Section C -

Transplant Vascular Diseases Volker Nickeleit, M.D. J. Charles Jennette, M.D.

Many different disease processes can affect the arteries, arterioles and capillaries in solid organ transplants. Vascular changes are clinically significant since they determine long-term graft function and survival. Cardiac allograft vasculopathy is associated with considerable mortality. Some arterial and arteriolar lesions are transplant specific, such as rejection episodes. Others are found in native organs as well as in transplants, such as hypertension induced injury or calcineurin inhibitor induced toxicity. Occasionally, in particular in heart allografts, different insults to the arterial tree occur simultaneously or are superimposed on one another (e.g. pre-existing hypertension induced donor arteriosclerosis with superimposed subsequent vascular rejection). Thus, the pathologist evaluating transplant specimens faces a challenging task. We will focus here primarily on arterial lesions encountered in kidney transplants since they are best characterized and most relevant for the practicing pathologist. The pathophysiologic chain of events leading to vasculopathies in cardiac, pancreas and renal allografts are very similar, however, cardiac transplant vasculopathies typically remain histologically undeteceted due to the superficial nature of the subendocardial surveillance biopsies. They often only become apparent at time of autopsy (see study case 11). For reasons only incompletely understood transplant vasculopathies observed in liver and lung allografts are less common and seem to be clinically less significant than those observed in kidney, heart or pancreas transplants. Although the complication rate of transplant biopsies in the hands of an experienced clinician is extremely low, a graft biopsy remains an invasive procedure associated with some risk and discomfort. Thus, once a biopsy is indicated, the biopsy sample should be large enough to provide sufficient information, in particular of the arterial tree in cases of a kidney or pancreas transplant biopsy. Unnecessary re-biopsies or inadequate diagnostic yields should be avoided. Ideally, renal transplant biopsies should consist of two tissue cores obtained with a 14 or 15 gauge needle in order to adequately sample arteries, i.e. to obtain approximately 3 to 4 medium sized arterial cross sections (branches of arcuate arteries or interlobular caliber vessels). If the sample is too small and arteries are not properly sampled, then the diagnosis and therapy may be inadequate. This is the reason why we generally discourage the use of fine needle aspiration or 18 gauge needles in the setting of kidney transplant biopsies. Small tissue segments may be frozen for additional studies, i.e. the detection of the complement degradation product C4d along (peritubular or myocardial) capillaries or MHC-class II (HLA-DR) in tubular epithelial cells by immunofluorescence microscopy. I) Calcineurin inhibitor induced toxicity 1) Introduction Cyclosporine A and tacrolimus are immunosuppressive drugs which both act via calcineurin inhibition and suppression of interleukin-2. Both drugs are nephrotoxic and the observed side effects are very similar. Since cyclosporine was introduced first and nephrotoxicity was already noticed by Calne and colleagues in 1978, the side effects induced by calcineurin inhibitors are commonly referred to as "cyclosporine type". Calcineurin inhibitor induced histologic changes are primarily seen in native and transplanted kidneys. For the classification of cyclosporine and tacrolimus induced nephrotoxicity we use the following terminology: 1) functional, and 2) structural toxicity. 'Pure' functional toxicity is found without morphological changes. It is due to vasospasms. Structural toxicity is characterized by morhological alterations and is almost always associated with kidney dysfunction. Structural and/or functional nephrotoxicity have been reported in patients with kidney, liver, heart, and bone marrow transplants, as well as in patients with autoimmune diseases treated with calcineurin inhibitors. Cyclosporine and tacrolimus nephrotoxicity is dose-dependent, however, it shows great inter-individual variations. Toxic side effects may be noted in some 'sensitive' patients in therapeutic dose ranges or if additional risk factors, e.g. ischemia, are present. Of note: the observed morphological changes in the vasculature induced by calcineurin inhibitors bear close similarities to those described above under "thrombotic microangiopathies". 2) Clinical findings Almost all patients treated with cyclosporine or tacrolimus present with deterioration of renal function, i.e. a rise in the serum creatinine. Occasionally, serum creatinine levels can double and patients may even present with 'acute renal failure'. Dysfunction is due to vasospasms of arterioles, a decrease in the glomerular filtration rate and renal plasma flow. Arterial hypertension is common; hyperkalemia, mild metabolic acidosis, hypomagnesemia, and hyperuricemia are rare. The most serious toxic side effect is a fully developed thrombotic microangiopathy with clinical signs of the 'hemolytic uremic syndrome' (HUS). HUS can develop at any time after the initiation of therapy, most often, however, it is seen during the early time course. 