Demonstration
protocol for the anatomopathological study of
lymphatic vessels in lymphedema
(Portuguese
PDF version)
Claudia
Stein Gomes1, Fernando Silveira Picheth1, Ezio Fulcheri2,
Corradino Campisi3, Francesco Boccardo3
1.
Division of Angiology and Vascular Surgery , Hospital Santa Casa de
Misericórdia de
Curitiba - PUCPR, Brazil
2. Institute of Pathological Anatomy, San Martino Hospital,
University of Genoa, Italy
3. Surgery Division (DISCAT), Section of Emergency Clinical
Surgery, Center of Lymphology and Microsurgery, San Martino Hospital,
University of Genoa, Italy
Correspondence:
Dr. Claudia Stein Gomes
Rua Padre Anchieta, 2004/1302
CEP 80730-000 - Curitiba - PR
Brazil
Tel.: +55 (41) 335.2135
Fax: +55 (41) 322.9892
E-mail: steingomes@sulbbs.com
ABSTRACT
Objective:
To suggest a study protocol for lymphatic vessels in order to obtain
the data necessary to better understand their morphology and paraphysiological
alterations, as well as any clearly pathological manifestations
of the lymphatic circulation in the pathology of lymphedema, both
of primary and secondary origin.
Methods: The protocol begins with a material sample, which
can be taken from the fibrous-fatty tissue surrounding the lymphatic
vessels or from an isolated lymphatic collector. Afterwards, a series
of steps are followed, including the fixation and inclusion of the
material, the preparation of 11 glass slides with different histochemical
and immunohistochemical stainings, and finally, the reading of the
slides in search of alterations ranging from the lymphatic vessel
components to the periadventitial matrix.
Results: According to the prevailing cell group on the vessel
walls and the periadventitial matrix, an actinic reaction can be
suspected, such as, for example, in cases where there is excessive
fibrosis and regression of contractile fibers, or even in the case
of a postsurgical chronic lymphedema, when there is evidence of
a degenerative process of the lymphatic walls.
Conclusions: The creation of a protocol that provides a better
understanding of such a complex pathology is necessary in order
to perform an encompassing study of the lymphatic vessels in different
pathological anatomy laboratories.
Key-words:
lymphedema, immunohistochemistry, histology.
Palavras-chave: linfedema, imunohistoquímica, histologia.
J
Vasc Br 2003;2(4):313-17
Lymphedema
is a pathology characterized by an increase in the volume of soft tissues
in the affected region that can evolve into large deformities such as
in cases of elephantiasis. Lymphedema is caused by a lack of lymph transport
by the lymphatic vessels (congenital or idiopathic), or is secondary
to inflammatory, infectious, irradiative or surgical processes. The
disease affects a large number of patients, mainly after oncological
interventions or after inflammatory and/or infection processes.
Treatment of this pathology includes manual lymphatic drainage methods,
pressotherapy and elastic compression. Surgical treatment varies from
excision of skin and subcutaneous tissue to more thorough treatment,
with microsurgical techniques for lymphatic venous anastomosis between
lymphatic collectors and a competent vein with the aid of an operation
microscope 1.
Through microsurgery, performed at inguinal level for lower limbs and
at the brachial level for upper limbs, where there are pre and post
lymphatic collectors lymph nodes with diameters ranging from 0.5 to
1 mm, it becomes possible to perform anatomopathological studies of
the perilymphatic and lymphoid tissues. Normally, some histopathological
lesions of the lymphatic vessels are identified and described 2,
which are the basis for the different types of lymphedema. These are
certain constrictive and dystrophic modifications of the vessels, generically
classified as vascular wall fibrosis and increased periadventitial matrix,
which are interpreted as indirect signals and sequelae of acute or chronic
inflammatory reaction. Nevertheless, these lesions do not justify the
pathological basis of the different conditions, nor can they explain
the polymorphous or undetermined clinical stages.
The lymphatic vessels are essentially composed of an endothelium with
its valvular apparatus, of a generally fine wall and of the adventitia.
The vessel wall is composed of a deep layer of the intima and a medium
layer that consist of a cellular part (fibroblasts and smooth-muscle
cells) and a non-cellular part (elastic fibers, collagen and proteoglycans).
The vasa vasorum is found in the adventitial layer. All components of
lymphatic vessels are covered by a perivascular sheath and are primarily
responsible for lymph transport.
The anatomopathological study of lymphatic vessels is not easily performed,
once the lymphatic vessels are small-caliber structures. With common
histological stainings, such as hematoxylin-eosin, one can only observe
the fibrosclerotic alterations on the vessel walls, quantify the component
of the periadventitial matrix and look for the elements of inflammatory
reaction. From this viewpoint, the study is purely morphological. In
addition, the use of specific stains is necessary to allow the lymphatic
vessels to be identified and differentiated from blood vessels. The
endothelium of the lymphatic vessels does not produce a sufficient quantity
of coagulation factor VIII to be evaluated as a histological section
with immunohistochemical methods 3. Only after an adequate
lysis with proteolytic enzymes (collagenase or trypsin) is it possible
to uncover the antigenic sites and provide evidence for factor VIII
in the endothelium of the lymphatic vessels.
