Heart Failure Reviews

, Volume 18, Issue 6, pp 673–681 | Cite as

Classification and histological, immunohistochemical, and molecular diagnosis of inflammatory myocardial disease

  • Cristina Basso
  • Fiorella Calabrese
  • Annalisa Angelini
  • Elisa Carturan
  • Gaetano Thiene


In the WHO 1996 classification of cardiomyopathies, myocarditis is defined as an “inflammatory disease of the myocardium associated with cardiac dysfunction” and is listed among “specific cardiomyopathies”. Myocarditis is diagnosed on endomyocardial biopsy (EMB) by established histological, immunological, and immunohistochemical criteria, and molecular techniques are recommended to identify viral etiology. Infectious, autoimmune, and idiopathic forms of inflammatory cardiomyopathy are recognized that may lead to dilated cardiomyopathy. According to Dallas criteria, myocarditis is diagnosed in the setting of an “inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes, not typical of ischemic damage associated with coronary artery disease”. The majority of experts in the field agree that an actual increase in sensitivity of EMB has now been reached by using immunohistochemistry together with histology. A value of >14 leukocytes/mm2 with the presence of T lymphocytes >7 cells/mm2 has been considered a realistic cut off to reach a diagnosis of myocarditis. The development of molecular biological techniques, particularly amplification methods like polymerase chain reaction (PCR) or nested-PCR, allows the detection of low copy viral genomes even from an extremely small amount of tissue such as in EMB specimens. Positive PCR results obtained on EMB should always be accompanied by a parallel investigation on blood samples collected at the time of the EMB. According to the recent Association for European Cardiovascular Pathology guidelines, optimal specimen procurement and triage indicates at least three, preferably four, EMB fragments, each 1–2 mm in size, that should immediately be fixed in 10 % buffered formalin at room temperature for light microscopic examination. In expected focal myocardial lesions, additional sampling is recommended. Moreover, one or two specimens should be snap-frozen in liquid nitrogen and stored at −80 °C or alternatively stored in RNA-later for possible molecular tests or specific stains. A sample of peripheral blood (5–10 ml) in EDTA or citrate from patients with suspected myocarditis allows molecular testing for the same viral genomes sought in the myocardial tissue.


Cardiomyopathy Endomyocardial biopsy Histology Myocarditis Molecular pathology Pathology Viruses 


Conflict of interest

The authors have no conflicts of interest or financial ties to disclose.


