Endomyocardial biopsy surveillance, with frequent biopsies in the early months and standardized treatments for particular clinical situations, is the gold standard procedure in follow-up and assessment of rejection.^1,2 Most biopsy samples are performed via the jugular or subclavian veins.¹ Access through the right femoral vein for access to the right ventricle is also feasible.

Cardiac rejection is classified by type of immune response, target tissue, and time of onset (Table 168.1). Acute or cellular rejection is an immune response aimed primarily at the cardiac myocyte³; it typically occurs within weeks to months of transplant, but can recur even years later if immunosuppressive treatment is insufficient. The histologic hallmarks are those of myocardial inflammation and myocyte necrosis. The second major type of rejection, antibody-mediated rejection (also known as humoral rejection), is manifest by immune complex deposition in small vessels, often in the absence of inflammation (Chapter 169). The immune target of chronic rejection, or graft vasculopathy, is also the endothelial cell, but primarily of arteries and, to a lesser extent, veins (Chapter 170).

One of the main challenges facing transplant biologists is clarifying the relationship of the three forms of rejection, especially in the context of identifying risk factors for and eventually preventing graft vascular disease, which is the main cause of graft failure after 1 year. A challenge in pathologic study of chronic rejection is the lack of sampling of larger vessels at biopsy and a paucity of autopsy studies.

Biopsies of the right ventricular septum should comprise a few (at least 2) pieces of myocardium. Ideally, the evaluated tissue will be comprised of myocardium tissue and devoid of thrombus or scarred tissue. Since patients are usually biopsied multiple times throughout the lifetime of the allograft, it is common to identify a prior biopsy site (see below). For mechanical reasons, the bioptome in repeat biopsies tends to sample similar regions of the interventricular septum.

The tissue can be fixed in formalin and paraffin embedded, or a piece can be frozen and saved for immunofluorescence study with the main purpose of performing C4d staining. Since this antibody is now available for paraffin immunohistochemistry, all the tissue can be fixed and processed routinely. Multiple sections should be taken for evaluation, and intervening unstained slides can be saved for further stains. Phenotyping of lymphocytes (CD3, CD4, CD8, CD20, CD68) is a way of confirming rejection, better evaluating the compartment of inflammation and differentiating rejection from Quilty effect or infection.^4,5 Masson trichrome can aid in the pathologic diagnosis. Immunohistochemical studies for CMV and silver stains for fungi can be performed in suspected cases.

Acute rejection occurs generally within weeks to months after transplant and results in clinical and electrocardiographic changes that do not reliably reflect the severity of myocardial damage. The gold standard for transplant rejection remains the endomyocardial biopsy, although antimyosin scintigraphy shows promise as a less invasive method to follow transplant rejection. Histologic diagnosis rests on the identification of one or more “infiltrates” of T lymphocytes and presence of myocyte damage; the definition of infiltrate has not been defined quantitatively, but includes an aggregate of cells that is in the interstitial or in a perivascular location. The cellular infiltrate in acute allograft rejection is primarily T lymphocytes, both CD4 and CD8 positive, as well as macrophages and occasional eosinophils.

In addition to establishing a diagnosis of cellular rejection, it is essential to grade the degree of inflammation. The grading of rejection is based on one primary feature: the presence or absence of myocyte necrosis. Because necrosis is rare in the absence of extensive inflammation, the degree of inflammation is also of importance in grading.

In 1990, the International Society of Heart Lung Transplantation (ISHLT) issued a grading system, which is based on the degree of inflammation and myocyte damage and presence of interstitial changes.⁶ The ISHLT simplified the grading system in 2004 (Table 168.2).⁷ The simplified system reflects a 3-tiered system of mild (inflammation without myocyte necrosis) (Figs. 168.1 and 168.2), moderate (inflammation with focal myocyte necrosis) (Figs. 168.3, 168.4, 168.5), and severe (extensive necrosis with other features such as edema, vasculitis, and

hemorrhage). The previous distinction between mild focal inflammation and mild diffuse inflammation (previous grades 1A and 1B, respectively) has been abandoned, as well as a single focus of “aggressive” inflammation (previous grade 2).