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ROLE OF ALLOGRAFT BIOPSY IN THE MANAGEMENT OF TRANSPLANTED PATIENT

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Presentation on theme: "ROLE OF ALLOGRAFT BIOPSY IN THE MANAGEMENT OF TRANSPLANTED PATIENT"— Presentation transcript:

1 ROLE OF ALLOGRAFT BIOPSY IN THE MANAGEMENT OF TRANSPLANTED PATIENT
Parmjeet Randhawa Professor, Division of Transplantation Department of Pathology University of Pittsburgh

2 OPTIMAL RENAL ALLOGRAFT BIOPSY TECHNIQUES
US guidance: serious AEs including death 2 cores obtained with 18 gauge needle ideal Evaluate adequacy of sample in biopsy suite Saving frozen tissue desirable for AMR, necessary for diagnosis ICGN EM is needed to characterize glomerular disease & demonstrate PTC-BMD Let me start off with a few words on optimal ----; All biopsies should be performed under ultrasound guidance to minimize the possibility of rare serious adverse events such as; traumatizing the hilar structures or puncturing the intestine; our program has had the mortifying experience of a patient bleeding to death after a routine biopsy, which ironically did not even show any significant pathologic change. Two cores ----; taking only one core can result in under-grading of acute rejection or missing it altogether in about 10% of cases, and for a disease like BKV nephropathy which preferentially affects the renal medulla, a single core obtained from the cortex has a false negative rate of about 35%). It is very desirable to evaluate ; Getting a report that reads ‘tissue inadequate for opinion’ and having to call the patient back to the hospital next day is not very good for the surgeon’s reputationb Saving frozen ELECTRON MICROSCOPY PERITUBULAR CAPILLARY-basement membrane duplication, which is a marker for early chronic rejection. 10 glomeruli, 2 arteries, but of course we must not follow this rule slavishly Xxxxxxxxxx John & Herzenberg j clin pathol : 1 core cortex sensitivity for acute rejection is 90%, becomes 99% with 2 cores; Medulla only sensitivity for acute rejection about 75%, specificity may be even lower as infection, obst, drugs more common here Pullman actual practices in nephropath: adv anat Pathology ; 18 or 16 gauge needles in common use; a few even used 20 gauge but is not optimal; most evaluated 9 slides for native kidneys, 8 for transplant (banff says 7); most did not do routine electron microscopy/immunofluorescence for transplant (26% immunofluorescence; 31% routine C4d in 2007; 75% who did C4d used frozen); adequacy 70% dissection scope, 20% naked eye, 7% hand lens, 4.5% conventional scope; immunofluorescence 51% michelle’s or zeus and 44% snap freeze 1

3 ADEQUACY CRITERIA FOR RENAL ALLOGRAFT BIOPSY
10 gloms, 2 arteries, examined in 7 slides Suboptimal biopsies can be diagnostic Cortex -severe tubulitis with no glomeruli -single artery with intimal arteritis Medulla -BKVN -diffuse C4d The adequacy criteria for a renal allograft biopsy are the presence of 10 glomeruli & 2 arteries, examined in the form of at least 7 slides ---(each with ‘multiple’ levels on it; Pittsburgh=2HE, 1PAS, 1TRI, 1MST, 2C4d, pullman questionnaire average 8 slides and 3 levels per slide) However, we should not follow this rule slavishly. Suboptimal biopsies can be diagnostic if there is severe------, there is a single artery , or if only the medulla is sampled, but that sample clearly shows BKVN or diffuse– Xxxxxxherzenberg j clin pathol : banff says 3-4 um thick, some prefer 2-3um; 18 gauge needles show less intimal arteritis; Pullman adv anat Pathology : most cores 8.4mm long as adequacy criterion 1

4 Determine if it is adequate Low power grade i, ci, ct, cv
GENERAL APPROACH Determine if it is adequate Low power grade i, ci, ct, cv Medium to high power evaluation needed to score g, t, v, cg, ah Synthesize findings & correlate clinical data A good general approach to follow in evaluating renal allograft biopsies is to: First determine if it is adequate for pathologic evaluation I use the low power of the microscope to grade inflammation abbreviated as i, chronic interstitial fibrosis designated as ci, chronic tubular atrophy referred to as ct, and chronic vascular dis as CV; this is not a very laborious task and I can usually do this in <15 seconds Medium to high power evaluation is needed to—-----this can take from 5 to 10 minutes per biopsy And finally we synthesize the findings & correlate ---- to reach the final anatomic diagnosis 1

5 Subcapsular inflammation and fibrosis should be ignored in scoring of the lesions (usually about 1 or two 20x fields, though in some cases it may be more extensive)