  1. 1.
    Richardson P, McKenna WJ, Bristow M, Maisch B, Mautner B, O’Connell J, Olsen E, Thiene G, Goodwin J, Gyarfas I, Martin I, Nordet P (1996) Report of the 1995 WHO/ISFC task force on the definition of cardiomyopathies. Circulation 93:841–842PubMedCrossRefGoogle Scholar
  2. 2.
    Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, Moss AJ, Seidman CE, Young JB (2006) Contemporary definitions and classification of cardiomyopathies. An American Heart Association Scientific statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; quality of Care and Outcome research and Functional Genomics and Translational Biology Interdisciplinary Working Groups: and Council on Epidemiology and Prevention. Circulation 113:1807–1816PubMedCrossRefGoogle Scholar
  3. 3.
    Elliott P, Andersson B, Arbustini E, Bilinska Z, Cecchi F, Charron P, Dubourg O, Kühl U, Maisch B, McKenna WJ, Monserrat L, Pankuweit S, Rapezzi C, Seferovic P, Tavazzi L, Keren A (2007) Classification of the cardiomyopathies: a position statement from the European Society of Cardiology working group on myocardial and pericardial disease. Eur Heart J 29:270–276PubMedCrossRefGoogle Scholar
  4. 4.
    Aretz HT, Billingham ME, Edwards WD, Factor SM, Fallon JT, Fenoglio JJ Jr, Olsen EG, Schoen FJ (1987) Myocarditis: a histopathologic definition and classification. Am J Cardiovasc Pathol 1:3–14PubMedGoogle Scholar
  5. 5.
    Billingham ME (1989) Acute myocarditis: is sampling error a contraindication for diagnostic biopsies? J Am Coll Cardiol 14:921–922PubMedCrossRefGoogle Scholar
  6. 6.
    Edwards WD, Holmes DR, Reeders GS (1982) Diagnosis of active lymphocytic myocarditis by endomyocardial biopsy: quantitative criteria for light microscopy. Mayo Clin Proc 57:419–425PubMedGoogle Scholar
  7. 7.
    Edwards WD (1984) Myocarditis and endomyocardial biopsy. Cardiol Clin 2:647–656PubMedGoogle Scholar
  8. 8.
    Edwards WD (1985) Current problems in establishing quantitative histopathologic criteria for the diagnosis of lymphocytic myocarditis by endomyocardial biopsy. Heart Vessels 1:138–142CrossRefGoogle Scholar
  9. 9.
    Chow LH, Radio SJ, Sears TD, McManus BM (1989) Insensitivity of right ventricular endomyocardial biopsy in the diagnosis of myocarditis. J Am Coll Cardiol 14:915–920PubMedCrossRefGoogle Scholar
  10. 10.
    Hauck AJ, Kearney DL, Edwards WD (1989) Evaluation of postmortem endomyocardial biopsy specimens from 38 patients with lymphocytic myocarditis: implications for role of sampling error. Mayo Clin Proc 64:1235–1245PubMedCrossRefGoogle Scholar
  11. 11.
    Thiene G, Bartoloni G, Poletti A, Boffa GM (1996) Tecniche, indicazioni ed utilità della biopsia endomiocardica. In: Baroldi G, Thiene G (eds) Biopsia endomiocardica—Testo atlante. Edizioni Piccin, Padova, pp 5–45Google Scholar
  12. 12.
    Burke AP, Farb A, Robinowitz M, Virmani R (1991) Serial sectioning and multiple level examination of endomyocardial biopsies for the diagnosis of myocarditis. Mod Pathol 4:690–693PubMedGoogle Scholar
  13. 13.
    Frustaci A, Bellocci F, Osen EG (1994) Results of biventricular endomyocardial biopsy in survivors of cardiac arrest with apparently normal hearts. Am J Cardiol 74:890–895PubMedCrossRefGoogle Scholar
  14. 14.
    Calabrese F, Angelini A, Carturan E, Thiene G (2006) Myocarditis and inflammatory cardiomyopathy: histomorphological diagnosis. In: Schultheiss HP, Kapp JF, Grotzbach G (eds) Chronic viral and inflammatory cardiomyopathy. Springer, Berlin, pp 305–321Google Scholar
  15. 15.
    Hauck AJ, Edwards ED (1992) Histopathologic examination of tissues obtained by endomyocardial biopsy. In: Fowles RE (ed) Cardiac biopsy. Futura, Mount Kisco, pp 95–153Google Scholar
  16. 16.
    Linder J, Cassling RS, Rogler WC, Wilson JE, Markin RS, Sears TD, McManus BM (1985) Immunohistochemical characterization of lymphocytes in uninflamed ventricular myocardium. Implications for myocarditis. Arch Pathol Lab Med 109:917–920PubMedGoogle Scholar
  17. 17.
    Schnitt SJ, Ciano PS, Schoen FJ (1987) Quantitation of lymphocytes in endomyocardial biopsies: use and limitations of antibodies to leukocyte common antigen. Hum Pathol 18:796–800PubMedCrossRefGoogle Scholar
