SCVP myocarditis criteria and definitions

Large Specimen and Endomyocardial Biopsy Myocarditis Criteria

Welcome to the Society for Cardiovascular Pathology (SCVP) web tutorial on defining myocarditis on biopsies and large specimens (ventricular apex excisions, explanted heart or autopsy hearts). This web tutorial will serve as an adjunct to the consensus papers published in the literature.  These criteria are based upon a rigorous 2-year exploration of myocarditis focusing on reproducibility and clinical significance of histopathologic and immunohistopathologic findings.  This work was initiated due to clear gaps in our criteria for diagnosing myocarditis with inconsistency between institutions and clinician unhappiness with the “borderline” diagnosis.

Myocarditis is a combination of an inflammatory infiltrate and, often myocyte injury. While there are many types of myocarditis (e.g. giant cell, eosinophilic), these consensus criteria define lymphocytic myocarditis exclusively. Further, these criteria are strictly for diagnosing myocarditis in pathologic specimens, which can be used by clinicians to inform them of the correct approach to treat the patient.  Lastly, these criteria are designed to be 1) an advancement over the Dallas Criteria and the ESC criteria for endomyocardial biopsies and 2) the first ever histopathologic myocarditis criteria for large surgical specimens (ventricular apex excisions, explanted heart or autopsy hearts).

Biopsy Criteria

The endomyocardial biopsy criteria comprise 4 grades of myocarditis: no myocarditis, mild myocarditis, moderate myocarditis, and severe myocarditis. Each grade has a specific set of criteria that have to be determined for the correct diagnosis.

  • * Single-cell hypereosinophilia; nuclear karyorrhexis/karyolysis; sarcoplasmic membrane scalloping.

A case without myocarditis (grade 0) has no evidence of clusters of lymphocytes (≥ 5) on either an H&E or a CD3 immunohistochemical stain for T lymphocytes.  Further, there are not more than 15 CD3+ cells per 40x high powered field (hpf).

The diagnosis of mild myocarditis (grade 1) can be made in one of two ways.  The first (1a)  is to identify a single focus of an interstitial cluster of 5 or more lymphocytes noted by a CD3 or H&E stained slide. This cluster of cells should be within the myocardium proper, adjacent to a myocyte and/or myocytes, and not along the endocardium or in areas of loose connective tissue (adipose/fibrosis).  Further, the cluster of cells should not be in an area of dense fibrosis or scar.  Lymphocytes in those areas are not evaluated for myocarditis but can be reported as evidence of chronic inflammation in the biopsy.  The second approach (1b) to make the diagnosis of mild myocarditis is to identify 15 or more CD3+ cells in a single 40x hpf within the biopsy material.  These should be around myocytes and not in the endocardium, dense fibrosis, loose connective tissue or scar.  Ideally, there will be a general pattern of increased CD3+ cells above the baseline throughout the biopsy, but that is not strictly part of the criteria.  Myocardial injury, noted by single-cell hypereosinophilia, nuclear karyorrhexis/karyolysis, and/or sarcoplasmic membrane scalloping can be present.  If present, it should be reported. Myocardial injury is not required for this diagnosis.

Moderate myocarditis (grade 2) is diagnosed by having multifocal myocyte inflammation with or without myocyte injury. There should be two or more clusters of lymphocytes as noted by a CD3 or H&E stained slide.  These clusters should be within the myocardium proper, adjacent to a myocyte or myocytes, and not along the endocardium or in areas of loose connective tissue (adipose/fibrosis).  Further, the cluster of cells should not be in an area of dense fibrosis or scar. Myocardial injury, noted by single-cell hypereosinophilia, nuclear karyorrhexis/karyolysis, and/or sarcoplasmic membrane scalloping can be present.  If present, it should be reported. Myocardial injury is not required for this diagnosis. It is expected that diffuse CD3+ lymphocytes will be increased in the setting of multifocal clusters, but there is no criteria based on counting these cells.