6 THE BANFF SCHEMA FOR RENAL ALLOGRAFT PATHOLOGY
REJECTION RELATED CATEGORIES -Acute or chronic antibody mediated rejection -Acute or chronic T-cell mediated rejection NON-REJECTION RELATED -Acute tubular necrosis -Drug toxicity, Donor derived pathology, -Infections, Recurrent disease -Technical & vascular complications A widely recognized schema for evaluating renal transplant biopsies is the Banff Schema for Renal Allograft Pathology In this schema causes of renal allograft dysf are divided into rejection related categories such as and non rejection related ---such as --- Amr has already been covered by dr colvin, donor derived Pathology and infections will be covered in tomorrows talks; technical and vascular complications are often diagnosis primarily on clinical grounds, although the biopsy findings can be supportive and help exclude non-technical causes of graft dysf; This morning I will restrict my discussion to—in yellow 1

7 ACUTE REJECTION Acute T-cell mediated rejection
Acute cellular rejection The first diagnostic category I want to consider is ACUTE T-cell mediated rejection. it used to be simply called acute cellular rejection, but since amr also has cellular infiltrates, ALBEIT OF A DIFFERENT KIND, the designation T-cell mediated rejection is more appropriate. 1

8 ACUTE T-CELL MEDIATED REJECTION
CTL Granzyme B Perforin GMP-17 (TIA-1) Fas Ligand CD95 APC Th IL-2 Allorecognition Direct Indirect IL-15 IL-2 Th Th Th Th No short cut needed-This cartoon by Dr David Rush illustrates the MECHANISMS OF – Allorecog OF APC ags by the direct & indirect paths result in Th producing ckines like il2 and 15 IL-2 results in prolif of tcells which ampflies itself by an autocrine pathway and damages the graft by at least 3 difft mechanisms: Some T-cells diff into ctl’s which can lyse target cells either by the fas-ligand fas pathway or the Gr/Perf cytolytic pathway 2. Other T-cells sec ifna and activate mac to produce tnfa which is a ctx ckine 3. Still other tcells produce il4 10 which can activate B-cells to produce donor specific anti-HLA Abs; NOTE THAT THIS LINK BETWEEN T-cells AND B-cells mediated immunity explains why acute cellular rejection and abm can occur at the same time XXXXXXXXX SHORT FORM: In this pathway antigen recognition leads to IL-2 mediated T-helper cell proliferation and release of WELL KNOWN cytokines such as GB, Perforin, FROM Tctx cells and tumor necrosis factor-α FROM MACROPHAGES; IN PARTICULAR I WOUD Like to point out that T-helper cells also secrete IL-4 and IL-10 which PROVIDE B-CELL HELP AND FACILITATE production of anti-HLA antibodies; thus the t-cell and antibody mediated pathways of acute rejection are linked to each other, and it is possible for biopsies to show MIXED T-cells and antibody mediated rejection; IFNg M TNFa IL-4 IL-10 anti-HLA Ab B Dr. David Rush

9 ACUTE T-CELL MEDIATED REJECTION
An alloimmune reaction mediated by cell mediated immunity Subclinical with normal creatinine Laboratory evidence of graft dysfunction Severe cases may show fever, graft tenderness, leukocytosis and eosinophilia

10 HISTOLOGIC CRITERIA FOR DIAGNOSIS OF ACUTE T-CELL MEDIATED REJECTION
Predominantly mononuclear infiltrate Presence of parenchymal damage Occurrence of subendothelial inflammation in venules, arteries DESPITE MUCH MOLECULAR LEVEL WORK IN recent years The dx criteria for acute cellular rejection are --

11 GRADING OF ACUTE TCMR-1 Severity of inflammation
Intensity of tubulitis Disruption of tubular architecture Presence of arteritis In the practice of Path is not simply enough to mention whether acute cellular rejection is present of absent An effort must be made to grade the severity of the process This grade determines both the URGENCY and the INTENSITY with which that episode of rejection must be rx The grading of acute rejection is based on

12 GRADING OF INTERSTITIAL INFLAMMATION
Limit assessment to cortex unless medulla alone sampled Ignore areas in continuity with capsule <10% area is assigned grade 0 Grades 1, 2, 3 are 10-25, 26-50, >50% Area is evaluated not the intensity Here are some practical tips for grading interstitial inflammation. Limit --- (diagnosis of rejection can be made on medullary biopsies if appropriate caution is exercised to prevent confusion with drug induced or infectious interstitial nephritis, including BKV nephropathy and bacterial pyelo) Ignore ----(I tend to ignore one 20 x field or even 2-3 fields if there is graded reduction as you go away from the capsule, and the remaining biopsy is free of inflammation) Less than 10%-- Grades 1, 2, 3 RESPECTIVELY correspond to of the biopsy area being affected by inflammation The Banff schema suggests that on the area of the biopsy affected be analyzed and that the intensity is not important (move on to figure) In the practice of Path is not simply enough to mention whether acute cellular rejection is present of absent An effort must be made to grade the severity of the process This grade determines both the URGENCY and the INTENSITY with which that episode of rejection must be rx The grading of acute rejection is based on

