Role of the histopathologist in assessment of the rejection patterns of renal allograft biopsies

Renal biopsies on transplanted kidneys are performed to determine whether the compromised graft function is due to rejection, nephrotoxicity caused by immunosuppressive drugs or other causes such as acute tubular necrosis, acute pyelonephritis, and obstruction of the vasculature or due to recurrent glomerular disease. A renal biopsy, whether it is from a native or a transplant kidney, is an invasive procedure used to obtain a morphological diagnosis and to get prognostic information to guide treatment. Thus, when a patient is “put at risk” by an invasive procedure, the goal should be to collect sufficient amount of tissue to render the best possible diagnosis (1). Histopathologist plays an important role in identifying the rejection pattern based on the morphological features of the allograft biopsy (2). Initial light microscopic assessment of allograft biopsies can be supplemented by immunohistochemical and immunofluorescence studies to arrive at a definitive diagnosis (3).

In a large multicenter study of more than 2,000 protocol biopsies, no patient deaths were reported (4).Similar results were more recently published from a large single center in Germany (5).Ultrasound guidance increases the probability of obtaining tissue from the cortex from 75 percent to more than 90 percent.Guidance by on-site examination with a dissecting microscope increases tissue adequacy to nearly 100 percent.
Open wedge biopsies typically require anesthesia and are not commonly undertaken after transplantation.Wedges are more frequently obtained during organ procurement to assess the suitability for transplantation or at the time of grafting, that is, implantation zero hour biopsies.
Surgeons often prefer wedge biopsies since bleeding can be better controlled and sampling generates large tissue fragments.
Before studying the light microscopic features it is important to assess the diagnostic adequacy of renal allograft biopsies.The criteria for assessing the diagnostic adequacy of renal allograft biopsies are given in table 1.

Rejection
Rejection episodes can be classified according to different schemes that guide therapeutic decisions.Frequently transplant physicians prefer a simple classification approach that is mainly based on the temporal occurrence of a rejection episode following surgery.
According to this concept "hyperacute" or "accelerated acute" rejection implies a very early event (within hours or days after transplantation), "acute" rejection is diagnosed within days or weeks, and chronic rejection is considered to be a late event occurring months or years after grafting.In hyperacute rejection, fibrin thrombi are present in renal vessels including the glomerular capillaries and peritubular venules (Fig. 1).The thrombosis is associated with acute ischaemic injury, focal hemorrhage, infiltration by neutrophils and in most severe cases frank infarcts.Immunofluorescence studies may show linear staining for immunoglobulins and complement along the walls of the capillaries and peritubular venules.Hyperacute rejection is produced by the interaction of circulating antibodies in the recipient with antigens on donor endothelial cells.The antibodies are often related to previous blood transfusions, pregnancies or an earlier transplantation.
Acute rejection may involve the interstitial compartment and/or vessels.When the vascular compartment has been involved it has been termed acute vascular or acute humoral rejection.The most severe changes occur in the small arteries, veins and arterioles (6).There is swelling and vacuolisation of endothelial cells with focal ulceration.This is associated with intimal infiltration by mononuclear cells and changes in the media such as swelling of medial smooth muscle cells.In addition there can be thrombosis.Occasionally a pattern of necrotiz-ing vasculitis may be seen.Glomeruli can show endothelial swelling, increased cellularity and occasional thrombosis.In addition there may be interstitial haemorrhages, tubular necrosis and infarctions.
Acute rejection involving the interstitial compartment has been termed acute interstitial or acute cellular rejection (7).Early stages will show oedema and focal infiltration of the interstitium and peritubular capillaries by lymphocytes.As the rejection continues the inflammatory infiltrate becomes diffuse and the lymphocytes will be accompanied by plasma cells, monocytes and macrophages (Fig. 2).Neutrophils may be present but not in abundance.
A characteristic finding of acute interstitial rejection is invasion of tubular epithelial cells by lymphocytes producing a lesion referred to as tubulitis (Fig. 3).The intensity of the infiltrate and tubular injury are features used to grade rejection.The infiltrate is more concentrated in the cortex than in the medulla.Glomerular and vascular changes are invariably present but may be mild (8).Although it is convenient for descriptive purposes to consider the two types of acute rejection separately, almost all the cases of acute rejection are really a combination of interstitial and vascular changes.Chronic rejection is not a distinct entity but rather the end stage of repeated episodes of acute rejection.Chronic rejection once initiated is irreversible.The glomerular lesions consist of ischaemic glomerular capillary collapse, thickening of the capillary walls and segmental and global sclerosis (9).Tubules show atrophy and there is diffuse interstitial scarring.The blood vessels, especially the interlobular and arcuate arteries show severe obliterative fibrointimal proliferation or mucoid widening of the media (Fig. 4).
Immunofluorescence studies may occasionally show linear or granular deposition of IgM, IgG and complement components (10).However these general clinical terms are rather imprecise, for example on histological grounds acute and chronic changes are often not sharply separated entities but rather represent an evolving sequence of morphological changes.
A renal allograft biopsy is generally considered to be the gold standard for the evalua- Most widely used and accepted is the so called Banff system, introduced in 1993.This system has significantly standardized the classification of renal transplant rejection (11).Over the past years it had undergone several modifica-tions.(Table 2) In this classification an adequate core biopsy must contain a minimum of 10 glomeruli and at least two arteries; a marginal sample is that with 7 to 10 glomeruli and one artery; and an unsatisfactory biopsy is a core with less than 7 glomeruli or no arteries.The above classification has undergone revisions in 2007 (12), to facilitate the establishment of therapeutic guidelines for clinical management (Table 3) Table 3-2007 update of the Banff classification scheme (12) 1.Normal 2. Antibody mediated changes (may coincide with categories 3,4,5 and 6), due to documentation of circulating anti-donor antibody, C4d and allograft pathology C4d deposition without morphological evidence of active rejection C4d+, presence of circulating anti-donor antibodies, no signs of acute or chronic T-cell mediated rejection or antibody mediated rejection.