  18. 18.
    Tazelaar HD, Billingham ME (1987) Myocardial lymphocytes. Fact, fancy or myocarditis ? Am J Cardiovasc Pathol 1:47–50PubMedGoogle Scholar
  19. 19.
    Maisch B, Bultmann B, Factor S (1998) Dilated cardiomyopathy with inflammation or chronic myocarditis: variability and consensus in the diagnosis. Eur Heart J 19:647 (Abs)CrossRefGoogle Scholar
  20. 20.
    Kuhl U, Noutsias M, Seeber B, Shultheiss HP (1996) Immunohistological evidence for a chronic intramyocardial inflammatory process in dilated cardiomyopathy. Heart 75:295–300PubMedCrossRefGoogle Scholar
  21. 21.
    Angelini A, Crosato M, Boffa GM, Calabrese F, Calzolari V, Chioin R, Daliento L, Thiene G (2002) Active versus borderline myocarditis: clinicopathological correlates and prognostic implications. Heart 87:210–215PubMedCrossRefGoogle Scholar
  22. 22.
    Maisch B, Richter A, Sandmoller A, Maisch B, Richter A, Sandmoller A (2005) Inflammatory dilated cardiomyopathy (DCMI). Herz 30:535–544PubMedCrossRefGoogle Scholar
  23. 23.
    Angelini A, Calzolari V, Calabrese F, Boffa GM, Maddalena F, Chioin R, Thiene G (2000) Myocarditis mimicking acute myocardial infarction: role of endomyocardial biopsy in the differential diagnosis. Heart 84:245–250PubMedCrossRefGoogle Scholar
  24. 24.
    Herskowitz A, Ahmed-Ansari A, Neumann DA, Beschorner WE, Rose NR, Soule LM, Burek CL, Sell KW, Baughman KL (1990) Induction of major histocompatibility complex antigens within the myocardium of patients with active myocarditis: a nonhistologic marker of myocarditis. J Am Coll Cardiol 15:624–632PubMedCrossRefGoogle Scholar
  25. 25.
    Basso C, Carturan E, Corrado D, Thiene G (2007) Myocarditis and dilated cardiomyopathy in athletes: diagnosis, management, and recommendations for sport activity. Cardiol Clin 25:423–429PubMedCrossRefGoogle Scholar
  26. 26.
    Basso C, Calabrese F, Corrado D, Thiene G (2001) Postmortem diagnosis in sudden cardiac death victims: macroscopic, microscopic and molecular findings. Cardiovasc Res 50:290–300PubMedCrossRefGoogle Scholar
  27. 27.
    Basso C, Burke M, Fornes P, Gallagher PJ, de Gouveia RH, Sheppard M, Thiene G, van der Wal A, Association for European Cardiovascular Pathology (2008) Guidelines for autopsy investigation of sudden cardiac death. Virchows Arch 452:11–18PubMedCrossRefGoogle Scholar
  28. 28.
    Calabrese F, Thiene G (2003) Myocarditis and inflammatory cardiomyopathy: microbiological and molecular biological aspects. Cardiovasc Res 60:11–25PubMedCrossRefGoogle Scholar
  29. 29.
    Calabrese F, Carturan E, Thiene G (2010) Cardiac infections: focus on molecular diagnosis. Cardiovasc Pathol 19:171–182PubMedCrossRefGoogle Scholar
  30. 30.
    Baughman KL (2006) Diagnosis of myocarditis: death of Dallas criteria. Circulation 113:593–595PubMedCrossRefGoogle Scholar
  31. 31.
    Bowles NE, Richardson PJ, Olsen EG, Archard LC (1986) Detection of Coxsackie-B-virus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1:1120–1123PubMedCrossRefGoogle Scholar
  32. 32.
    Chapman NM, Tracy S, Gauntt CJ, Fortmueller U (1990) Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization. J Clin Microbiol 28:843–850PubMedGoogle Scholar
  33. 33.
    Hilton DA, Variend S, Pringle JH (1993) Demonstration of Coxsackie virus RNA in formalin-fixed tissue sections from childhood myocarditis cases by in situ hybridization and the polymerase chain reaction. J Pathol 170:45–51PubMedCrossRefGoogle Scholar
  34. 34.
    Jin O, Sole MJ, Butany JW, Chia WK, McLaughlin PR, Liu P, Liew CC (1990) Detection of enterovirus RNA in myocardial biopsies from patients with myocarditis and cardiomyopathy using gene amplification by polymerase chain reaction. Circulation 82:8–16PubMedCrossRefGoogle Scholar
  35. 35.
    Grasso M, Arbustini E, Silini E, Diegoli M, Percivalle E, Ratti G, Bramerio M, Gavazzi A, Vigano M, Milanesi G (1992) Search for Coxsackievirus B3 RNA in idiopathic dilated cardiomyopathy using gene amplification by polymerase chain reaction. Am J Cardiol 69:658–664PubMedCrossRefGoogle Scholar
  36. 36.
