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Immunology Letters 131 (2010) 67–72
Contents lists available at ScienceDirect
Immunology Letters
journal homepage: www.elsevier.com/locate/
Seroma fluid subsequent to axillary lymph node dissection for breast cancer
derives from an accumulation of afferent lymph
Erika Montalto a , Salvatore Mangraviti b , Gregorio Costa a , Paolo Carrega c , Barbara Morandi d ,
Gaetana Pezzino a , Irene Bonaccorsi a , Antonino Cancellieri e , Maria Cristina Mingari d,f ,
Mario Mesiti g , Guido Ferlazzo a,∗ , Giovanni Melioli b
a
Laboratorio di Immunologia e Biotecnologie Terapeutiche, Dipartimento di Patologia Umana, Università di Messina, Via Consolare Valeria 1, Messina - 98125, Italy
Laboratorio Centrale di Analisi, Istituto Giannina Gaslini, Genova, Largo G. Gaslini 5, Genova - 16148, Italy
c
Laboratorio di Immunologia Clinica e Sperimentale, Istituto Giannina Gaslini, Largo G. Gaslini 5, Genova - 16148, Italy
d
Dipartimento di Medicina Sperimentale, Università degli Studi di Genova, Via Leon Battista Alberti 2, Genova - 16132, Italy
e
Unità di Chirurgia Generale ad Indirizzo Oncologico, Università di Messina, Via Consolare Valeria 1, Messina - 98125, Italy
f
Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, Genova - 16132, Italy
g
Dipartimento di Protezionistica Ambientale, Sanità Sociale ed Industriale, Università di Messina, Via Consolare Valeria 1, Messina - 98125, Italy
b
a r t i c l e
i n f o
Article history:
Received 12 January 2010
Received in revised form 1 March 2010
Accepted 10 March 2010
Available online 16 March 2010
Keywords:
Seroma
Breast neoplasms
Lymph
Lymphadenectomy
Interleukin-6
a b s t r a c t
Seroma is a frequent complication of breast cancer surgery, the etiology of which remains indefinite. It
represents a subcutaneous accumulation of fluid frequently reported after surgical procedures such as
axillary lymph node dissection.
Despite previous studies have associated seroma fluid to an inflammatory exudate, the surgical removal
of draining lymph nodes may indicate that seroma might not represent a mere exudate but rather an
accrual of lymph drained from tributary tissues. To verify this hypothesis, seromas were collected at different intervals of time in patients operated upon for axillary lymph node removal. Fluids were analyzed
in details by flow cytometry and biochemical assays for their cellular content and for their molecular
features and relevant cytokine content.
Lymphocytes and other peculiar blood mononuclear cells were present, while erythrocytes, platelets
and granulocytes were absent or extremely rare. The protein concentration resulted lower (median
64%) than in peripheral blood. However, specific proteins related to locoregional tissues resulted highly
concentrated (e.g. up to 500% for ferritin and 300% for lactate deydrogenase and exclusive presence of
interleukin-6) whereas all enzymes and proteins synthesized in the liver or other organs (e.g. alkaline
phosphatase, ALT, ␥GT, prealbumin, transferrin, ceruloplasmin, C3 and C4, ␣2 macroglobulin from liver;
apolipoproteins from liver and gut; amylase and lipase from pancreas) were represented in reduced
concentrations, thus ruling out that seroma proteins derive directly from blood serum. As a whole, this
comprehensive cytological and molecular analysis provided evidences that seroma is constituted by
serum ultrafiltrated-derived extracellular fluid of regions located upstream of removed lymph nodes. This
fluid is then enriched by proteins and cells collected in the drained regions. Remarkably, seroma fluids
collected in the same patient at different time points (up to 50 days following surgery) displayed similar
biochemical features, clearly indicating that fluid composition was not significantly affected by postsurgical locoregional flogosis. Finally, the period of seroma formation indicates that lymph accumulates
in the axillary region during the interval of time needed for afferent lymphatic vessels to re-anastomose
with the efferent ducts.
Therefore, seroma fluid represents a font of biological material suitable for investigating the biology of
breast cancer, healing tissues and lymph.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author at: Laboratory of Immunology and Biotherapy, University
of Messina, Italy. Tel.: +39 0 90 221 2040; fax: +39 0 90 221 2043.