13 Here is an example of what I mean; both the upper panel and the lower panel show these blue circles representing lymphocytic occupying 100% of the biopsy field. According to the Banff schema both these biopsies should receive an inflammatory infiltrate score of 3; Generally speaking the model works because area of involvement & intensity of inflammation are generally proportional; however, in some biopsies allowance has to be made for the fact that the inflammation is extremely sparse; eg in the biopsies like the one in the upper panel I assign the grade i1 instead of i3. this is admittedly a subjective process; the cut off between i0 and i1 inflammation is hard to define, xxxxxxxx (it is difficult to describe precisely in words; when is see a biopsy, I try to assign it into four equal quartiles that encompass the best and the worst cases I have seen in my career, and use that in the grading process.

14 INFLAMMATION IN AREAS OF SCARRING
Inflammation in areas of scarring was considered not to be important in official versions of the Banff classification for the first 15 years. However, it is now realized scoring of the total inflammation in a biopsy including both fibrotic and non-fibrotic areas is a better predictor of graft outcome. xxxxxxxxxxxxxx For biopsies with at least mild ci, total i-score >25% is only 67% graft survival (versus 94% if total i-score <25%) (conventional i-score in non-atrophic areas has no prognostic value). Case is made that once total i-score assigned,scoring tubulitis may not add additional value!!! (t1 versus t2 similar molecular profile; tubulitis grading less reproducible than i-grading) xxxxxxxxx I was never comfortable with this recommendation as clearly patients with chronic rejection are not immune to superimposed to acute rejection.-----total i-score (including areas ci ct) better correlation with abnormal tctx burden (transcript levels) 0.85 versus 0.73 (r values) higher area under the curve in ROC analysis, better predictor of graft survival 94% vs67% with total i-score <25% versus >25% (cf conventional i-score drop was not to 67 and was of borderline significance) Total i-score matched only by cg in ability to predict graft survival; it was surpassed by diagnosis of ANTIBODY MEDIATED REJECTION;------Grading tubulitis in general more problematic than grading i-score; the two are usually in parallel; hence adding tubulitis scoring in scarred area may not mean add value

15 IMPLICATIONS OF LESION SCORING IN AREAS WITH FIBROSIS
DeKAF study 265 bx from 7 centers (7.5+/-6.1 y) 72% biopsies had tatr scores>0(conventional t = 0) 50% had iatr scores >0 (conventional i = 0) Inclusion of modified scores in data analyses yielded prognostic information Matas et al. Am J Transplant 2010: 10: 315 THE implications of lesion scoring in biopsies with scarring is shown by this so called dekaf study which evaluated (DeKAF delayed kidney allograft function study)(all cases were new onset late graft dysf (exclude BKN, GN) 72% 50% 40% Inclusion xxxxxxxxxxxxxxxxxx 40% ptci scores >0 though i-score was 0—does banff allow peritubular capillary score scarred areas? (Late onset)Delayed Kidney Allograft Function Study 265 New Exclude defined entities such as bkn, glomerulonephritis)-creatinine rise >25% or new onset proteinuria defined as alb/creatinine ration >0.2 or prot/creatinine ratio >0.5) G, i (other scores excluded because they were tightly correlated with the scores listed here)(i&t ci&ct had r=0.8) xxxx\ Miscellaneous: tubulitis-atrophy score almost identical to total i-score in 3 clusters and gave same prognostic information on univariate analysis Local pathology reading said not to differentiate clusters based on the argument that all had similar % of biopsies with diagnosis of chronic allograft nephropathy However review of data indicates % acute rejection clearly different; and % transplant glomerulopathy and calcineurin inhibitor toxicity diagnoses also seem to differ although no statistic analysis is even attempted (cluster 1 definitely less acrCGcni; others less different but then their % graft survival difference is also less pronounced) (this same group states in another paper that CAN in LATE POST-TRANSPLANT BIOPSIES 7.5+/-6.1 Years provides no prognostic information!– obviously it would if there were enough cases with >70% scarring) C4d and DSA similar proportion in 5 clusters with varying pathology (was low only in cluster 1 with mild ct)