The objective of this study is to suggest a protocol for the study of
lymphatic vessels in order to obtain the data necessary for a wider
understanding of the morphology and the paraphysiological (compensating)
or frankly pathological (degenerative) alterations of the lymphatic
circulation in primary and secondary lymphedema.
Therefore, in order to understand the pathology of the lymphatic vascular
system, it is important that specialists shift from a morphological
study to a morphofunctional study, which provides evidence for the functional
characteristics of the vessel walls in terms of residual contractile
capacity or hypertrophic and hyperplastic reaction of the smooth-muscle
components.
METHOD
Sampling
The material obtained from the surgical intervention can be of two types:
an isolated segment from the lymphatic collector or some fibrous-fatty
tissue which surround the lymphatic vessels. The material should be
fresh and should arrive as quickly as possible to the anatomic pathology
laboratory. If possible, this material should also be marked with surgical
thread in one of its extremities to serve as an orientation for the
study.
The pathologist should maintain the material in a closed container with
neutral formalin 4 and avoid the cooptation and distortion
of lymphatic vessels
Fixation
The fixation should be brief, taking no more than 12 hours, to avoid
lesion to the antigenic sites.
Embedding
When dealing with a piece of fibrous-fatty tissue and lymphatic vessels,
paraffin embedding should be made after the material is cut into macroscopic
sections.
When dealing with a segment from the lymphatic collector, it should
be maintained in an erect position and embedded in agar 5
before being embedded in paraffin.
Slide
preparation
Routinely, 11 glass slides are prepared. These slides are stained in
the following manner:
- First slide: hematoxylin-eosin stain;
- Second slide: Masson's trichrome stain;
- Third slide: silver impregnation method for reticular fibers;
- Fourth slide: Weigert's elastic stain
- Fifth slide: Van Gieson stain;
- Sixth slide: immunohistochemical stain with smooth-muscle (antiactin)
antibodies;
- Seventh slide: immunohistochemical stain with antivimentin antibodies;
- Eight slide: immunohistochemical stain with antidesmin antibodies;
- Ninth slide: CD 31 stain;
- Tenth slide: CD 34 stain;
- Eleventh slide: hematoxylin-eosin stain.
Slide
Reading Analysis
The slide analysis is performed in order to make a quantitative and
distributive evaluation of the lymphatic vessel cells and the perilymphatic
tissue cells.
The first stains that are studied to identify the lymphatic vessel are
the CD 31 and CD 34 stains.
The endothelium of the blood vessels is selectively stained by the immunohistochemical
staining with the anti-CD 34 antibody, whereas the endothelium of the
lymphatic vessel is usually negative for this staining. However, the
endothelium of lymphatic and blood vessels is stained with the anti-CD
31 antibody 3,6.
Afterward, the morphological structure of the vessel is evaluated using
the hematoxilin-eosin stain: the diameter and thickness of the wall,
valves and periadventitial matrix (Figure 1) are all assessed. As for
the vessel lumen, it may be with a reduced, normal or increased caliber.
The wall may be fine, normal, thickened or fibrotic. The valvular apparatus
may be absent, normal or prominent and weakened. Finally, the periadventitial
matrix may be slightly, partially or fully evident (Figure 2). This
algorithm should serve as a preliminary guide in the microscopic observation
of the serial section, allowing for the observation of basic morphological
parameters. In such a way, the identification of signs of phlogosis,
when present in the lymphatic vessels, suggest an inflammatory lymphedema
2,7.
 Figure
1- Cross-section view of a lymphatic vessel. After identification of
the lymphatic vessel, the following are evaluated: (A) diameter, (B)
thickness of the wall, and (C) presence of valvular structure, as shown
by the arrows.
Figure
2-Cross-section view of a lymphatic vessel showing its (a) lumen with
endothelial cells, (b) wall with muscular fibers and (c) periadventitial
matrix, as shown by the arrows.
Using
the Masson, Weigert and Van Gieson trichrome processes, it is possible
to obtain a more refined structural evaluation of the vessels, including
an observation of the components of the intercellular matrix such as
collagen, elastic fibers and reticular fibers 2. Using
immunohistochemical techniques such as the Avidin Biotin Peroxidase
Complex (ABC) method, it is also possible to study the cellular part
of the lymphatic wall 2,3,6. With the antivimentin
antibody, it is possible to evidence the presence of fibroblasts, fibrocytes
and also smooth-muscle cells. Desmin only stains the myofibroblasts,
and smooth-muscle actin stains the myofibroblasts and also the smooth-muscle
cells of tunica intima and media. For the study of the smooth-muscle
cells present on the lymphatic vessel wall, observations are made in
terms of: quantity, whether it be average, scarce or increased; distribution
of fine bundles, whether they be large or fragmented; and typology,
whether it is be fragile, hypertrophic or dysplastic. Therefore, depending
on the predominant cell group in the vessel wall and in the periadventitial
matrix, a regression of contractile fibers may be suspected, as, for
example, in cases in which there is a high degree of fibrosis or chronic
postsurgical lymphedema resulting from the predominance of a degenerative
process of the lymphatic wall 2.