E-mail address: [email protected] (G. Ferlazzo).
0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.imlet.2010.03.002
Afferent lymph stream moves from peripheral interstitial spaces
to the draining lymph node stations through lymphatic vessels [1].
Following lymph nodes, the efferent lymph moves trough lymphatic vessels to thoracic duct and finally to the venous circulation.
It is common notion that lymph from thoracic duct is character-
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68
E. Montalto et al. / Immunology Letters 131 (2010) 67–72
ized by a high concentration of lipids [2] derived from food intake
and absorbed by the gut, thus suggesting that lymph may be considered a continuous sampling of peripheral anatomic districts
[3,4].
Seroma is a subcutaneous accumulation of noninfected fluid,
frequently observed in certain medical conditions after abdominal
surgery [5] and axillary lymph node dissection for breast cancer [6].
The pathophysiology of seroma is largely unknown [as reviewed in
7]. Although seroma was originally considered the results of a pure
lymphatic obstruction, thus far most of the studies have suggested
that seroma is an essudate, with specific characteristics largely
superimposable to those defined by Light rules [8–13]. Indeed,
most of these studies analyzed seroma fluid content a few days
following surgery, when a strong inflammatory component in the
axillary region is conceivable. Nevertheless, the surgical removal
of axillary lymph nodes corresponds to a physical interruption of
lymphatic vessels draining lymph from interstitial spaces: in this
context, subcutaneous accumulation of sterile seroma fluid could
also be considered as an accumulation of afferent lymph in the
absence of a guided lymph flow to locoregional lymph nodes. In the
case of seroma consequent to axillary lymph node dissections, the
district drained by sliced lymphatic vessel was hosting a breast cancer, with all the possible variation of protein composition related
to different cancer stages, different inflammation (including postsurgical flogosis), different surgical procedures and, in some cases,
neo-adjuvant therapies.
Very few papers have described the characteristics of afferent lymph: in an animal model [14], it has been shown that
lymph of pre-mammary gland contained 7, 6, and 10 times less
protein, albumin, and globulin, respectively, than plasma. Glucose concentrations were equivalent and in lymph, only 6,9%
of serum cholesterol and 50% of triglyceride and calcium were
found.
Gamma-glutamyl-transaminase and aspartate transaminase
were substantially higher in plasma than in lymph. Thus, substantial differences are evident when plasma is compared to lymph. In
another studies in humans [15], it has been described that afferent lymph had selective features, in particular the concentration
of protein was significantly lower than in plasma (<25%) and specific molecules (i.e. interleukin (IL)-6 and IL-8) were extremely well
represented in lymph and virtually absent in human plasma.
In this report, we describe a series of biochemical and cellular characteristics of seroma fluid collected in women operated
upon breast cancer and provide evidences strongly favouring the
hypothesis that this fluid represents afferent lymph, a biological
fluid otherwise extremely difficult to investigate in humans.
2. Materials and methods
2.1. Patients
Eleven seroma samples were collected in seven different
patients operated upon breast cancer and removal of axillary lymph
nodes. Seroma was collected using needle aspiration between days
15 and 50 after axillary dissection. In three patients more than 1
sample was obtained at 1–2-week interval, while in other patients
only one sample was obtained. Aliquots of seroma were used to
identify cell subsets present in the fluid. The remaining fluid was
immediately centrifuged, cell pellet was removed and aliquots of
supernatants were stored frozen at −80 ◦ C until analysed. In all
patients, a sample of serum was obtained at the first collection
of seroma fluid. An informed consent to the study of the biological characteristics of the fluid was given by all patients while no
ethical committee permission was required because the draining
procedure is routinely performed in the treatment of seroma.
2.2. Cytologic analysis
The population of cells present in the seroma fluid was studied using flow cytometry. Briefly, samples were analysed on a
FACScanto II (BD, Montaing View, CA) and the percentage of granulocytes, lymphocytes and monocytes were calculated using a linear
forward scatter and side scatter dotplot after electronic removal
of debris. Anti-CD3 (T lymphocytes), -CD14 (monocytes), -CD15
(granulocytes) monoclonal antibodies were also employed for the
analyses.