3) Histology Calcineurin inhibitor induced microangiopathies are found in the peripheral vascular tree, most characteristically in afferent arterioles. The arteriolar lesions can extend in severe cases downstream into the glomerulus and upstream into small arteries with up to 2 layers of smooth muscle cells (close to the branching points between interlobular arteries and pre-arterioles). Larger arteries with more than 2 layers of medial cells, i.e. arcuate type arteries and their branches, are typically spared. The earliest morphological changes noted by light microscopy are vacuolization/ballooning of smooth muscle cells (due to dilatation of the endoplasmic reticulum), and occasional single cell necrosis of endothelial cells and/or medial smooth muscle cells. Such changes are common, vastly reversible on dose reduction, and generally do not carry any prognostic significance. The fully developed calcineurin inhibitor induced arteriolopathies reveal nodular, PAS positive, protein (hyaline) deposits deep in the arteriolar wall replacing individual necrotic myocytes. Often, these protein deposits are arranged in a necklace like pattern along the outer/adventitial aspect of the vessels. If myocyte and endothelial cell necrosis progress, hyaline deposits can extend transmurally into the subendothelial compartment. In most severe cases arteriolar walls are circumferentially devoid of medial smooth muscle cells and show transmural hyaline deposits instead. Rarely, a calcineurin inhibitor induced arteriolopathy presents with mucoid (TMA-like) thickening of the intimal layer. Severe thrombotic microangiopathies with fibrin and platelet thrombi are uncommon events, seen in less than 1% of patients. Mild and moderate forms of calcineurin inhibitor induced arteriolopathies with intramural hyaline nodules may undergo remodeling, i.e. "healing", once the administration of cyclosporine or tacrolimus is stopped. This phenomenon has been observed in repeat biopsies. Medial protein deposits can vanish and the medial layer of affected arterioles may show thickening with irregularly (re)arranged smooth muscle cells and an increase in extracellular matrix. Important: arteriolar lumens remain patent. In contrast, severe arteriolopathies with circumferential and transmural hyaline deposits lead to arteriolar scarring/occlusion and nephron loss. The morphology of calcineurin inhibitor induced arteriolopathies is typical for toxic side effects but not pathgnomonic. Findings have to be interpreted in the appropriate clinical context. The differential diagnosis includes: 1) "thrombotic microangiopathies/HUS", e.g. recurrent disease or de-novo disease induced by agents other than calcineurin inhibitors, 2) diabetic arteriolosclerosis, and 3) hypertension induced arteriolosclerosis with hyalinosis. HUS or "nodular" diabetic arteriolosclerosis have to be excluded based on clinical information since the histologic examination does not allow for the proper identification. Hypertension induced arteriolosclerosis typically shows subendothelial hyaline deposits overlying an intact, although occasionally atrophic, medial smooth muscle layer. Nodular hyaline deposits in the media are generally absent in the setting of "benign nehrosclerosis"; they only become prominent in patients suffering from marked hypertension with "malignant nephrosclerosis". Associated findings : Calcineurin inhibitor induced glomerulopathies often accompany severe forms of arteriolopathies (in our experience approximately 60% of cases with circumferential and transmural hyaline deposits). The glomerular lesions can be interpreted as the extension of arteriolar endothelial cell injury into the glomerulus. The more severe the arteriolopathy, the more often glomerular changes are noted. Glomerular injury induced by calcineurin inhibitors usually shows a focal and segmental distribution pattern. In some cases, only a single glomerulus might be affected. Glomeruli can show signs of a thrombotic microangiopathy with intracapillary thrombus formation, generally extending from afferent arterioles into the glomerular capillaries. These changes are rare (seen in less than 1% of patients). More frequent are structural abnormalities of the glomeruli with double contouration of the peripheral basement membranes and segmental mesangiolysis. The glomerular changes are identical to (remote) forms of thrombotic microangiopathies seen in native kidneys. Striped interstitial fibrosis and tubular atrophy are often found in cases of calcineurin inhibitor induced arteriolopathies. These changes represent 'non-specific', secondary phenomena caused by nephron loss; they are not pathgnomonic for calcineurin inhibitor toxicity. Toxic changes in tubules, i.e. isometric vacuolization of the cytoplasm of proximal tubular cells, can accompany toxic arteriolopathies. Toxic tubular changes are fully reversible on dose reduction. Immunohistology: The protein deposits along arterioles contain IgM and/or complement factors (C3, C1q, C5b-9, C4d). Glomeruli do not show diagnostically relevant deposits. In up to 20% of affected kidneys scant fibrin accumulation may be found along the vasculature. This immunohistochemical staining profile is considered to be non-diagnostic. It can also be observed in cases of hypertension induced arteriolosclerosis or thrombotic microangiopathies. Calcineurin inhibitor induced toxicity is not associated with diagnostically significant C4d deposits along peri-tubular capillaries. If C4d is noted then a second diagnosis of concurrent acute rejection should be entertained. 4) Outcome Functional calcineurin inhibitor induced toxicity is fully reversible on dose reduction; it is of no prognostic significance. Mild and moderate forms of structural toxicity with 'cyclosporine type arteriolopathies' do not affect long-term kidney function; generally the dose of calcineurin inhibitors is lowered. Severe structural toxicity with circumferential and transmural hyaline deposits, vascular occlusion or overt signs of 'thrombotic microangiopathies' with fibrin thrombi are serious complications requiring therapeutic intervention, such as a change to calcineurin inhibitor free immunosuppressive drug regimens. Long-term kidney function may be impaired due to advanced nephron loss and proteinuria may develop due to secondary focal and segmental glomerulosclerosis. II) Rejection / Vascular Rejection - Introduction Acute rejection episodes involving a solid organ transplant, such as liver, kidney, pancreas or heart, can involve the interstitial compartment and/or blood vessels. In the setting of heart, pancreas and renal allografts, rejection episodes affecting arteries, i.e. vascular