Acute antibody mediated rejection
C4d+, presence of circulating anti-donor antibodies, morphological evidence of acute tissue injury, such as i.ATN like minimal inflammation ii.Capillary and/or glomerular inflammation and/or thromboses iii.Arterial-v3 Chronic active antibody-mediated rejection C4d+, presence of circulating anti-donor antibodies, morphological evidence of chronic tissue injury such as glomerular double contours, peritubular capillary basement membrane multilayering, interstitial fibrosis/tubular atrophy and or fibrous intimal thickening in arteries.
Renal allograft biopsies need to be handled accurately in the laboratory.The cores should be divided for light and immunofluorescence (IF) microscopy.Electron microscopic studies are typically limited to cases in which a glomerulonephritis is suspected.The largest tissue portion should always be processed for light microscopic studies.Classically the laboratory should prepare 2-3µm thick sections for hematoxylin & eosin (at three levels), periodic acid schiff, trichrome and elastic tissue stains.Tissue for IF microscopy can be collected by cutting off one half of one (fresh) biopsy core and examining under a dissecting microscope for the presence of glomeruli.The

3 .
Borderline changes: "Suspicious" for acute rejection Cases with foci of mild tubulitis (t1-1 to 4 mononuclear cells/tubular cross section) and interstitial inflammation (i1/mild -10% to 25% of parenchyma affected) 4. Acute/active rejection Type IA: Cases with significant interstitial inflammation (i2 or i3 >25% of parenchyma affected) and foci of moderate tubulitis (t2 >4 mononuclear cells/tubular cross section or group of 10 tubular cells) Type IB: Cases with significant interstitial inflammation (>25% of parenchyma affected) and foci of severe tubulitis (t3>10 mononuclear cells/tubular cross section or group of 10 tubular cells) Type IIA: Cases with mild to moderate intimal arteritis (v1) Type IIB: Cases with severe intimal arteritis comprising >25% of the luminal area (v2) Type III : Cases with "transmural arteritis" and/or arterial fibrinoid change and necrosis of medial smooth muscle cells (v3 with accompanying lymphocytic inflammation) 5. Chronic/ sclerosing allograft nephropathy Grade I : Mild interstitial fibrosis and tubular atrophy (less than 25% of the cortical area) without(a) or with(b) specific changes suggesting chronic rejection Grade II : Moderate interstitial fibrosis and tubular atrophy (26 -50% of cortical area) (a) or (b) Grade III : Severe interstitial fibrosis and tubular atrophy(>50% of cortical area) and tubular loss (a) or (b) 6. Changes unrelated to rejection (v-vascular, i-interstitial, t-tubular, ptc-peritubular capillaries, mild-1, moderate -2, severe -3) 27 IIA. Cases with mild to moderate intimal arteritis (v1) IIB.Cases with severe intimal arteritis comprising >25% of the luminal area (v2) III.Cases with transmural arteritis and/or arterial fibrinoid change and necrosis of medial smooth muscle cells with accompanying lymphocytic inflammation (v3) Chronic active T-cell mediated rejection, "chronic allograft arteriopathy" (arterial intimal fibrosis with mononuclear cell infiltration, formation of neo-intima) 5. Interstitial fibrosis and tubular atrophy, no evidence of any specific etiology (may include non-specific vascular and glomerular sclerosis, but severity graded by tubulo-interstitial features) Grade i. Mild interstitial fibrosis and tubular atrophy (<25% of cortical area) ii.Moderate interstitial fibrosis and tubular atrophy (26 -50% of cortical area) iii.Severe interstitial fibrosis and tubular atrophy/loss (>50% of cortical area) 6.Other : changes not considered to be due to acute or chronic rejection 29 According to the 2007 update of Banff classification scheme, -Category 1 is similar to the Banff 97 classification.