2.3. Cytokine assay
Cytokines were evaluated in both seromas and autologous sera
by flow cytometry employing a fluorescent bead immunoassay
(FlowCytomix, Bender MedSystems, Vienna, Austria) according to
manufacturer instructions.
2.4. Biochemical analysis
The following laboratory tests were performed in all seroma
samples. Total protein, albumin, total immunoglobulin A (IgA), total
immunoglobulin G (IgG), total immunoglobulin M (IgM), Alpha1
antitrypsin (AAT), aptoglobin (APG), apo-liproprotein A1 (APOA1),
Apolipoprotein B (APOB), ferritin, transferrin, alpha1 acid glycoprotein (A1AG), prealbumin, ceruloplasmin, Complement-C3 (C3),
Complement-C4 (C4), were performed using a turbidimetric assay.
Alkaline phosphatase (ALP), aspartate-leucine transferase (ALT),
aspartate amino transferase (AST), lactate dehydrogenase (LDH),
gamma-glutamyl transpeptidase (GGT), creatine kinase (CK), lipase
and amylase were assayed on the same instruments using optimized enzyme kinetics. Ions chloride (Cl), potassium (K), sodium
(Na), bicarbonate, phosphor (P) and calcium (Ca) were assayed
using a potenziometric approach. Nitrogen, glucose, total bilirubin,
triglycerides and cholesterol were assayed using enzymatic kinetics. All these tests were performed on Cobas 800 Roche analyser
using the protocol for serum and accordingly to the producer’s recommendations. Capillary electrophoresis for the identification of
serum fractions was performed on a Sebia Hydrasis. Fibronectin
and alpha2 macroglobulin (␣2M) were assayed on a Siemens BNII
nephelometer. Finally beta2 microglobulin (␤2M), was assayed on
a Siemens Immulite 2000 using chemiluminescence. The same protocols were also used to detect the presence of all analytes on
autologous serum samples.
3. Results
In this study, we observed that seroma fluids were, in some
cases, still accumulating 45 days after axillary lymph node dissection, i.e. when surgical wound healing had been fully reached and
no sign of local inflammation was present. These evidences support
the notion that seroma fluids is not necessarily associated to an
inflammatory exudate secondary to flogosis of the axillary region
caused by the surgical procedure.
The cellular component of seroma fluid was studied using flow
cytometry and immunophenotyping. Fig. 1 shows the results of
a representative analysis. The percentage of lymphocytes ranged
between 58.4 and 93.8% (median value 84.7%), the percentage
of CD14+ monocytes and other CD3− CD19− large mononuclear
cells ranged between 3.5 and 20.5% (median value 7.5%) and
the percentage of granulocytes ranged between 0.1 and 44.0%
(median value 0.9%). These results were different from results
obtained analyzing the same populations in peripheral blood,
which were, as expected, 20–30% for lymphocytes, 2–8% for
CD14+ monocytes and 55–70% for granulocytes (Fig. 1). Remarkably, CD14+ monocytes of peripheral blood are substituted in
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E. Montalto et al. / Immunology Letters 131 (2010) 67–72
69
Fig. 1. Cytofluorimetric analysis of cells derived from a seroma and peripheral blood of a representative patient. Gra90ent in seroma and well represented in blood. On the
contrary, lymphocytes (L) are well represented in both samples. CD14 positive monocytes (M) of peripheral blood are substituted, in seromas, by cells with heterogenous
physical characteristics (forward scatter and side scatter) only partially expressing CD14.
seroma by cells with more heterogenous physical characteristics (flow cytometric forward scatter and side scatter) and only
partially expressing CD14. Lymphocyte subsets in seroma displayed a peculiar composition as well, being T lymphocytes ranging
between 79 and 95% (median value 87.1), B cells from 1.5 to 12.8%
(median value 4.4) and NK cells from 1.4 to 6.6% (median value
2.3).
Tables 1 and 2 show the results of the biochemical analysis of
11 seroma fluids analyzed in this study. Of note, the concentrations of ions was largely superimposable to those observed in the
peripheral blood. The presence of a physiologic osmolarity is in
agreement with the cytometric evidence of living cells with intact
physical properties in the seromas.