rejection episodes, are of particular therapeutic and prognostic importance. Rejection can be classified in different ways. Frequently, transplant physicians prefer a simplistic approach based on the time-span post transplantation. Thus, the term "hyperacute" or "accelerated acute" rejection implies a very early event within hours or days after transplantation, "acute" rejection is seen within days or weeks, and "chronic" rejection is a late event occurring months or years after grafting. The gold standard for the characterization of rejection episodes, however, is not the clinical presentation but rather the detection of typical histological changes in a graft biopsy. Integration of the different viewpoints - clinical versus histological - is problematic and sometimes even confusing since morphological alterations cannot always be easily translated into the timeframe of "hyperacute, acute and chronic". For example, histological changes characteristic for so-called "acute rejection" may be encountered many years after transplantation in non-compliant patients. On the other hand, morphological features typical for "chronic rejection" may already be seen a few weeks post grafting. Frequently, "acute rejection" concurs with "chronic rejection". A rejection episode may be of cellular, humoral or mixed etiology. Consequently, rejection (including "vascular rejection") is best classified based on morphological findings. In this context, "vascular rejection" is used as a general, descriptive term comprising various arterial and capillary changes occurring during rejection episodes. The term "vascular rejection" does not characterize a specific etiology or morphologic phenotype. Since the basic principles of "vascular rejection" are very similar in different organ systems (in particular in heart, pancreas and kidney grafts), we will discuss for didactic purposes the various morphological phenotypes of vascular rejection seen in renal allografts. Banff '97 and CCTT (coordinate clinical trial in transplantation) classification categories of rejection will be highlighted. 1) Adjunct Immunohistochemical Markers of Rejection: the Complement Degradation Product C4d The understanding and classification of rejection has undergone substantial changes in recent years. For the first time, additional immunohistochemical markers are incorporated into the diagnostic decision making process – similar to the practice long established for the work-up of malignant neoplasms. The immunohistochemical detection of C4d in transplant biopsies – pioneered by H. Feucht from Munich more than 10 years ago - has fundamentally changed our understanding, since we now have a marker for "humoral rejection". This marker is widely used during the work-up of renal and to a lesser extent also of heart allograft biopsies. C4d is the split product of the activated complement factor C4, a component of the classical complement cascade that is typically activated subsequent to the binding of antibodies to their receptors and activation of the complement factor C1. C4d transiently binds covalently to cell surfaces and extracellular matrix proteins, such as basement membrane components, at the site of C4 activation. C4d can easily be detected by immunohistochemistry both in fresh frozen and formalin fixed tissue samples. Its detection in transplant biopsies is generally considered to be an indirect sign, a "foot print", of an active antibody mediated alloresponse (immunoglobulins and activated complement factors are generally not detected by immunohistochemistry, likely due to rapid shedding or internilization of the molecules). C4d is often found in cases of "vascular rejection". For pathologists, the detection of C4d is an important tool not only to accurately diagnose an antibody mediated allo-response ("C4d positive") but also to exclude other differential diagnoses closely mimicking rejection, such as thrombotic microangiopathies, surgical complications associated with thrombus formation or severe ischemia/reperfusion injury ("C4d negative"). Nowadays, the search for C4d is considered "standard of care" for the adequate evaluation and diagnostic work-up of kidney transplant biopsies. The results of C4d staining are part of the classification of renal allograft rejection according to the updated Banff '97 classification scheme. C4d in renal allografts: Of diagnostic relevance is the focal or diffuse, strong accumulation of C4d along peritubular capillaries in the renal cortex and/or medulla. (In heart allograft biopsies, C4d is found along intramyocardial capillaries). Only non-fibrotic and non necrotic parenchymal regions should be evaluated. The minimal threshold level to call a biopsy 'positive' is the strong, circumferential detection of C4d in 10 or more capillaries surrounding adjacent tubules. (Minimal threshold levels in cardiac biopsies are undetermined). C4d deposits in other locations (e.g. along the endothelium of arteries, in glomeruli, arterioles with hyalinosis or along atrophic tubules) are regarded to be non-diagnostic. It is important to remember that the accumulation of C4d marks an independent humoral allo-response. Consequently, C4d deposits can be detected in combination with various histologic changes. The association between C4d and morphological signs of acute "cellular" rejection defined by the CCTT criteria is statistically significant. C4d is found in 24% - 43% of tubulo-interstitial rejection episodes, in 45% of biopsies with transplant endarteritis, in 50% of severe vascular rejection episodes with fibrinoid vascular wall necrosis or thrombosis, and in 50% - 60% of cases with glomerular rejection (i.e. transplant glomerulitis or –glomerulopathy). Sometimes, the presence of markedly dilated peritubular capillaries filled with mononuclear cell elements or abundant polymorphonuclear leukocytes may be a morphological indicator of C4d positivity/humoral rejection. "Acute pure humoral rejection", also termed "C4d positive capillary transplant vasculopathy", a