Severe myocarditis (grade 3) is diagnosed when there is extensive and heavy predominantly lymphocytic infiltrate with myocyte injury.  A CD3 is not required to make this diagnosis, but is recommended. For this diagnosis, there should be an extensive, multifocal or diffuse infiltrate.  Myocyte injury should be obvious.  The infiltrate can include macrophages and rare eosinophils.  Giant cells or extensive numbers of eosinophils would suggest different diagnoses (giant cell myocarditis, sarcoidosis and/or eosinophilic diseases).

Biopsy Decision Tree

How to approach a biopsy

This decision tree is a step-by-step guide to evaluate an endomyocardial biopsy for myocarditis.

 

Step one

The first step is to evaluate an H&E slide. Either a florid myocarditis with numerous lymphocytes and myocyte injury is seen or it is not.  If a florid myocarditis pattern is noted, this would be grade 3 severe myocarditis.  One should look for giant cells (either on the H&E or on a CD68 IHC) or numerous eosinophils, as these could represent other forms of myocarditis.  Otherwise, the diagnosis of severe myocarditis can be rendered.  If there is not florid myocarditis, the reviewer advances to step two.

Step two

The remaining H&E stains and a CD3 are reviewed for lymphocyte clusters (5 or more cells around myocytes, as described elsewhere).  If a clusters is present, determine if there is a single cluster or more than one cluster.  Clusters should be separated, but can appear on the same biopsy piece or across biopsy pieces.  They can also appear on different levels of the tissue. A CD3 stain may be helpful in confirming a cluster or clusters are present.  If there are two or more clusters, this is grade 2 moderate myocarditis.  If there is only a single cluster, this is grade 1 mild myocarditis.  In reviewing the H&E slides make note of any myocyte injury, this does not change the diagnostic level, but it should be reported out for the case.  If no lymphocyte clusters are present, advance to step 3.

Step three

The CD3 stain should be evaluated for excessive lymphocytes.  A medium to low power pass can be made of the tissue to identify areas with the most lymphocytes.  Then a single 40x hpf should be used to count lymphocytes within the tissue.  Lymphocytes should be within the myocardium proper, adjacent to myocytes or along small perivascular spaces between myocytes.  They should not be along the endocardium, in scars, within blood vessels, or in large areas of loose connective tissue (adipose/fibrosis). Fifteen or more CD3+ cells within a hpf is grade 1, mild myocarditis.  One can interpret more than one hpf to find the region with the most positive cells. If there are no hpf with 15 or more CD3+ lymphocytes, the diagnosis of grade 0, no myocarditis is given. In the setting of no myocarditis, other diagnoses can be rendered depending on the other findings of the specimen.

Biopsy Adequacy

Specimen considerations

A minimum of five pieces of myocardium should be obtained for evaluation of myocarditis (citation).  Any fewer than three pieces would be considered insufficient/inadequate. At least four levels should be taken on every case with two or more sections per slide for H&E staining.  In addition, a CD3 immunohistochemical stain for T lymphocytes is necessary for any case that does not have severe (grade 3) myocarditis.  The expert panel also recommends a CD68 immunohistochemical stain for macrophages and giant cells (useful for identifying giant cell myocarditis or healing injury) and a Masson Trichrome, Movat Pentachrome or similar to determine the extent of fibrosis (useful for determining the extent of fibrosis which may indicate chronicity).

A useful approach for preparing slides to evaluate a myocarditis case is as follows.

Slide number Stain
1 H&E
2 Masson Trichrome or Movat Pentachrome
3 H&E
4 CD3
5 H&E
6 CD68
7 H&E

These biopsies have 5 and 4 pieces respectively and would be adequate to evaluate for myocarditis.