The average protein concentration in seromas were 64% (range
52–76%) of serum proteins. When these proteins were fractionated and studied using capillary electrophoresis, it was evident that
the proteins belonging to the seroma had a highly similar composition of serum proteins, even if more diluted. Indeed, while
the percentages were very similar, the concentration of different
fractions was lower than that observed in patients’ sera. As far as
immunoglobulins concerns, electrophoretic data were supported
also by immunometric quantization of these proteins: indeed,
IgG and IgA were more diluted in seroma (55 and 28% of serum
counterpart) than expected on the basis of serum proteins (64%,
see before), while IgM were much more diluted (18% of serum
concentration). Along this line, small molecules (such as nitrogen, glucose, bilirubin, triglycerides, cholesterol) were only slightly
modified respect to serum level, while large proteins (represented
by AAT, APG, APOA1, APOB, transferrin, A1AG, prealbumin, ceruloplasmin, C3, C4, ␣2M and fibronectin) resulted as diluted as total
serum proteins. Of note, ␤2M (272% of serum counterpart), ferritin (523%), and the enzymes CK (149%), LDH (302%) and AST
(92%), all belonging to the proteins strictly related to the “damaged” or inflamed breast tissue following a surgical operation, were
significantly increased, especially if considered the total protein
dilution consistently observed in seroma fluids. These data are
particularly relevant because other biomarkers, such as lipase, spe-
cific of pancreas (47%) and ALT, mainly derived from liver (28%),
were as diluted as expected on the basis of total protein dilution
in seroma. Remarkably, as shown in Fig. 2, there was a significant inverse correlation between the molecular weight of proteins
and the concentration of these proteins in the seroma fluid, thus
suggesting that a specific mechanism of “physical” filtration was
operative throughout seroma fluid accumulation. Nevertheless,
proteins and enzymes derived from peripheral tissues (␤2M, ferritin, CK, LDH and AST) resulted highly concentrated in seroma
fluids.
In addition, in 3 patients, seroma samples were collected
at least twice at interval of time of 10–20 days (3 samples
collected in pt5 and 2 samples in pt6 and pt7). Remarkably,
fluid compositions were highly comparable when collected from
the same patient either 1 week after surgery or 40 days after
surgery (see Tables 1 and 2). This evidence further supports
Fig. 2. Correlation between seroma protein concentrations and their molecular
weight. On horizontal axis, the log of molecular weight. On the vertical axis, the
percent variation of protein concentration. The line represents the observed serum
values. Five proteins resulted more concentrated than predicted on the basis of the
exponential correlation line: (A) ferritin, (B) ␤2 microglobulin, (C) lactate dehydrogenase, (d) creatine kinase, and (e) aspartate amino transferase.
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E. Montalto et al. / Immunology Letters 131 (2010) 67–72
Table 1
Ions and low molecular weight molecules contained in seromas and comparison with related sera (% of serum concentration). PT: patient.
Chlorine
Potassium
Sodium
Calcium
Phosphor
Bicarbonate
Nitrogen
Glucose
Bilirubin
Triglycerides
Cholesterol
PT1
PT2
PT3
PT4
PT5.1
PT5.2
108
4.61
142
4.0
3.72
30.9
56
91
0.59
32
101
107
3.64
143
3.5
3.66
35.7
40
99
0.36
24
65
96.9
3.71
125
3.6
3.59
30.1
37
78
0.51
19
59
106
3.6
139
3.7
3.6
27.9
40
115
0.36
28
83
101
3.57
134
3.8
3.53
32.7
38
74
0.82
35
97
104
3.54
140
3.9
3.7
36.3
47
78
0.5
25
105
PT5.3
99.9
3.51
133
4.0
3.34
23.8
41
84
0.47
20
95
Fig. 3. Levels of IL-6 detectable in seromas but not in the sera of the same patients.
Data shown represent median values of results obtained in nine patients analyzed.
the notion that seroma content is not strictly related to postsurgical inflammation in the axillary region, which is expected
in the early days following surgery. Finally, we evaluated the
content of IL-6 in the seroma since this cytokine has been
reported to be highly concentrated in human lymph but not
in the serum of the same donors [15]. As shown in Fig. 3,
we found high level of IL-6 (median value 825 pg/mL, range
5800–514 pg/mL) but not of IL1␤ and IL-12 (the latter not shown),
two other prototypical inflammatory lymphokines released by activated monocytes/macrophages. As previously reported [15], these
cytokines were present in very limited amount, if any, in the sera
of the same patients.