newly defined entity characterized by strong C4d positivity in early, severely dysfunctioning grafts lacking any signs of cellular rejection, is a rare event seen in our experience in less than 10% of all C4d positive biopsies (see below). Clinical observations and prognosis: During the post transplantation period, C4d is detected in 30% of all diagnostic graft biopsies (35% of all biopsied patients). It is typically detected early after transplantation (median: 38 days post grafting). Occasionally, C4d can also be found years after grafting (in our experience, as late as 15 years). C4d is a dynamic marker since it can accumulate and disappear within days (approximately 4 – 8 days). At present, the clinical significance of C4d deposits in renal allografts is only incompletely understood. As a general rule, C4d positivity in the setting of cellular rejection or significant allograft dysfunction indicates serious rejection episodes requiring aggressive treatment. Often, long-term prognosis is poor. C4d was found to be the strongest independent predictor of poor graft outcome. In contrast, C4d can also occasionally be detected in stable renal grafts with normal histology. This latter observation is of undetermined clinical significance. It does not indicate the immediate necessity for aggressive therapeutic intervention. C4d deposits in heart allografts: Only relatively little is known about C4d deposits in heart allografts. It seems that C4d is not only detected in the setting of "rejection", but also in surveillance biopsies of stable grafts (see USCAP 2005, abstract 258, AM Safley et al). If histologic signs of "pure humoral rejection" are detected, i.e. swelling of endothelial cells, dilatation of capillaries and intravascular accumulation of monocytes, then the detection of C4d can help to establish a specific diagnosis of "acute pure humoral rejection" or "C4d positive capillary transplant vasculopathy". Whether C4d positivity is associated with the development of cardiac allograft vasculopathy is currently unknown. Appendix - Detection of C4d: The complement degradation product C4d can easily be detected in fresh frozen tissue samples by immunofluorescence microscopy. We use a mouse monoclonal antibody which is commercially available from Quidel (San Diego, CA; dilution 1:50, 30 min incubation at room temperature followed by incubation with a secondary goat anti-mouse affinity purified and FITC labeled anti IgG antibody (Jackson ImmunoResearch Laboratories, PA), dilution 1:40, 30 minutes at room temperature - as previously published). C4d can also be detected in formalin fixed and paraffin embedded tissue sections employing a rabbit polyclonal antibody (Biomedica Gruppe, Vienna, Austria). We use the steam antigen retrieval technique (30 minutes), followed by a 30 minute incubation with the primary antibody at 37 degrees Celsius (dilution 1:50) and subsequent avidin/biotin histochemical staining procedures as previously published. This antibody cross reacts with cynomolgus monkeys. 2) Vascular Rejection: Morphological Variants A) Transplant Vasculopathy with Massive Intravascular Coagulation/thrombosis and Vascular Immunoglobulin Deposition Synonyms: Hyperacute, peracute rejection, Banff '97 class I rejection, not classified according to the CCTT scheme. Definition: Vascular immunoglobulin deposition, endothelial cell damage and occlusive thrombi in large and small arteries and capillaries. Macroscopic Findings: Following the restoration of blood flow during surgery, the kidney graft develops a reddish mottled aspect, increases in size and rapidly turns reddish-blue. Nephrectomy specimens are usually markedly swollen, hemorrhagic and infarcted. Histology: The histology is characterized by fibrin thrombus formation in large arteries (i.e. renal artery and branches) and small arteries (i.e. interlobar, arterioles) as well as glomerular capillaries. Fibrin thrombi in large arteries can show a polymorphonuclear leukocyte reaction along the endothelial cell surface, a "non-specific" change which should not be misinterpreted as a pathgnomonic sign for an antibody mediated rejection phenomenon. Despite intravascular thrombosis, vascular walls often remain viable, even in areas of infarction. If renal allografts survive for days, early transplant biopsies reveal thrombus formation in only few vessels. This focality is due to the unaltered fibrinolytic activity of the host organism resulting in thrombolysis in the transplant. Thrombus formation causes acute ischemic type injury of tubular epithelial cells (so-called acute tubular necrosis, ATN) or in more severe cases infarction. In the interstitial compartment foci of hemorrhage and polymorphonuclear leukocytes are conspicuous. Polymorphonuclear leukocytes are found either as a demarcation phenomenon between viable and infarcted areas (as for example typically seen in myocardial infarcts), or adjacent to tubules with severe ATN. If polymorphonuclear leukocytes are primarily found in dilated peritubular capillaries in viable regions of the renal parenchyma, they may be used as an indicator for an antibody mediated rejection episode (see below). Significant lympho-histiotiocytic infiltrates, i.e. signs of cellular rejection, are generally lacking, although they can become more conspicuous if grafts survive for a few weeks. Ultimately, massive thrombosis leads to extensive infarction and graft loss. Immunohistology: During the early phases of rejection linear deposits of IgG and IgM together with complement factors are present along the vascular endothelium of arteries, glomeruli, and peritubular capillaries. In nephrectomy specimens with complete necrosis, immunoglobulin deposits may be scant or even lacking. Transplant vasculopathies with massive intravascular coagulation are generally C4d positive, although experience is very limited due to the rare occurrence of these events (less than 0.2% of all rejection episodes). Etiology: Preformed circulating recipient antibodies bind to