    Petitjean J, Kopecka H, Freymuth F, Langlard JM, Scanu P, Galateau F, Bouhour JB, Ferrière M, Charbonneau P, Komajda M (1993) Detection of enterovirus in endomyocardial biopsy by molecular approach. J Med Virol 41:260PubMedCrossRefGoogle Scholar
  37. 37.
    Martin AB, Webber S, Fricker FJ, Jaffe R, Demmler G, Kearney D, Zhang YH, Bodurtha J, Gelb B, Ni J (1994) Acute myocarditis. Rapid diagnosis by PCR in children. Circulation 90:330–339PubMedCrossRefGoogle Scholar
  38. 38.
    Pauschinger M, Bowles NE, Fuentes-Garcia FJ, Pham V, Kühl U, Schwimmbeck PL, Schultheiss HP, Towbin JA (1999) Detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction. Circulation 99:1348–1354PubMedCrossRefGoogle Scholar
  39. 39.
    Calabrese F, Valente M, Thiene G, Angelini A, Testolin L, Biasolo MA, Soteriou B, Livi U, Palù G (1999) Enteroviral genome in native hearts may influence outcome of patients who undergo cardiac transplantation. Diagn Mol Pathol 8:39–46PubMedCrossRefGoogle Scholar
  40. 40.
    Calabrese F, Rigo E, Milanesi O, Boffa GM, Angelini A, Valente M, Thiene G (2002) Molecular diagnosis of myocarditis and dilated cardiomyopathy in children. Clinico-pathologic features and prognostic implications. Diagn Mol Pathol 11:212–221PubMedCrossRefGoogle Scholar
  41. 41.
    Kuhl U, Pauschinger M, Noutsias M, Seeberg B, Bock T, Lassner D, Poller W, Kandolf R, Schultheiss HP (2005) High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 111:887–893PubMedCrossRefGoogle Scholar
  42. 42.
    Kühl U, Pauschinger M, Seeberg B, Lassner D, Noutsias M, Poller W, Schultheiss HP (2005) Viral persistence in the myocardium is associated with progressive cardiac dysfunction. Circulation 112:1965–1970PubMedCrossRefGoogle Scholar
  43. 43.
    Caforio AL, Calabrese F, Angelini A, Tona F, Vinci A, Bottaro S, Ramondo A, Carturan E, Iliceto S, Thiene G, Daliento L (2007) A prospective study of biopsy-proven myocarditis: prognostic relevance of clinical and aetiopathogenetic features at diagnosis. Eur Heart J 28:1326–1333PubMedCrossRefGoogle Scholar
  44. 44.
    Akhtar N, Ni J, Stromberg D, Rosenthal GL, Bowles NE, Towbin JA (1999) Tracheal aspirate as a substrate for polymerase chain reaction detection of viral genome in childhood pneumonia and myocarditis. Circulation 99:2011–2018PubMedCrossRefGoogle Scholar
  45. 45.
    Carturan E, Milanesi O, Kato Y, Giacometti C, Biffanti R, Thiene G, Calabrese F (2008) Viral detection and tumor necrosis factor alpha profile in tracheal aspirates from children with suspicion of myocarditis. Diagn Mol Pathol 17:21–27PubMedGoogle Scholar
  46. 46.
    Dunn JJ, Chapman NM, Tracy S, Romero JR (2000) Genomic determinants of cardiovirulence in coxsackievirus B3 clinical isolates: localization to the 5′ non-translated region. J Virol 74:4787–4794PubMedCrossRefGoogle Scholar
  47. 47.
    Höfling K, Kim KS, Leser JS, Chapman NM, Willian S, Gauntt CJ, Tracy S (2000) Progress toward vaccines against virus that cause heart disease. Herz 25:286–290PubMedCrossRefGoogle Scholar
  48. 48.
    Lam KM, Oldenburg N, Khan MA, Gaylore V, Mikhail GW, Strouhal PD, Middeldorp JM, Banner N, Yacoub M (1998) Significance of reverse transcription polymerase chain reaction in the detection of human cytomegalovirus gene transcripts in thoracic organ transplant recipients. J Heart Lung Transplant 17:555–565PubMedGoogle Scholar
  49. 49.
    Pauschinger M, Phan MD, Doerner A, Kuehl U, Schwimmbeck PL, Poller W, Kandolf R, Schultheiss HP (1999) Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis. Circulation 99:889–895PubMedCrossRefGoogle Scholar
  50. 50.
    Mahrholdt H, Goedecke C, Wagner A, Meinhardt G, Athanasiadis A, Vogelsberg H, Fritz P, Klingel K, Kandolf R, Sechtem U (2004) Cardiovascular magnetic resonance assessment of human myocarditis: a comparison to histology and molecular pathology. Circulation 109:1250–1258PubMedCrossRefGoogle Scholar
  51. 51.
    Donoso Mantke O, Meyer R, Prösch S, Nitsche A, Leitmeyer K, Kallies R, Niedrig M (2005) High prevalence of cardiotropic viruses in myocardial tissue from explanted hearts of heart transplant recipients and heart donors: a 3-year retrospective study from a German patients’ pool. J Heart Lung Transplant 24:1632–1638PubMedCrossRefGoogle Scholar