4. Discussion
The composition of seroma fluid has been rarely described in
published reports and considered as an adverse effect to be avoided
by therapeutic strategies [16]. Nevertheless, the importance of
defining accurately the origin of seroma fluid in a pathophysiology context is also paralleled by the major interest that such a
fluid has in clinical pathology. Indeed, in breast cancer, the origin
of the fluid itself (anatomical regions including tissues that gave
shelter to neoplastic cells) is particularly exciting because both specific biomarkers and immuno-competent cells possibly primed by
the autologous tumor could be detected: already in 1990, interest in this fluid has been raised and the “potential benefit of an
immunotherapeutic approach” of cells derived from seroma [17]
was suggested.
In a recent paper, the composition of seroma fluid as well as
the composition of lymph have been reported, suggesting that
the serous fluid formed under the flap in the early post-operative
period after an abdominoplasty is reminiscent of an inflamma-
PT6.1
PT6.2
PT7.1
PT7.2
Average
% of serum
concentration
Units
110
3.89
141
3.9
3.73
28.3
31
73
0.72
22
114
109
4.11
141
4.0
4.12
27.6
35
72
0.62
14
127
103
3.09
140
4.0
4.13
27.2
49
45
0.88
47
116
93.8
3.02
125
4.1
3.96
27.1
39
70
0.83
33
114
103.5
3.7
136.6
3.9
3.7
29.8
41.2
79.9
0.6
27.2
97.8
0.99
0.85
0.98
0.84
0.79
1.32
1.50
1.32
1.21
0.29
1.09
mEq/L
mEq/L
mEq/L
mEq/L
mEq/L
mEq/L
mg/dL
mg/dL
mg/dL
mg/dL
mg/dL
tory exudate but subsequently it slowly turns into a fluid with
characteristics similar to those of lymph [5]. Nevertheless, in opposition to our study, this previous report analyzed seroma collected
only at early intervals of time, which therefore did not allow a
final assessment of seroma characteristics in the absence of postsurgical inflammation.
Our study clearly showed that ions as well as small molecules
had a concentration largely superimposable to that of plasma, while
larger molecules (such as proteins with immunological, or enzymatic activities) were characterized by a concentration that was
inversely related to the molecular weight, suggesting a mechanism in seroma formation which acts partially excluding largest
molecules. In this context, immunoglobulins had a prototypic
behavior, because IgG, IgA and IgM are synthesized by plasmacells in virtual any district of the body but the concentration of
IgG (the smallest Ig) was 55% (similar to total protein dilution, corresponding to 64%), IgA had an intermediate molecular weight and
an intermediate concentration (28% of IgA in plasma) and IgM, the
heaviest immunoglobulin were significantly diluted (18% of IgM in
plasma).
In addition, a number of proteins, represented by A1AG (related
to inflammation), LDH, AST and CK (related to lysis of all human
cells, including muscle cells), ferritin (the tissutal iron depot) and
␤2M (related to the proliferation or lysis of cells expressing HLAclass I molecules, i.e. again all human nucleated cells), appeared
concentrated in the seroma (from the same concentration of plasma
for AST to a 5-fold increase for ferritin). On the contrary, other
molecules, produced in specific districts such as ALT, ALK, GGT,
prealbumin, AAT, APT, transferrin, ceruloplasmin, C3 and C4, ␣2M
(from liver); apolipoproteins (from liver and gut); amylase and
lipase (from pancreas) and fibronectin (stromal cells) and therefore not related to the post-surgical tissue damage and remodeling,
resulted as diluted as total proteins, even if a gradient related to
the molecular weight was evident. These results clearly indicate
that seroma content is not directly derived from plasma but it is
rather representative of the protein repertoire of related anatomical regions.
Electrophoretic analysis did not add any significant information
regarding the nature of seroma proteins, being the different fractions diluted but present in proportions superimposable to serum.