the endothelium of arteries and capillaries in the graft and lead to rapid and massive coagulopathy and thrombosis in all caliber arteries and capillaries. Transplant vasculopathy with massive intravascular coagulation is the "prototype" for an antibody mediated rejection phenomenon. Outcome: In general ominous. Plasmapheresis may be tried in protracted cases. Comment: Rejection episodes with widespread thrombi are very rare events (less than 0.2 %) that typically occur immediately post transplantation (rarely a few weeks after grafting). B) Necrotizing Transplant Vasculopathy Synonym: Accelerated acute rejection; category IV (type III) or category II (type III) acute rejection (Banff '97 update); type III acute rejection (CCTT) Definition: Transmural "fibrinoid" arterial wall necrosis affecting all caliber arteries (most often interlobar, arcuate, interlobular caliber vessels). Macroscopic Findings: The kidneys are grossly enlarged and show hemorrhagic and anemic infarcts side by side ("mottled appearance"). In the most severe forms, the nephrectomy specimens have a dark red and swollen appearance, closely resembling cases of transplant vasculopathy with massive intravascular coagulation and immunoglobulin deposition. Histology: Arterial lesions are patchy in nature, generally only involving some of the examined arterial cross sections. Affected vessels exhibit segmental, less often, circumferential fibrinoid necrosis which can be transmural (from the intimal to the adventitial layer) or medial (involving only the inner part of the media with destruction of the internal elastic lamina). The intimal surface is usually (but not always!) devoid of endothelial cells and covered with fibrin/platelet aggregates. However, occlusive fibrin thrombi – resembling cases of transplant vasculopathy with massive intravascular thrombosis - are generally not detected. In many cases, fibrinoid arterial necrosis is associated with intimal and transmural mononuclear cell infiltrates (i.e. lymphocytes and histiocytes). Severe arterial injury results in patchy interstitial hemorrhage or even focal infarction. If the rejection episode responds to treatment, necrotic arterial walls can form microaneurysms and show remodeling phenomena with infiltration of myofibroblasts, deposition of collagen and scar formation. Elastic tissue stains characteristically reveal segmental destruction and loss of the lamina elastica interna. Associated findings : The glomeruli can show different abnormalities, ranging from focal and segmental to diffuse and global: i) non-specific ischemic glomerular collapse, ii) massive glomerular capillary dilatation with blood stasis, iii) fibrin thrombi, or iv) transplant glomerulitis. Immunohistology: Typically fibrin can be found in necrotic vessel walls and in the interstitium. Specific IgG or IgM deposits along the endothelium of arteries or capillaries are generally lacking. In our experience approximately 50% of cases with necrotizing transplant vasculopathy are C4d positive. Etiology: The specific etiology of fibrinoid arterial wall necrosis is undetermined. Antibodies (de-novo ?) likely cause injury in C4d positive cases. Arteries may also show signs of a cell mediated injury with intramural lymphocytic and histiocytic infiltrates. Outcome: Generally poor; most grafts are lost within weeks post biopsy. Comment: Necrotizing transplant vasculopathy is typically seen within the first weeks after grafting. It is an uncommon form of rejection (seen in approximately 1% to 4% of all rejection episodes occurring within the first weeks post grafting). According to the updated Banff '97 classification scheme, C4d positive cases are classified as "category II (type III)" and C4d negative cases as category IV (type III) acute rejection. Necrotizing transplant vasculopathy can also be seen many years after transplantation in non-functioning grafts/ patients off immunosuppression; it is then typically accompanied by transplant endarteritis and sclerosing transplant vasculopathy. C) Capillary Transplant Vasculopathy, C4d Positive Synonym: Acute pure humoral rejection; category II (types I and II) acute rejection (Banff '97 update); not classified according to CCTT. Definition: Diffuse capillary injury with intracapillary accumulation of polymorphonuclear leukocytes and mononuclear cell elements; occasionally capillary fibrin thrombi are found; strong accumulation of C4d; detection of circulating donor specific antibodies in the plasma. Macroscopic Findings: The kidneys are grossly enlarged, dark blue with extensive hemorrhagic infarcts closely resembling cases of transplant vasculopathy with massive intravascular coagulation and immunoglobulin deposition. Histology: In typical cases focal or diffuse injury of peritubular capillaries is noted. The capillaries are dilated and filled with polymorphonuclear leukocytes and/or monocytes, as well as occasional fibrin thrombi. Capillary leakage results in foci of interstitial hemorrhage and edema containing scattered inflammatory cells, i.e., lymphocytes, histiocytes and polymorphonuclear leukocytes. The impaired capillary blood flow causes acute ischemic tubular injury, i.e. ATN, and sometimes even frank necrosis. Signs of typical cellular rejection with conspicuous tubulitis are lacking. Arteries are generally uninvolved. (Similar changes have been described in early myocardial graft biopsies from dysfunctioning heart transplants. Endomyocardial biopsies can show swollen endothelial cells, intracapillary macrophages, and interstitial edema.) Associated findings : Often capillary injury not only involves peritubular but also glomerular capiIlaries, which can contain mononuclear and polymorphonuclear inflammatory cells (signs of transplant glomerulitis), and fibrin thrombi. In most severe cases glomeruli reveal mesangiolysis and segmental tuft necrosis. Immunohistology: The most characteristic finding, which led to the recognition of this entity, is the strong and diffuse, linear accumulation of C4d