Issue 4 - August 2010 - Pacini Editore

168
Further elements to look for are:
a) inflammatory infiltrate types (lymphocytic, polymorphous,
granulomatous, eosinophilic) and extention using semiquantitative
or quantitative evaluation;
b) myocellular damage (not only necrosis or myocytolysis but
also other alterations, such as cytoplasmic vacuolization,
apoptosis, and atrophy);
c) pattern of fibrosis (interstitial, perivascular, subendocardial)
and its extent using semi-quantitative or quantitative evaluation.
Histo-morphological stains:
• Azan-Mallory trichrome is useful to highlight and quantify
fibrosis.
• Weigert-Van Gieson, which highlights elastic fibres in
brown/black, and allows for evaluation of vessel wall structure
and endocardial fibroelastosis.
Immunohistochemical tests: immunohistochemical analysis
represents a fundamental corollary to traditional histology
enabling the characterization of inflammatory infiltrate and,
when this is present in very small quantities, its identification.
Antibody panel to use: CD45, CD68/PGM1, CD3, CD4, CD8,
CD20, HLA-ABC, HLA-DR.
Morphometric quantification: it is desirable to quantify
inflammatory infiltrate (currently a lymphocytic infiltrate
> 7 T lymphocytes/mm 2 is considered as pathologic) using
computerized morphometry on immunohistochemical
sections stained with anti-CD3 antibody. Morphometry on
Azan-Mallory trichrome stained sections may allow precise
quantification of fibrosis.
Molecular tests and in particular techniques of gene amplification,
such as polymerase chain reaction (PCR) or nested-
PCR, because of their high sensitivity, allow the amplification
of viral DNA or RNA, thus detecting any viral genome
present in the small samples of EMB tissue. Nowadays,
sequence analysis and the identification of replicating virus
forms are increasingly utilized to characterize infective agents
precisely 5-9 . If myocarditis is clinically suspected, at least the
following most frequent cardiotropic virus genomes must be
checked for in myocardial tissue: enterovirus, adenovirus,
cytomegalovirus, Epstein Barr virus, herpes simplex virus, Influenza
A and B viruses; B19 parvovirus and C hepatitis virus.
In the setting of positive PCR results blood samples collected
at the time of the biopsy should be tested: if positivity for the
same virus is present in both myocardial tissue and blood, it is
necessary to quantify its load with quantitative PCR analysis
to exclude any haematic contamination of the myocardial
specimen. Gene sequencing is a more sophisticated technique
allowing the characterization of the infective agents as well as
its virulence and cardiotropism. Different serotypes can bear
a different virulence and cardiotropism and guide prognosis
and therapeutic interventions. However the detection of viral
genome does not necessarily imply a direct pathogenetic role,
since it could be an innocent by stander. Also a negative PCR
does not exclude viral disease. Final diagnosis of myocarditis
must be the results of an integrated clinical, instrumental,
morphological and molecular approach.
Key points
• The accurate diagnosis of myocarditis requires a representative
number of specimens to be subjected to complete
traditional histopathological, and immunohistochemical
(lymphocyte types, HLA, C3-C4) and molecular virological
(a study of the presence/persistence of RNA and DNA virus
genome) analysis.
5 th triennial congress of the italian society of anatomic Pathology and diagnostic cytoPathology
• The application of immunohistochemical methodologies
(which allows the identification of inflammatory infiltrates,
their more adequate quantification and the evaluation of
myocardial expression of immunological activation markers)
increases interpretative capacity, especially in cases
of prevalently autoimmune mechanism responsible for
“chronic myocarditis”.
• It is mandatory to apply molecular tests, especially gene
amplification techniques such as the Polymerase Chain
Reaction (PCR), quantitative (real time-PCR) or qualitative
(nested-PCR), which, nowadays, because of their high
sensitivity, allow the identification even of a small number
of copies of any viral genome present in the EMB.
• The exclusion of a viral aetiology is an essential requirement
in considering a myocarditis as immuno-mediated
(both antibody- and cell- mediated) and choosing the most
appropriate therapeutic strategy 8 9 .
• Etiological characterization is important also in assessing
prognosis.
references
1 Maron BJ, Towbin JA, Thiene G, et al. Contemporary definitions and
classification of the cardiomyopathies. An American Heart Association
Scientific statement from the Council on clinical cardiology, heart
failure and transplantation Committee; quality of care and outcomes
research and functional genomics and translational biology interdisciplinary
working Groups; and Council on epidemiology and prevention.
Circulation 2006;113:1807-16.
2 Aretz HT, Billingham ME, Edwards WD, et al. Myocarditis: a histopathological
definition and classification. Am J Cardiovasc Pathol
1987;1:3-14.
3 Angelini A, Crosato M, Boffa GM, et al. Active vs borderline myocarditis:
clinicopathological correlates and prognostic implications.
Heart 2002;87:210-5.
4 Calabrese F, Rigo E, Milanesi O, et al. Molecular diagnosis of myocarditis
and dilated cardiomyopathy in children. Clinico-pathologic
features and prognostic implications. Diagn Mol Pathol 2002;11:212-
21.
5 Calabrese F, Thiene G. Myocarditis and inflammatory cardiomyopathy:
microbiological and molecular biological aspects. Cardiovasc
Res 2003;60:11-25.
6 Calabrese F, Carturan E, Thiene G. Cardiac infections: focus on molecular
diagnosis. Cardiovascular Pathology 2010;19:171-182.
7 Caforio AL, Calabrese F, Angelini A et al. A prospective study of
biopsy-proven myocarditis: prognostic relevance of clinical and aetiopathogenetic
features at diagnosis. Eur Heart J 2007;28:1326-33.
8 Documento di consenso sulla biopsia endomiocardica promosso
dall’Associazione per la Patologia Cardiovascolare Italiana. G. Ital
Cardiol 2009;10(Suppl):1-9.
9 Kuhl U, Pauschinger M, Noutsias M, et al. High prevalence of
viral genomes and multiple viral infections in the myocardium of
adults with “idiopathic” left ventricular dysfunction. Circulation
2005;111(7):887-93.
Diagnostic and terapeuetic work-up for
cardiovascular diseases: the role of pathologists
E. Arbustini, M. Grasso, M. Diegoli, A. Agozzino, M. Concardi
Centre for Inherited Cardiovascular Diseases, Transplant Research
Area, IRCCS Foundation Policlinico San Matteo, Pavia, Italy
Background. The progression of knowledge on the pathologic
basis of cardiovascular diseases and the development of biotechnological
tools for pathological and molecular studies are
progressively increasing the number of specific vs. descriptive
diagnoses. Disease-specific diagnostic work-up tailored on
phenotypes (percorsi diagnostico-terapeutici assistanziali:
PDTA) constitute the tool; in this scenario pathology may play
a fundamental role for diagnosis, prognostic stratification,