On the other hand, also cellular analysis, represented almost exclusively by mononuclear cells, supports the hypothesis that seroma
fluid is an accumulation of afferent lymph. Further supporting this
hypothesis, the presence of large mononuclear cells only partially
expressing CD14 is reminiscent of “veiled cells” detectable in afferent lymph [18]. Also, the finding of extremely high levels of IL-6
in seroma is strongly suggestive for an accumulation of afferent
lymph. Indeed, this cytokine has been reported to be specifically
contained in human afferent lymph, but not in the autologous
serum [15]. IL-6 consistently detectable in seroma may derive from
tissue stromal cells, which abundantly release this cytokine [1]
Total proteins
Albumin
IgA
IgG
IgM
Alpha-1 antitrypsin
Aptoglobin
Apolipoprotein A1
Apolipoprotein B
Ferritin
Transferrin
Alpha1 acid
glycoprotein
Prealbumina
Ceruloplasmin
Complement-C3
Complement-C4
Fibronectin
Alpha2
macroglobulin
Beta2
microglobulin
Alkaline
phosphatase
Aspartate-leucine
transferase
Aspartate amino
transferase
Lactate
dehydrogenase
Gamma-glutamyl
transpeptidase
Creatine kinase
Lipase
Amylase
Albumin
(electrophoresis)
Alpha1
(electrophoresis)
Alpha2
(electrophoresis)
Beta1
(electrophoresis)
Beta2
(electrophoresis)
Gamma
(electrophoresis)
39
6
14
405
5
67
8
32
63.1
4.4
6.3
7.9
3.6
14.7
54
4
22
801
8
57
18
61
61.3
4.8
8.9
7.5
3.2
14.3
1.81
14
12
41
5
11.8
39
11
16
57
11
32.9
122
3.58
3.93
2344
70
528
34
80
42
49
16
255
160
67
PT2
5.3
2960
92
702
56
137
111
96
23
713
154
72
PT1
14.2
3.1
6.6
6
4.9
55
14
30
65.2
6
146
9
2
21
1.89
15
9
29
7
8.6
28
3.81
2421
88
480
29
69
29
43
14
195
130
76
PT3
10.9
3.8
7.8
6.6
5.2
32
6
16
65.7
6
395
12
2
33
3.26
14
11
35
5
20.2
38
3.6
2282
77
350
15
101
22
39
30
315
136
58
PT4
13.1
4
6.3
9.7
5.7
294
11
31
61.2
9
706
20
5
90
2.81
11
14
58
8
13.2
92
4.39
2502
77
570
14
107
106
72
30
623
141
80
PT5.1
11.9
3.6
7.7
8.3
5
258
10
35
63.5
8
546
19
3
41
2.68
14
14
50
6
24.4
72
4.73
2771
65
576
11
113
92
82
26
906
156
78
PT5.2
12.4
3.7
7.3
8.1
4.3
184
9
33
64.2
7
423
13
4
25
2.34
13
14
47
6
20.3
66
4.33
2594
63
528
10
94
69
76
23
725
146
73
PT5.3
Table 2
Protein content and electrophoretic results of seromas and comparison with related sera (% of serum concentration).