along peritubular capillaries. In addition – although it is not considered to be of additional diagnostic significance – C3d is found. The detection of linear immunoglobulin deposits (IgG, IgM) along capillary walls is an exceptionally rare event. In areas of hemorrhage and necrosis fibrin deposits are conspicuous. Glomerular C4d deposits are considered to be of no diagnostic significance. Etiology: C4d positive capillary transplant vasculopathy is caused by circulating donor specific anti-class I or frequently anti-class II antibodies. In contrast to transplant vasculopathy with massive intravascular thrombosis and vascular immunoglobulin deposition, the donor specific antibodies are often formed de novo post transplantation. Little is known about specific antigens on the capillary endothelium targeted by the antibodies. Outcome: Graft survival varies. It is poor in cases with marked thrombosis and necrosis and seems more favorable in grafts lacking capillary thrombi and interstitial hemorrhage. However, clinical experience with this form of rejection is currently limited. Comment: C4d positive capillary transplant vasculopathy, i.e.acute pure humoral rejection, is a newly recognized entity and much remains to be learned in the future. These antibody mediated rejection episodes are infrequent and account for far less than 5% of all rejection episodes in our experience. Acute pure humoral rejection is typically diagnosed within the first weeks post transplantation. Histological signs of cellular rejection, i.e. tubulitis and tubular MHC-class II expression, are characteristically lacking. C4d positive capillary transplant vasculopathy is common in kidney transplants originating from ABO incompatible donors. Therapeutic attempts are made with high dose IVIG treatment and/or plasmapheresis or immunoabsorption. In our opinion, C4d positive capillary transplant vasculopathy, i. e. acute pure humoral rejection, has to be clearly separated from other histological changes associated with C4d deposits, in particular, C4d positive cellular rejection episodes (tubulo-interstitial, transplant endarteritis). From a pathophysiological point of view, such C4d positive rejection episodes do not qualify as "pure humoral rejection" but rather reflect a mixed cellular and humoral etiology. However, this distinction is not always made. Mixed humoral and cellular rejection episodes likely require specific, currently undetermined anti-rejection therapy. D) Transplant Endarteritis Synonym: Infiltrative and proliferative transplant vasculopathy; acute transplant vasculitis; endovasculitis; endothelialitis; category IV (type II) acute rejection (Banff '97 update); type II acute rejection (CCTT) Definition: Infiltration of lymphocytes and macrophages through the activated endothelial cell layer into the subendothelial compartment/intima of arteries. De novo intimal collagen types I and III deposits are inconspicuous. The media and the internal elastica of the affected arteries remain unaltered. Macroscopic Findings: Nephrectomy specimens showing pure transplant endarteritis are extremely rare. The kidneys are of normal size or slightly enlarged and may exhibit small anemic infarcts. Histology: Transplant endarteritis progresses through various stages. Infiltrative stage: The earliest changes are detected along the endothelial cell layer. The endothelial cells are activated with nuclear enlargement, cytoplasmic swelling and vacuolization. The most striking form of 'activation' is an arcade-like detachment of the endothelial cell from the underlying basement membrane. Per definition, mononuclear cell elements, i.e. lymphocytes and histiocytes, are present under the activated endothelial cell layer. Endothelial cell necrosis is generally inconspicuous, although, it can be occasionally noted in association with small intimal fibrin deposits. Occlusive thrombi, however, are typically absent. During the course of transplant endarteritis, i.e. in days or few weeks, the macrophages in the intima can transform into foam cells. Proliferative stage: The infiltrative stage imperceptibly evolves into the proliferative stage characterized by spindle-shaped cell elements with large nuclei (i.e. myofibroblasts), which progressively increase in number. Proliferation markers, such as KI-67, reveal a high proliferative activity in the inflamed intima. While the number of myofibroblasts increases over time, usually the number of lymphocytes and macrophages decreases. Foam cells, however, typically persist. The accumulation of myofibroblasts is associated with the deposition of "early" extracellular matrix proteins, in particular fibronectins and collagen type IV. Of note: "scar" collagens I and III are absent (their deposition marks the transformation of transplant endarteritis into sclerosing transplant vasculopathy, see below). During the infiltrative and proliferative phases, endothelial cells characteristically remain enlarged and activated. Infiltrative/proliferative transplant vasculopathy mostly involves the entire intimal circumference of an artery, however, segmental changes can be found, often at arterial branching points. Since the inflammatory process is limited to the intima, both the lamina elastica interna and the media remain unchanged. Transplant endarteritis – like all vascular lesions seen in rejection - is a focal phenomenon that can be superimposed on pre-existing lesions, such as arterionephrosclerosis originating from the donor. Associated findings : Transplant endarteritis may be accompanied by transplant glomerulitis and/or by transplant glomerulopathy. Focal or diffuse tubulo-interstitial rejection with tubulitis is common. Immunohistology: Many cells infiltrating the arterial intima are CD68 positive monocytes / macrophages. In addition, T lymphocytes are detected among which CD8-cells are more common than CD4-cells (about 2:1). Activated T-cells positive for IL-2R and TNF-R are rare. Even during the early infiltrative stages of transplant