DISCUSSION
In medical
literature, there are few published works that refer to the study of
lymphatic vessels in peripheral lymphedema 2,7-12.
This results from the fact that there are no major surgical treatment
centers for this pathology where surgeons work in connection with anatomic
pathology laboratories.
The findings of the Center of Lymphology and Microsurgery of the University
of Genoa, Italy, which has a vast experience in the microsurgical treatment
of limb lymphedema through lymphatic venous anastomoses, allowed the
development of major anatomofunctional studies on lymphatic vessels,
which resulted from biopsies performed during surgical interventions
(as demonstrated in the numerous reports published by Campisi et al.
13-18).
Currently, there is even a classification proposed by these pathologists
from the research group of Genoa for the lymphatic and lymph node alterations
found in patients with secondary lymphedema who were submitted to microsurgical
treatment for lymphatic venous anastomosis 2. This
classification was proposed thanks to a adequate technique which allowed
the presentation of the diagnosis based on both the simple morphology
(diameter of the lymphatic vessels, presence of fibrosis or inflammatory
signals) and the functional morphology (evaluation of the contractility
and activity of the wall) of the lymphatic vessels.
In the near future, there is a possibility for the expansion of research
with a larger number of cases and with samples taken from other segments
of the affected limb. With such information, a detailed study of the
distinct features of this disease can be developed.
We believe that, in order to perform an ample study of the lymphatic
vessels in diverse anatomic pathology laboratories, a protocol must
first be created for a better understanding of this complex pathology.
REFERENCES
1.
Campisi C, Boccardo F. Linfedemas - Tratamento por técnicas microcirúrgicas.
In: Brito CJ, Duque A, Merlo I, Murilo R, Lauria F Fº, editores.
Cirurgia Vascular. Rio de Janeiro: Revinter; 2002. p. 1246-77.
2. Dellachà A, Fulcheri E, Boccardo F, Campisi
C. Patologie latenti dei vasi linfatici come possibili substrati del
linfedema cronico secondario. Linfologia 1998;2:20-4.
3. Culling CFA, Allison RT, Barr WT. Cellular Pathology
Technique. 4th ed. Woburn (MA): Butterworth-Heinemann; 1985.
4. Carson F, Martin JK, Lynn JA. Formalin fixation for
electron microscopy: a re-evaluation. Am J Clin Pathol 1973;49:365-73.
5. Ventura L, Bologna M, Ventura T, Colimberti P, Leocata
P. Agar specimen orientation technique revisited: a simple and effective
method in histopathology. Ann Diagn Pathol 2001;5(2):107-9.
6. Lapertosa G, Baracchini P, Fulcheri E, Tanzi R. Small
blood vessels or lymphatic channels with neoplastic microemboli: a comparative
immunohistochemical study. Verh Dtsch Ges Path 1986;70:358-64.
7. Badini A, Fulcheri E, Campisi C, Boccardo F. A new
approach in histopathological diagnosis of lymphedema: pathophysiological
and therapeutic implications. Lymphology 1996;29 Suppl :190-8.
8. Campisi C, Badini A, Boccardo F. Anatomo-pathological
bases in the management of primary lymphedema and microsurgical implications.
Lymphology 1994;27 Suppl :546-9.
9. Badini A, Fulcheri E. Vantaggi dell'immunoistochimica
nella diagnostica istopatologica del linfedema. Minerva Cardioangiol
1997;45:17-24.
10. Pfister G, Saesseli B, Hoffmann U, Geiger M, Bollinger
A. Diameters of lymphatic capillaries in patients with different forms
of primary lymphedema. Lymphology 1990;23(3):140-4.
11. Rada IO, Tudose N, Fedorac R. Fibrosclerosis of
tunica media in the prenodal lymphatic vessels of patient with lymphedema.
Morphol Embryol (Bucur) 1986;32(2):93-7.
12. Kinmonth JB, Wolfe JH. Fibrosis in the lymph nodes
in primary lymphoedema. Histological and clinical studies in 74 patients
with lower-limb oedema. Ann R Coll Surg Engl 1980;62:344-54.
13. Campisi C, Zattoni J, Siani C, et al. Twenty year
clinical experience in the microsurgery management of lymphedema. Lymphology
1994;27 Suppl :651-7.
14. Campisi C. Lymphatic microsurgery: legend or reality?
Phlebolymphology 1994;7:11-15.
15. Campisi C. The modern surgery of lymphedema. Lymphology
1996;29 Suppl :210-21.
16. Campisi C, Boccardo F. Frontiers in lymphatic microsurgery.
Microsurgery 1998;18:462-71.
17. Campisi C, Boccardo F. Role of microsurgery in
the management of lymphoedema. Int Angiol 1999;18(1):47-51.
18. Degni M. New techniques of lymphatic-venous anastomosis
for the treatment of lymphedema. J Cardiovasc Surg (Torino) 1978;19(6):577-80.
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