7.7
2.9
6.6
8.4
5.8
85
16
48
68.6
12
394
14
4
33
3.1
17
14
47
12
12.8
50
4.24
2706
28
289
12
109
56
74
29
513
167
95
PT6.1
7.5
2.3
7.1
6.9
4.8
64
17
48
71.4
11
451
14
4
40
3.02
17
15
45
11
18.9
52
4.61
2934
29
300
13
106
55
82
32
755
176
84
PT6.2
12.6
2.7
6.7
8.3
5.1
75
41
58
64.6
11
1229
25
9
41
2.46
18
16
62
13
9.7
68
5.1
2911
74
581
38
104
109
79
38
590
174
99
PT7.1
12.8
2.6
7.1
7.8
4.9
60
38
70
64.8
10
808
16
4
20
2.93
17
17
49
10
14.6
57
5.05
3020
70
574
34
83
70
71
32
739
175
97
PT7.2
12.0
3.2
7.1
7.8
5.0
112
17
42
64.9
8
573
16
4
40
3
15
14
47
9
17
62
4.5
2677
67
498
24
100
69
69
27
575
156
80
Average
0.80
0.67
1.20
0.82
1.28
1.49
0.47
0.84
1.06
0.42
3.02
0.92
0.24
0.49
2.72
0.68
0.31
0.34
0.33
0.71
0.27
0.64
0.70
0.28
0.55
0.18
0.85
0.68
0.47
0.30
5.23
0.55
1.08
% of serum
concentration
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%
%
%
%
U/L
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%
U/L
U/L
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U/L
U/L
mg/dL
mg/dL
mg/dL
mg/dL
mg/dL
mg/dL
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g/dL
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Author's personal copy
E. Montalto et al. / Immunology Letters 131 (2010) 67–72
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Author's personal copy
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E. Montalto et al. / Immunology Letters 131 (2010) 67–72
and then collected through lymphatic vessels in the axillary cavity. On the other hand, the macrophages present within seroma
might represent another source of IL-6 but this is unlikely since
both IL-1␤ and IL-12, other cytokines actively secreted by activated
macrophages, were barely or not measurable in seroma fluids.
On the basis of these results, it seems likely that seroma fluid,
collected in breast cancer patients following removal of the draining lymph nodes of the axillary chains, represents a continuous
sampling of the extracellular fluid from upstream tissues through
afferent lymphatic vessels. These vessels are interrupted by surgery
and some weeks are necessary for draining lymphatic network
reconstitution. Indeed, it has been recently described in an animal
model that a period of 6–8 weeks is needed for afferent lymphatic
vessels to re-anastomose with the efferent duct following surgical removal of draining lymph nodes [19]. Meanwhile, fluid leaking
from lymphatic vessels should accumulate in the region of lymph
node removal.
This accumulation can last for several weeks and, during this
period, sliced lymphatic vessels continuously drain the interstitial
fluids from locoregional tissues. This hypothesis is supported by
the evidence that all the enzymes and proteins synthesized in the
liver or other organs, are represented in reduced concentrations,
whereas only proteins that can be derived from drained tissues are
concentrated in the seroma. In previous reports, seroma has been
considered an inflammatory exudate consequent to an increased
blood vessel permeability. If this would be the case, we should
expect all plasma proteins equally represented in the fluid, without
the specific segregation we are here reporting. In addition, in those
previous studies, seroma was classified as an exudates using either
the original Light’s criteria [8] or the criteria by Heffner et al. [20].
Nevertheless, these criteria were established for differentiating trasudates (e.g. fluid accumulation related to left ventricular failure,
or cirrhosis) from exudates (fluid accumulation related to inflamed
tissues because of increased blood vessel permeability). Therefore, it is unlikely that these criteria can be useful to differentiate
an exudate from lymph. Indeed, lymph is defined as the extracellular fluid produced continuously by filtration from the blood,
enriched with peripheral tissue catabolites, cells and debris, collected in afferent lymphatic vessels and then conveyed into lymph
nodes, efferent vessels and finally circulating blood [1]. Therefore,
lymph composition will differ according to anatomical origin and
the pathophysiology of the drained tissue. In breast cancer patients,
where draining lymph nodes have been removed, lymph vessels
should leak the fluid, i.e. lymph, which they physiologically transport. Cells belonging to the drained tissues discharge molecules
(such as enzymes and other tissue or disease related biomarkers)
that mixed with the original plasma-derived ultrafiltrate, can be
harvested in seromas.
In conclusion, seroma fluid has a cellular and protein composition that supports the hypothesis that seroma is constituted
by accumulation of afferent lymph. This fluid can be useful not
only to evaluate the presence of residual molecules specific of
the surgically removed cancer tissue, but also to obtain cells with
special immunological features, for attempting the discovery of
novel biomarkers belonging to inflamed tissue, surgical wound and
locoregional immune response.
Acknowledgments
Research in our laboratories is supported by: Associazione
Italiana per la Ricerca sul Cancro (AIRC); Fondazione Banco di
Sicilia; Ministero Italiano della Salute, Progetto Strategico Oncologia; Regione Sicilia, Ricerca Sanitaria Regionale 2007; Regione
Liguria, Ricerca Sanitaria Regionale 2008.
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