endarteritis, scattered alpha-smooth muscle actin positive myofibroblasts are detected, often in small clusters close to the internal elastic lamina. Myofibroblasts are of utmost importance since they do not respond to conventional anti-rejection therapy and serve as machineries for intimal scar formation. Currently, it is undetermined whether myofibroblasts originate from the medial smooth muscle cell layer, from circulating progenitor cells or alternatively from trans-differentiated histiocytes. The intimal inflammatory process typically lacks a B-cell component (very rarely, individual CD-20 positive cells or plasma cells can be detected). Activated endothelial cells and infiltrating mononuclear cells upregulate MHC-class II (HLA-DR) and adhesion molecules such as ICAM or VCAM. Approximately 40% to 50% of rejection episodes with transplant endarteritis are C4d positive, i.e. show C4d along peritubular capillaries. In contrast, the accumulation of C4d along the endothelium of arteries seems to be a non-specific phenomenon, also found in native kidneys. Etiology: Transplant endarteritis is a cell mediated type of injury, mainly driven by T-cells and macrophages. C4d positive cases seem to represent mixed "cellular" and "humoral" rejection episodes that should be specifically diagnosed. Outcome : Transplant endarteritis – in comparison to tubulo-interstitial rejection – carries a less favorable longterm prognosis. The detrimental impact on transplant survival becomes already apparent after one year of follow-up. Transplant endarteritis is often associated with the subsequent development of sclerosing transplant vasculopathy (i.e. so-called chronic vascular rejection). Comment: Transplant endarteritis is a common phenomenon, seen in approximately 30% of rejection episodes occurring during the first year. We have diagnosed it as early as 6 days and as late as 14 years post transplantation. Transplant endarteritis may often be underdiagnosed due to small (inadequate) biopsy samples lacking arteries. The diagnosis of transplant endarteritis carries great prognostic and therapeutic significance. Patients usually do not benefit from conventional bolus steroid therapy, but rather require potent treatment with ATG or OKT3. Treatment is most efficient during the early, infiltrative phase when myofibroblasts are absent. However, even aggressive anti-rejection therapy often does not result in the complete histological clearance of the intimal inflammatory process, which can persist as smoldering inflammation. In our experience, this phenomenon is seen in 22% of cases. "Smoldering" intimal inflammation results in the persistent stimulation of myofibroblasts and consequently in progressive intimal sclerosis, i.e. sclerosing transplant vasculopathy. Thus, transplant endarteritis is a major risk factor for so-called "chronic vascular rejection". Approximately 40% to 50% of cases with transplant endarteritis are C4d positive. Some studies showed that outcome of these rejection episodes is poor, potentially requiring currently undetermined new treatment strategies. (Transplant endarteritis also occurs in epicardial arteries of heart allografts, however, it remains typically undetected) E) Sclerosing Transplant Vasculopathy Synonym: Chronic vascular rejection; chronic transplant vasculopathy; category V rejection (Banff '97 update); not classified according to CCTT. Definition: Rejection induced arterial intimal thickening mainly due to the de-novo deposition of collagens types I and III; absence of intimal elastosis. Macroscopic Findings: Nephrectomy specimens with pure sclerosing transplant vasculopathy are very rare; they are most often found at time of autopsy. The kidneys are reduced in size, pale pink, granular and exhibit scars. Arteries at the hilum and in the peri-pelvic adipose tissue are stenosed. (Coronary arteries and their branches often show diffuse hardening and concentric thickening with stenoses; the hearts are frequently dilated and may show acute infarcts). Histology: The lumen of arteries is narrowed by concentric or sometimes eccentric intimal fibrosis, which is most pronounced at arterial branching points. The pattern of intimal fibrosis in cases of sclerosing transplant vasculopathy displays some distinct, diagnostically helpful features: 1) Most characteristic is the lack of elastic lamellae, i.e. an absence of marked intimal elastosis. Typically the inner elastic lamina remains intact. (The elastica only gets disrupted subsequent to inflammation and necrosis of the media) 2) The fibrotic intima contains scattered, irregularly arranged myofibroblasts with enlarged "activated" nuclei. 3) The fibrotic intima may contain scattered mononuclear inflammatory cell elements, i.e. histiocytes and lymphocytes. 4) Sometimes foam cells are present. 5) During intimal remodeling, myofibroblasts may occasionally form a new rudimentary media under the endothelial layer. Such a so-called neo-media formation is almost pathognomonic for "chronic vascular rejection". (Neo-media formation appears to be more prominent in renal allografts) 6) The endothelial cell nuclei are typically enlarged, hyperchromatic and slightly polymorphic. These features help to establish a specific diagnosis of sclerosing transplant vasculopathy/chronic vascular rejection. The differential diagnosis of sclerosing transplant vasculopathy includes arteriosclerosis induced by hypertension or diabetes mellitus. Arteriosclerosis shows a hypocellular intima with only rare, small, regularly arranged myofibroblasts embedded in a dense, fibrotic intima rich in elastic lamellae, i.e., fibro-elastosis. Neither the accumulation of foam cells nor neo-media formation is a feature of arteriosclerosis. Sclerosing transplant vasculopathy can be associated with other changes. Frequently, scattered lymphocytes are found in the intima, likely a sign of an ongoing, smoldering rejection. A diagnosis of concurrent transplant endarteritis should be rendered if clusters of lymphocytes and histiocytes