  52. 52.
    Schenk T, Enders M, Pollak S, Hahn R, Huzly D (2009) High prevalence of human parvovirus B19 DNA in myocardial autopsy samples from subjects without myocarditis or dilative cardiomyopathy. J Clin Microbiol 47:106–110PubMedCrossRefGoogle Scholar
  53. 53.
    Wang X, Zhang G, Liu F, Han M, Xu D, Zang Y (2004) Prevalence of human parvovirus B19 DNA in cardiac tissues of patients with congenital heart diseases indicated by nested PCR and in situ hybridization. J Clin Virol 31:20–24PubMedCrossRefGoogle Scholar
  54. 54.
    Stewart GC, Lopez-Molina J, Gottumukkala RV, Rosner GF, Anello MS, Hecht JL, Winters GL, Padera RF, Baughman KL, Lipes MA (2011) Myocardial parvovirus B19 persistence: lack of association with clinicopathologic phenotype in adults with heart failure. Circ Heart Fail 4:71–78PubMedCrossRefGoogle Scholar
  55. 55.
    De Salvia A, De Leo D, Carturan E, Basso C (2011) Sudden cardiac death, borderline myocarditis and molecular diagnosis: evidence or assumption? Med Sci Law 51(Suppl 1):S27–S29PubMedCrossRefGoogle Scholar
  56. 56.
    Bültmann BD, Klingel K, Sotlar K, Bock CT, Baba HA, Sauter M, Kandolf R (2003) Fatal parvovirus B19-associated myocarditis clinically mimicking ischemic heart disease: an endothelial cell-mediated disease. Hum Pathol 34:92–95CrossRefGoogle Scholar
  57. 57.
    Bock CT, Klingel K, Kandolf R (2010) Human parvovirus B19-associated myocarditis. N Engl J Med 362:1248–1249PubMedCrossRefGoogle Scholar
  58. 58.
    Chimenti C, Russo A, Pieroni M, Calabrese F, Verardo R, Thiene G, Russo MA, Maseri A, Frustaci A (2004) Intramyocyte detection of Epstein-Barr virus genome by laser capture microdissection in patients with inflammatory cardiomyopathy. Circulation 110:3534–3539PubMedCrossRefGoogle Scholar
  59. 59.
    Heidecker B, Kittleson MM, Kasper EK, Wittstein IS, Champion HC, Russell SD, Hruban RH, Rodriguez ER, Baughman KL, Hare JM (2011) Transcriptomic biomarkers for the accurate diagnosis of myocarditis. Circulation 123:1174–1184PubMedCrossRefGoogle Scholar
  60. 60.
    Park JH, Kim DS, Cho YJ, Kim YJ, Jeong SY, Lee SM, Cho SJ, Yun CW, Jo I, Nam JH (2009) Attenuation of coxsackievirus B3 by VP2 mutation and its application as a vaccine against virus-induced myocarditis and pancreatitis. Vaccine 27:1974–1983PubMedCrossRefGoogle Scholar
  61. 61.
    Pinkert S, Westermann D, Wang X, Klingel K, Dörner A, Savvatis K, Grössl T, Krohn S, Tschöpe C, Zeichhardt H, Kotsch K, Weitmann K, Hoffmann W, Schultheiss HP, Spiller OB, Poller W, Fechner H (2009) Prevention of cardiac dysfunction in acute coxsackievirus B3 cardiomyopathy by inducible expression of a soluble coxsackievirus-adenovirus receptor. Circulation 120:2358–2366PubMedCrossRefGoogle Scholar
  62. 62.
    Barry SP, Townsend PA, Latchman DS, Stephanou A (2007) Role of the JAK-STAT pathway in myocardial injury. Trends Mol Med 13:82–89PubMedCrossRefGoogle Scholar
  63. 63.
    Calabrese F, Carturan E, Chimenti C, Pieroni M, Agostini C, Angelini A, Crosato M, Valente M, Boffa GM, Frustaci A, Thiene G (2004) Overexpression of tumor necrosis factor (TNF) alpha and TNF alpha receptor I in human viral myocarditis: clinicopathologic correlations. Mod Pathol 17:1108–1118PubMedCrossRefGoogle Scholar
  64. 64.
    Leone O, Veinot JP, Angelini A, Baandrup UT, Basso C, Berry G, Bruneval P, Burke M, Butany J, Calabrese F, d’Amati G, Edwards WD, Fallon JT, Fishbein MC, Gallagher PJ, Halushka MK, McManus B, Pucci A, Rodriguez ER, Saffitz JE, Sheppard MN, Steenbergen C, Stone JR, Tan C, Thiene G, van der Wal AC, Winters GL (2012) 2011 consensus statement on endomyocardial biopsy from the Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology. Cardiovasc Pathol. 21:245–274Google Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Cristina Basso
    • 1
  • Fiorella Calabrese
    • 1
  • Annalisa Angelini
    • 1
  • Elisa Carturan
    • 1
  • Gaetano Thiene
    • 1
  1. 1.Pathological Anatomy, Department of Cardiac, Thoracic and Vascular SciencesUniversity of Padua Medical SchoolPaduaItaly

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