After a slide is visually scanned at low power (4x, 10x objectives) a pathologist typically moves to high power on areas that contain an inflammatory infiltrate. Generally this is with a 20x objective. At this power, one is investigating primarily for myocyte injury in areas of lymphocytic infiltrate. There is no strong indication to use the high power objectives on areas of normal appearing myocytes observed at low power. This section of the tutorial takes you through the observations made at high power. It will cover the topics of myocyte injury, identification of the infiltrating cells, and interstitial findings.
Confirmation of myocyte injury
Once the presence of a lymphocytic infiltrate is established, the next step would be to look for myocyte injury. Myocyte injury (ie damage/necrosis) that is associated with acute rejection may be difficult to recognize, and other changes can be mistaken for myocyte injury (poor fixation). Myocyte injury is often subtle and best appreciated by observing the same area across multiple levels. Some morphological features of myocyte injury include: vacuolization, perinuclear halo, myocyte nucleus without surrounding cytoplasm, lymphocyte encroachment onto the myocyte with irregular myocyte borders, and myocyte dropout (areas of heavy inflammation and matrix material where a myocyte should have been). Hypereosinophilia and nuclear pyknosis are also good indicators of myocyte injury. In the revised working formulation, milder types of acceptable myocyte injury include myocytolysis and coagulative necrosis.
Another clue for myocyte injury is a change in the sarcomere structure. If one can see normal myofibril patterning in the myocyte, that myocyte is not injured. If that patterning is gone and the cytoplasm becomes hypereosinophilic and clumpy, there is a good chance that myocyte is injured. Masson trichrome stain can highlight early myocyte injury, in which case injured myocytes are shrunken and have more of a blue-gray color instead of the normal eosinophilia seen in normal myocardium.
All of these changes must be seen in the presence of adjacent lymphocytes. If lymphocytes are not present, then the myocyte injury is not caused by cellular rejection and would be the result of some other process such as ischemic injury that can be seen in the early weeks following transplantation.
It is always a good idea to look at multiple levels of the potentially injured area as confirmation of the myocyte injury as sometimes a single histologic section can be misinterpreted as injury.
Identification of interstitial cells
If one looks beyond the myocytes, one can see a variety of additional cell types in the biopsy material. The most important would be the cellular infiltrate of acute cardiac rejection, which is principally T cells (CD4+ and CD8+), with fewer macrophages and occasional eosinophils. A substantial increase in activated B cells and natural killer cells has been seen in moderate to severe rejection.
Not all nuclei seen within the interstitium represent lymphocytes, however, and high magnification may be needed to identify the exact nature of the "infiltrate" to exclude cellular rejection. Other cellular components of the myocardial interstitium include fibroblasts and myofibroblasts, primitive mesenchymal cells, dendritic cells, mast cells, endothelial cells and pericytes. Scattered lymphocytes and occasional macrophages can also be seen in normal myocardium. Generally, only lymphocytes cluster in the myocardium.
Nature of the inflammatory infiltrate
The type of inflammatory cells seen in endomyocardial biopsies from cardiac transplant recipients is important in distinguishing an infiltrate as being due to rejection versus another process, as well as helping to determine the grade of rejection. Acute cellular rejection is always characterized by the presence of lymphocytes (T cells). Activated lymphocytes, which are more prominent in higher levels of rejection, have increased cell cytoplasm.
In the appropriate setting, a mixed inflammatory cell infiltrate with neutrophils and eosinophils may be indicative of higher grades of cellular rejection (3R). Neutrophils can also be seen in heart biopsies that have ischemic necrosis which can be present for several weeks after transplantation.
Eosinophils can be found in moderate (2R) or severe (3R) grades of rejection in which there is also myocyte necrosis. Generally they are rare and scattered. Eosinophils are almost never a feature of mild (1R) rejection and their presence should serve to increase one's awareness that moderate or severe (2R & 3R) rejection, maybe present.
Eosinophils can also be seen in hypersensitivity myocarditis most often related to pharmacologic agents. In this scenario, the eosinophils make up a large ratio of the infiltrating cell mix and often cluster. The inflammatory infiltrate in hypersensitivity myocarditis also includes lymphocytes, plasma cells, and macrophages. Although focal myocytolysis can be seen in hypersensitivity myocarditis, myocyte necrosis is not a prominent feature. Eosinophils in EMB can also be seen in parasitic infection.
Analysis of the interstitium
The interstitium should be evaluated at high power for the presence of cellular material and edema. The presence of a debris field, nuclear debris or other amorphous ("dirty") basophilic and eosinophilic material, in areas of heavy infiltrate is consistent with myocyte injury and cellular apoptosis. Early after transplantation, it is more often associated with peri-transplant ischemic injury. However, in late biopsies it is a useful histologic finding in areas concerning for rejection.
Interstitial edema in a cardiac transplant biopsy may be a sign of antibody-mediated rejection. However, separation of myocytes can be seen as an artifact of handling and processing of the biopsy and therefore simulate edema. With true edema, myocytes are separated by whispy eosinophilic strands of matrix. Additionally, in the setting of antibody-mediated rejection, other findings should also be present, such as prominent capillary endothelial cells, interstitial macrophages, and/or hemorrhage.
Myocytolysis (for graft CAD)
Focal or diffuse subendocardial vacuolization of myocytes, or myocytolysis, may be a marker for reversible ischemic injury. Myocytolysis is a clearing of the sarcoplasm and nuclei, with nuclear enlargement and prominent nucleoli. If seen in endomyocardial biopsy samples from cardiac transplant recipients who are more than a year from transplantation, this finding could be a marker for accelerated coronary artery narrowing that occurs in transplanted hearts.
Endothelial cell changes (in AMR)
Criteria for making the diagnosis of antibody-mediated acute rejection are presented elsewhere in this tutorial. Among the findings on H & E-stained slides that may suggest AMR, the low power prominence of capillaries and venules may be helpful. High magnification is useful for examination of endothelial cells, which in AMR, are large and prominent ("swollen"), with enlarged and hyperchromatic nuclei. These findings support the presence of AMR and warrant further investigation to asses for the presence of immunoglobulin and complement.
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