-Category 2 includes antibody mediated changes which could be acute antibody-mediated rejection or chronic active antibody mediated rejection.The involvement of antibodies is proved by the detection of circulating anti-donor antibodies and the complement degradation product C4d -Category 3 includes borderline changes suspicious for acute T-cell mediated rejection (13).-Category 4 consists of T-cell mediated rejection which could be acute or chronic.-Category 5 includes chronic changes of graft rejection and category 6 remains same as in the previous classification.Minor modifications to Banff 2007 classification (table 3) were done in 2009 (Banff 2009 meeting report) and 2011 (Banff 2011 meeting report) as indicated below.Banff 2009 C4d deposition without morphologic evidence of acute rejection was included under "indeterminate acute antibody mediated rejection" -Category 2. Update Journal of Diagnostic Pathology 2013 (8); 1:22-33 There were no changes to the scoring ( t1, t2, t3, i1, i2, i3, v1, v2, v3) and grading categories.Banff 2011 The 11th Banff meeting was held in 2011 with a focus on refining diagnostic criteria for antibody-mediated rejection (ABMR).The major outcome was the acknowledgement of C4d (-) negative ABMR in kidney transplants.Banff classification remains unchanged.A new Banff working group was created to define diagnostic criteria for ABMR in renal transplants independent of C4d.Results are expected to be presented at the 12th Banff meeting to be held in 2013 in Brazil.Antibody-mediated rejection and C4d staining The pioneering work by H. Feucht et al (14), more than a decade ago which led to the detection of the complement degradation product C4d in renal allograft biopsies contributed to major changes in the understanding and classification of kidney transplant pathology.C4d is the degradation product of the activated complement factor C4, a component of the classical complement cascade that is typically initiated by binding of antibodies to specific target molecules.There is covalent binding of the degradation product C4d to endothelial cell surfaces and extracellular matrix components of vascular basement membrane near the sites of C4 activation.C4d is also found in intracytoplasmic vacuoles of endothelial cells.Since C4d is only very rarely observed along peritubular capillaries in native kidneys, the accumulation of C4d along the walls of peritubular capillaries in the renal cortex and medulla is considered to be "transplant specific".It has been reasonably concluded that the immunohistochemical detection of C4d in renal allografts is a "foot print" of an antibody response.In the majority of C4d positive allograft recipients, circulating donor specific anti-bodies against MHC class I or class II can be often found (15).C4d is scored in renal allograft biopsies based on immunohistochemical or immunofluorescence studies using either formalin-fixed and paraffin-embedded biopsy samples or alternatively fresh frozen tissue (Fig.5).However, formalin fixation and paraffin embedding results in decreased sensitivity to detect C4d and immunofluorescence studies (Fig.6) are regarded as the "gold standard" for identification of complement degradation product C4d.A, strong, diffuse and circumferential staining of peritubular capillaries in non-fibrotic and non-necrotic cortical and/or medullary regions is generally considered to be diagnostic.C4d deposits in other locations, including vessels with arteriolosclerosis, atrophic tubular basement membranes, endothelial surfaces of large arteries and glomerular basement membrane are currently considered to be nondiagnostic and should not be used in the clinical decision making (16 Updated Banff classification scheme of renal allograft rejection has introduced a scoring scheme for C4d staining results (Table