-MHC restricted cytotoxicity
than CD16+ NK cells (24-26). Accordingly, we have found an increased proportion
of CD56+/CD11c+ and CD56+/CD11b+ cells in our patients and previous studies
have shown an impaired release of g-IFN by mitogen stimulated cultures
[14, 16], and a reduced cytotoxic activity against K562 target cells of
CD56+ lymphocytes from CFS subjects . Whether the expansion of
CD16-/CD56+ NK subsets in our CFS patients is a sort of "rebound" response
to diminished levels of CD16+ and CD57+ mature NK cells, or
is directly related to the action of a still unknown pathogen, remains
to be established. The umbalanced distribution of NK subsets evidenced
in our cohort of CFS patients, represents an intriguing finding which
can explain at least in part the reduced levels of NK activity detected
in CFS by different investigators [7-9]. Abnormalities in NK cells although
reported previously in CFS patients have not been confirmed by more
recent studies . The reason for such discrepancies is not clear at
the moment even though patient selection, disease status, immunophenotyping
techniques and different antibody combinations employed to evaluate
NK subsets, might explain divergent results on NK cells enumeration in
studies of CFS patients. As suggested by Strauss et al. the use of fresh
samples or cryopreserved cells may represent one of such biases . The
issue of NK function in CFS remains therefore an open matter of debate
warranting further careful investigations. Our results suggest that the
reduced NK functional activity in CFS may be in part related to an absolute
or relative increase of the less efficient CD16-/CD56+ subset of NK cells.
The increased expression of adhesion molecules and of activation markers
detected by us on CD56+ cells, further supports the hypothesis of a preferential
recruitment of such NK subset in CFS subjects.
We have also detected a reduction in the
relative proportion of CD4+/CD45RA+ "naive T cells", along with normal
numbers of CD4+/CD45RO+ in italian patients with CFS. These results are
in agreement with a recent report by Straus et al , even though the
absolute number of CD4+ T cells and the CD4/CD8 ratios appeared normal
in our patients. Accordingly, the rate of expression of the adhesion
molecule ICAM-1 (CD54) on CD4+ T cells appeared increased in our patients,
as also shown by a previous report by Gupta et al. ; even though we
did not perform CD45RO/CD54 double-labeling experiments, it is possible
to speculate that CD4+/CD54+ T cells in our patients were of CD45RO type
as clearly indicated by Straus et al. . If so, it could be proposed
as suggested by Strauss et al. that CD4+/CD45RO+ lymphocytes owing to a
high expression of surface molecule mediating cell adhesion and tissue
trafficking, may be responsible, following migration into specific tissues,
for at last some of the typical symptoms of CFS, i.e. myalgies, arthralgia
and lymph node tenderness. Cytokine produced focally by such cells within
specific tissues, might in fact mediate mild inflammatory response .
Further experiments are needed to verify such an hypothesis.
The increase in CD5+/CD19+ B cells detected
in some of our patients also deserves some speculations. Relative expansions
of such subset of B lymphocytes have been previously reported in other
series of CFS patients (8, 12) and in infectious mononucleosis . None
of our patients displaying increased CD5+/CD19+ cells had a positive serology
for EBV but all presented with adenopathy, which appeared sustained in
two biopsied cases by a lymphoid follicular hyperplasia. Since CD4+/CD45RA+
T cells are implicated in the control of B cell proliferation and differentiation
[29, 30], it could be speculated that the significant reduction of such
T cell subset as detected in different studies of CFS patients [8, 17,
31] and by us, might in turn result in a dysregulation of the B lymphocyte
compartment. As a matter of fact our CFS subjects showing a relative increase
of CD5+ B cells, presented the lowest numbers of CD4+/CD45RA+ T cells (data
not shown). In addition, decreased levels of CD4+/CD45RA+ T cells
have been reported to correlate with the extent of disease and the
presence of autoimmune phenomena in patients with lymphoproliferative disorders
sustained by CD5+/CD19+ B cells . Finally, selective loss of
CD45RA positive cells has been shown to play a role in the pathogenesis
of autoimmune disorders [33-36], including those accompanied by the expansion
of the CD5+/CD19+ subsets of B cells [37-38]. It could be therefore proposed
that the increased level of CD5+ B cells detected by us and other investigators
in CFS patients, is somehow related to the loss of regulating CD45RA+ T
cells, and that the expansion of such specific B cell pool might in part
contribute to CFS symptoms, which often overlap with those of autoimmune
diseases characterized by abnormalities of CD5+ B lymphocytes [37-41].
We conclude that CFS is a new disease
present in Italy, with clinical characteristics similar to those reported
in patients from other countries and associated to a number of heterogeneous
disfunctions of the immune system. Further investigations are needed in
order to better define immunological abnormalities detected in patients
with CFS with a special regard to the derangement in NK cell subpopulatons
reported in our study and to the defect in CD45RA T cells described by
different groups of investigators.
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LEGENDS TO THE FIGURES
Figure 1. Relative expression of
cell adhesion (CD11b, CD11c, CD54) and activation (CD38) molecules on CD56-positive
NK subpopulations. Flow cytometry data are expressed as a percent of double
positive cells. Closed bars (healthy subjects); shaded bars (CFS patients).
* p < 0.05.
Figure 2. Relative expression of
CD29, CD45RA and CD45RO antigens on CD4-positive lymphocytes. Flow cytometry
data are expressed as a percent of double positive cells. Closed bars (healthy
subjects); shaded bars (CFS patients). * p < 0.05.
Figure 3. (A) Relative expression
of adhesion molecules (CD11b, CD11c, CD54) on CD3-positive lymphocytes.
(B) Relative expression of intercellular adhesion molecule-1 (ICAM-1/CD54)
on CD4- and CD8-positive lymphocyte subpopulations. Flow cytometry data
are expressed as a percent of double positive cells. Closed bars (healthy
subjects); shaded bars (CFS patients).
* p < 0.05.
Figure 4. Representative dot-plots
showing two-color immunofluorescence analysis of peripheral CD4-positive
lymphocytes from an healthy control subjects (CNT; left panels) and a patient
with active CFS (CFS; right panels). Four regions are identified by setting
quadrant markers at the highest levels of non specific fluorescence. X-axes
represents log-green fluorescence; Y-axes represents log-red fluorescence.
Double-positive cells are identified in the upper-right region.
Figure 5. The percentage of CD5+/CD19+
B cells in peripheral blood of eleven CFS patients. Closed bars, CD19+
cells; shaded bars, CD19+/CD5+ cells.
Figure 6. Representative dot-plot
showing two-color immunofluorescence analysis of circulating CD19-positive
B cells from a CFS patient. Four regions are identified by setting quadrant
markers at the highest levels of non specific fluorescence. X-axes represents
log-green fluorescence (CD19); Y-axes represents log-red fluorescence (CD5).
Double-positive cells (CD5+/CD19+) are identified in the upper-right region.