Use of H&E and CD3 Stains

These criteria utilize an H&E and CD3 immunohistochemical stain to evaluate the presence of clusters or diffuse infiltrates of T lymphocytes as described. H&E stained slides are useful to determine the cellular features of myocyte injury including single-cell hypereosinophilia; nuclear karyorrhexis/karyolysis; and sarcoplasmic membrane scalloping. The CD3 stain is particularly useful in counting a diffuse infiltrate and can also help visualize lymphocyte clusters.  It is recommended to be performed in all evaluations for myocarditis, but it is essential when making the diagnosis of mild and moderate myocarditis (grades 1 and 2), due to the more patchy/heterogeneous patterns of mild and moderate myocarditis. Clusters of inflammatory cells may only be present on H&E stained slides, but not on the CD3 stained slide, which is why both slide types are valuable.  For severe myocarditis, the diagnosis can be made on H&E stained slides without a need for CD3 staining.  In severe myocarditis, a CD68 stain is recommended to help identify giant cells as giant cell myocarditis is a mimicker of severe lymphocytic myocarditis.

High powered field

These criteria use high powered field (hpf) taken as a single 40x field.  As different microscopes have slightly different objectives, the area of a single hpf can vary.  This has been discussed. In practical terms, these differences are real but negligible for most modern microscopes.  Below is a general table of field numbers, diameters at 40x and their equivalent area in mm2.  The field number is reported on most microscope lenses (FN or OFN).  If your microscope falls outside of this range, you may consider tweaking your counts to adjust to this general range.

Field Number Field Diameter (mm) hpf Area (mm2)
20 0.5 0.20
22 0.55 0.24
23 0.575 0.26
25 0.625 0.31
27 0.675 0.36

Biopsy Criteria Compromises

Myocyte injury

Myocyte injury was a critical aspect of the Dallas criteria to define myocarditis. Conversely, myocyte injury was not a feature of the ESC criteria, in which the presence of a certain number of leukocytes was all that was necessary to define myocarditis. These viewpoints are diametrically opposed. A challenge of using myocyte injury as an essential criteria to define myocarditis is the difficulty in agreement as to what histology defines myocyte injury.  It can be very difficult to discern in many cases and expert cardiovascular pathologists can disagree on its presence or what features to use when evaluating injury.

Therefore, in these new criteria, definitive myocyte injury was chosen to be an essential feature of severe myocarditis only.  In that histologic subtype of myocarditis, it would be expected with the extensive infiltrate of lymphocytes.  The criteria have been written such that myocyte injury could be seen in both mild and moderate myocarditis cases. These types of myocarditis overlap significantly with the ‘borderline myocarditis’ term of the Dallas criteria, where myocyte injury was not seen.  Therefore, it will be of interest to find out how often myocyte injury is reported in these intermediate grades and if the presence of injury with mild or moderate diagnoses of myocarditis portend a worse outcome for the patient.

The presence of myocyte injury in the absence of any inflammation should not be considered as a form of myocarditis.  Other causes such as single cell drop out due to a stress cardiomyopathy, elevated catecholamine levels, septic shock, or other causes should be considered.

Cluster thresholds for myocarditis levels

The number of clusters used to define mild vs moderate myocarditis is one or more than one cluster.  Why is this the defining threshold?  Why not 1-2 clusters vs. 3 or more clusters?  Or 1-3 cluster vs. 4 or more clusters? There is no literature to support any one of these levels.  The expert consensus was that a single cluster may be a random finding in a heart, with little significance.  However, the presence of two or more clusters is less likely to be a random finding and is more likely to be a meaningful discovery.

As all heart biopsies represent a tiny fraction of the heart, one never knows how representative it is of a heterogeneous process which is more frequent in the subepicardial and left ventricular regions, where the biopsies are not performed. Is a single cluster the “tip of the iceberg” with much more inflammation in the heart, or is it a random collection of cells, that might be seen infrequently in older individuals?

In an analysis of nearly 100 digitized heart biopsies evaluated for myocarditis, only 3 cases had exactly two cluster of lymphocytes. Therefore, despite the importance of this arbitrary threshold, it is unlikely to be a common decision point. Nonetheless, further evaluation of this threshold is warranted.

