This section of the tutorial takes you through the observations made at low power (2x to 10x). It will cover topics such as observing cellularity, the interstium, myocytes and vasculature.
The first low power biopsy assessment is to assure that adequate myocardium is present to evaluate for cellular rejection. As many heart transplant patients undergo multiple repeat biopsies, part or all of a biopsy piece may consist of a prior biopsy site. If organizing thrombus, granulation tissue, or areas of fibrosis with absent or disorganized myocytes are observed, a prior biopsy site is likely to be present. For adequacy, at least 3 biopsy pieces must be present, each with at least 50% of the surface area occupied by myocardium not altered by prior biopsy site. The presence of other non-myocardial tissues (valve leaflet, chorda tendinea, adipose tissue, etc.) should also be noted.
Overall biopsy cellularity
After determining biopsy adequacy, the major goals of low power examination are to: 1) note increased cells in the biopsy specimen; 2) determine the location and pattern of the cellular infiltrate; 3) get an overall feel for the amount of inflammatory infiltrate and 4) identify specific areas of increased cellularity to view at high power. The histological changes of acute cellular rejection are not uniform, and a low power scan allows the pathologist to focus on the worst lesional areas. Examination of tissue at several levels is critical for diagnostic accuracy.
Increased cellularity in EMB is readily identifiable in most cases. The distribution pattern of cellular infiltrates may be either focal or diffuse. At low power, it is generally possible to determine if the inflammation is perivascular or interdigitating among myocytes (perimyocytic) in a "chicken wire fence" pattern. Sometimes, this distinction, like many other morphological features, is subjective.
The location of infiltrates, and their relationship to myocytes, may suggest their underlying pathologies. The lymphocytic infiltrates in acute cell-mediated rejection (ACR) are found around cardiomyocytes (perimyocytic) or around small arterioles (perivascular). If the infiltrate is located primarily within the endocardium or in fibrous tissue, alternate etiologies such as ischemic injury, Quilty infiltrates or previous biopsy site should be considered. Endocardial lymphocytic infiltrates may also run along the surface, or near the endocardial surface in a linear fashion. These can be difficult to distinguish from a Quilty effect and tend to be be seen in association with perimyocyte infiltrates in higher grades of ACR.
Large, nodular, collections of lymphocytes along the endocardium, are typical of "Quilty" effects. For Quilty lesions in the myocardium or extending into the myocardium from the endocardium, low power observation of dense lymphocytic infiltrates with pushing borders and a relatively sharp interface with adjacent myocytes should suggest the correct interpretation. All perimyocytic lymphocytic infiltrates should be evaluated at higher magnification for evidence of myocyte injury.
The identification of "myocyte injury" is sometimes a challenging task during EMB examination. The "injury" covers a wide spectrum of changes with cell death at one end and subtle degenerative change at the other. Most of these changes need to be visualized at high power for accurate diagnosis.
Circumstantial evidence of myocyte injury, seen at low power includes encroachment of inflammatory cells onto myocytes or frankly overlying myocytes. The infiltrating inflammatory cells cause irregularity of myocyte borders and distortion of the normal myocardial architecture. In practice, inflammatory cells inside or close to myocytes are very convenient biomarkers that call for more careful examination. Caution should be used in any attempt to evaluate lymphocytes in relation to myocytes in areas of crush artifact from the bioptome.
Occasionally, ischemic injury (peri-operative or late ischemia due to allograft vasculopathy) induces foci of cardiomyocyte coagulation necrosis. Small foci involving few myocytes may not be distinguishable from ACR at low power. Larger foci will be readily evidenced by coagulative necrosis, typically with myocytes devoid of nuclei and variable degrees of myocyte resorption. Regardless of size, ischemic foci are often accompanied by a modicum of surrounding macrophages, lymphocytes and rarely neutrophils. In some cases these areas may be sharply demarcated and stellate in shape. The necrotic myocytes can be highlighted by trichrome staining which will stain these myocytes purplish.
Vacuolated myocytes may reflect myocytolysis secondary to chronic ischemia. However, contraction band artifact, particularly when seen in myocytes cut in cross-section may mimic myocytolysis. Careful low power assessment for the "checkerboard" pattern of myocytes randomly sectioned through condensed contractile elements, alternating with hyperlucent, lytic appearing areas should raise red flags and suggest a diagnosis of chronic ischemia.
Other than increased interstitial cellularity (discussed above), low power changes that can be observed in the myocardial interstitium are mainly edema and fibrosis. Edema is seen in the setting of severe ACR (Grade 3R), heart failure and/or leaky, injured vasculature with antibody mediated rejection. The interstitium has a pale basophilic, slightly bubbly appearance, evident in perivascular and/or perimyocyte spaces. The findings should be correlated with high power evaluation of the microvasculature for features of antibody mediated rejection. Edema should not be confused with artifactual, biopsy or histological preparation-induced spaces. These can be be small or large empty areas between myocytes without any of the wispy extracellular matrix seen in edema.
Increased interstitial fibrous tissue is most often in the form of broad areas of loss of myocytes with replacement fibrosis. These lesions are usually related to biopsies obtained within, or directly adjacent to, prior biopsy sites. Specific features of biopsy site changes are discussed elsewhere. Scarring related to chronic and/or healed myocardial ischemia may be difficult to distinguish from prior biopsy sites. Low power observation of "stellate shaped" scars should prompt high power evaluation for additional features of chronic ischemia. Regardless of the presence of these findings, interstitial fibrosis is not a feature of ACR.
Presence of vasculature
Evidence of cardiac allograft vasculopathy can rarely be noted on EMB and can easily be confused with the artifactual telescoping of blood vessels. Large vessels, particularly when associated with adipose or neural tissue should prompt high power evaluation for evidence of perforation. Pathologic changes in the vasculature are best evaluated at high power.
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