are noted under activated endothelial cells. Often, sclerosing transplant vasculopathy is superimposed on pre-existing arteriosclerosis (donor organ disease). In these latter cases remote, dense fibro-elastosis is detected along the inner aspect of the thickened intima and typical changes of chronic vascular rejection are superimposed towards the endothelial surface. Associated findings : Transplant glomerulopathy and transplant glomerulitis are often found, i.e. signs of glomerular rejection (in our experience in approximately 40% of cases). If sclerosing transplant vasculopathy is complicated by transplant endarteritis, the interstitial compartment usually shows evidence of tubulo-interstitial rejection. Chronic vascular rejection frequently results in arterial stenosis, ischemia, tubular atrophy and interstitial fibrosis, which can, however, be surprisingly minimal in some instances. Immunohistology: Immunohistology is unrevealing. In some cases deposits of IgM, complement factor C3, C4d and C5b-9 are present in arterial walls (non-diagnostic). In general, C4d is not detected along peritubular capillaries. If capillary C4d accumulation is noted (in approximately 30% of cases) then this observation indicates an "active" rejection episode. C4d positive cases of sclerosing transplant vasculopathy often show additional histologic evidence of active rejection, such as transplant endarteritis and/or glomerulitis. Etiology: Sclerosing transplant vasculopathy, i.e. chronic vascular rejection, is the scarring stage of rejection episodes involving the arterial tree. It is most frequently found subsequent to rejection episodes showing transplant endarteritis and can develop within few weeks. Intimal inflammation including the influx of macrophages induces the proliferation of myofibroblasts that synthesize extracellular matrix proteins, in particular scar collagens types I and III. Matrix synthesis is promoted by various cytokines and growth factors including platelet derived growth factor (PDGF), transforming growth factor beta (TGF-beta), basic fibrobast growth factor (bFGF) and others. Thus, sclerosing transplant vasculopathy is an immune mediated type of injury, initially driven by lymphocytes and macrophages. Likely, also circulating donor specific antibodies play a currently undefined role in the pathogenesis, last not least based on the detection of C4d along capillaries in some of the cases. Wether the intimal myofibroblasts are donor derived (from the medial smooth muscle layer) and/or recipient derived (from circulating progenitoir cells) is currently undetermined. Outcome : Grafts with sclerosing transplant vasculopathy, in particular, those with concentric changes or additional evidence of active rejection have an unfavorable prognosis. Frequently, graft function is lost within months to few years. Comment: Sclerosing transplant vasculopathy does not respond to treatment ("intimal scar"). However, if signs of active rejection, such as transplant endarteritis or C4d deposits, are additionally noted, therapeutic attempts can be made (anti-lymphocytic preparations, high dose tacrolimus or mycophenolate-mofetil). Cardiac allograft vasculopathy: Cardiac allograft vasculopathy is a major cause for morbidity and mortality after heart transplantation. It shares most histologic features with sclerosing renal transplant vasculopathy, however, due to the lack of histologic data much less is known about the evolution of cardiac allograft vasculopathy in humans. There is clear evidence that immunologic phenomena, i.e. rejection episodes, also promote myofibroblastic proliferation and scar formation in arteries of the heart. This process can be further aggrevated by hypertension, hyperlipidemia and diabetes mellitus. Since epicardial arteries are also prime targets for non-immunologic injuries, namely atherosclerosis induced by hypertension, diabetes mellitus or hyperlipidemia, "chronic vascular rejection" of the heart can be associated with typical atherosclerotic plaques/atherosclerotic disease. (Although hypertension induced arterial intimal fibroelastosis is common in intrarenal arteries, atherosclerotic plaque formation with calcification and accumulation of lipids are typically absent.) Often cardiac allograft vasculopathy involves epicardial arteries in a diffuse fashion, and it can sometimes even extend into small intramyocardial arterial branches. In contrast, pure atherosclerotic disease of epicardial arteries is typically focal in nature and commonly accentuated at vascular branching points. Selected References (Section C) Rejection Alexander RT, Lathrop S, Vollmer R et al. Graft vascular disease after cardiac transplantation and its relationship to mean acute rejection score. Arch Pathol Lab Med 2005, 129: 1283-1287 Ashraf S, Parrott NR, Dyer P, Roberts I, Johnson RW. Clinical response and temporal patterns of acute cellular rejection: relationship to chronic transplant nephropathy. Transpl Int 1998; 11 Suppl 1: S5-9 Billingham ME. Cardiac transplant atherosclerosis. Transplant Proceed 1987; 4 (suppl. 5 August): 19 Billingham ME. Chronic rejection in human allografts. In' Transplant vascular sclerosis', Orosz CG, Sedmak DD, Ferguson RM eds, Springer Verlag/ RG Landes Company, Austin 1995 Busch GJ, Reynolds ES, Galvanek EG, Braun WE, Dammin GJ. Human renal allografts. The role of vascular injury in early graft failure. Medicine 1971; 50: 29-83 Cary N. Heart transplantation. In 'Pathology and Immunology of Transplantation and Rejection', Thiru S and Waldmann H, eds, Blackwell Science, Oxford, 2001 Colvin RB. Kidney. In: R.B. Colvin, A.K. Bhan, R.T. McCluskey (eds). Diagnostic Immunopathology (ed. 2). New York : Raven Press 1995: pp 329-365 Colvin RB, Cohen AH, Saiontz C et al.: Evaluation of pathologic criteria for acute renal allograft rejection: Reproducibility , sensitivity, and clinical correlation. J Am Soc Nephrol 1997; 8: 1930-1941 Colvin RB: Renal transplant pathology. 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