High powered field (hpf) vs area (mm2)

A significant amount of effort was spent exploring the need for using hpf or a defined area in mm2 for a CD3+ diffuse lymphocytic infiltrate. The benefit of hpf is that anyone on any microscope can use their 40x objective and count the single field.  The challenge of hpf is that different microscopes have slightly different fields of view, such that some hpfs are larger than others.  As a result, there will not be a one-to-one correlation with the use of hpf.

The benefit of a defined area in mm2 is that this is an exact area, that should be more reproducible.  The challenge, however, is to both determine what that area is for a given ocular and to consistently use that size.  For example, if a 40x ocular’s field number is 25, the area of the view is 0.31mm2.  If criteria are set at 0.25mm2 (as was debated) then a pathologist should review only 80% of the field to match the 0.25mm2 area.  Which part of the field should be ignored?  What biases will be introduced? If the criteria are set to 1mm2, then the pathologist with the field number 25 ocular should review and count cells in 3 full fields and 7% of the final field.  Neither of these options is as exact as would be hypothesized with a defined area.

For groups that exclusively use digitized slides, an exact area in mm2 can be consistently determined.  However, digital signout is currently the exception to the rule in the evaluation of endomyocardial biopsies for myocarditis.  Therefore, the expert committee thought this was not going to be a widely useful approach.

Therefore, the use of a single 40x hpf field, due to its inherent simplicity, was favored over a defined area in mm2.  As practice patterns change, and digital pathology becomes more mainstream, this approach can change.

A second part of this controversy was how much area should be reviewed in which to count a diffuse infiltrate of CD3+ cells. It was reasoned that myocarditis is a heterogeneous disease and, as a result, a meaningful lymphocytic infiltrate can be in a small area at a high density, but that infiltrate may not be frequent elsewhere.  Therefore, focusing on a single 40x field would allow the pathologist to identify a significantly elevated number of lymphocytes in a small area.  The approach also reduces the time spent counting cells, which would be increased with more complicated plans such as counting 4 fields and taking the average, or similar schemes.

15 cells as a threshold for a diffuse infiltrate.

After significant discussion, disagreement, and evaluation of CD3+ staining across a range of specimens, it was settled that 15 or more CD3+ cells in a single 40x hpf would be considered mild myocarditis.  Although the expert committee initially focused on lymphocyte clusters for making the diagnosis of myocarditis, evidence brought forth showed reasonable examples of an increased lymphocyte infiltrate in which no clusters of 5+ cells were detected.  The question, then, was at what number of lymphocytes over what area would constitute a significant number of cells over baseline?

It was established that CD3+ lymphocytes are rare, but present cells in noninflamed hearts. In general, they range between 0-4 cells/hpf. However, randomly up to

Large Specimen Criteria

Apical Core

Apical Core criteria go here.

Myectomy

Myectomy information goes here.

Explanted heart

Explanted heart information goes here.

Autopsy heart

Autopsy heart information goes here.

Large Specimen Image Gallery

Large Specimen Image Gallery

Any pictures we want unique to large specimens go here.  This can include what is and what is NOT myocarditis.

Other images

Large Specimen Adequacy

Apical core

A discussion of what constitutes adequacy by tissue type.

Myectomy

A discussion of what constitutes adequacy by tissue type.

Explant

A discussion of what constitutes adequacy by tissue type.

Autopsy heart

A discussion of what constitutes adequacy by tissue type.

Areas for Research

With new criteria come new opportunities to determine how they will behave across pathologists and how they will impact patient care.  Here are some questions that are in need of supporting data.

How do biopsy criteria correlate with patient outcome?

Do the different myocarditis levels correlate with patient mortality/transplant? Is myocyte injury a critical indicator of worse patient outcomes? Do criteria correlate with ejection fraction at the time of biopsy? MRI findings?  Blood biomarkers?

How reproducible are criteria levels between pathologists?

How do these criteria change the cause of death in autopsy cases?

 

Ancillary Studies

Viral PCR

A short discussion of viral PCR?

Imaging

A short discussion of MRI and Lake Louise Criteria?

Anti-heart antibodies

A short discussion of auto antibodies?