16 GRADING OF TUBULITIS Define area of maximal involvement
Avoid tubules cut tangentially Grades 0-3 (0, 1-4, 5-10, >10) Look for disrupted tubules (2 foci, i2, i3) Atrophic tubules historically ignored Do not overlook non-atrophic areas or lymphocytes with blast transformation The points to keep in mind while grading tubulitis are: We should define Avoid- Grades 0-3 respectively correspond to none, 4 or less, 10 or less, and more than 10 (11 or more) lymphocytes per cross section respectively We must look for disrupted tubules, and if there are 2 foci of disruption and the inflammation grade is i2 or i3, the tubulitis must be graded as t3 (which is the highest possible grade) Atrophic tubules have been historically ignored in the grading of tubulitis though I just mentioned this finding does predict worse graft outcome Certainly if ct is present, we must not overlook non-atrophic tubules or lymphocytes with blast transformation that can allow a diagnosis of acute tcmr even in the presence of interstitial fibrosis and tubular atrophy

17 GRADING OF INTIMAL ARTERITIS
Look closely if interstitial hemorrhage, glomerulitis, or PTC inflammation Caveat added to report if < 4 arteries Grade v1 (<25%) (mild to moderate) Grade v2 (>25%) (severe) Grade v3 fibrinoid change, necrosis, transmural i (transmural intimal arteritis) Regarding the grading of intimal arteritis We must look closely A Caveat is added to <4 arterial PROFILES; eg we might say biopsy shows interstitial hemorrhage, but no intimal arteritis is seen in a single artery available for evaluation Grade v1 intimal arteritis refers to an artery with intimal lymphocytic infiltrates but less than 25% luminal occlusion In grade v2 more than 25% of the lumen is compromised (severe intimal arteritis) Grade 3 is characterized by transmural inflammation or fibrinoid change in the vessel wall (transmural intimal arteritis)

18 GRADING OF TCMR IN BANFF SCHEMA
Borderline: criteria higher grade not met Type IA: t2 (i2 or i3) Type 1B: t3 (i2 or i3) Type IIA: v1 (any i or t score) Type II B: v2 Type III: transmural inflam/fibrinoid PTC C4d indicates concurrent AMR ONCE I, t, and v have been scored AN OVERALL grade of tcmr is assigned to the biopsy -----(the words type and grade are equivalent; type was preferred by some participants at the 1997 meeting, because at that time there was not enough data eg to show that 1B had a worse prognosis than 1A; or if isolated v1 lesion with scarce inflammation is worse than 1b) The term BORDERLINE CHANGES SUSPICIOUS FOR ACUTE REJECTION is something will discuss in the next slide: in essence it is a biopsy that looks like rejection in terms of inflammation and tubulitis being present but criteria for a higher grade are not satisfied (I will come back to this in the next slide) Type 1 A rejection is characterized by grade t2 tubulitis; the inflammation score is i2 or i3 Type 1 B rejection must have at t3 tubulitis (at least 2 foci if tubular disruption criterion being applied) AND inflammation score of grade i2 or i3 Type 2a rejection is defined by the presence of grade v1 intimal arteritis; accompanied by any inflammation or tubulitis score; sometimes we see biopsies with intimal arteritis with little or no inflammation; such cases are referred to as ‘isolated v1 lesions’; a multicentric study organized by the banff panel is currently looking into its significance (we tend to play it safe and treat it with steroids and careful clinical monitoring) Type 2b rejection requires the presence of grade v2 intimal arteritis And finally Type III rejection requires transmural inflammation or fibrinoid change; this is a virtual death sentence for the graft; the graft loss rate is in the % range Peritubular capillary C4d deposition in any of these categories makes it necessary to diagnose concurrent amr (v3 lesion, according to some authorities occurs only in amr, though I often see it in the context of severe tubulitis in allograft nephrectomy specimens; with IIa & IIb the inflammation is often indistinguishable from that seen in 1a and ib, & CERTAINLY CD3 positive T-cells CELLS WILL OCCUR IN PLENTY; therefore in my opinion most of these cases should be called mixed tcmr and amr with intimal arteritis)YET xxxxxxxxCooperative Clinical Trials in Transplantation (now merged with banff); >5% cortex, 3 foci tubulitis within 10 consec fields of worst I, and 2/3 other features (edema, activated L, tubular injury)

19 BORDERLINE CHANGES SUSPICIOUS FOR ACUTE REJECTION
Looks like rejection but criteria I, II not met i1 with any t grade OR t1 with any i Most often t1 tubulitis and i1 inflammation For biopsies with i2,3 look for t2,t3,v1 Theoretically i1,t2,t3 & i2,i3,t1 allowed Some cases evolving or treated higher AR Coming back to b-line change---The category borderline change deserves separate discussion, as it is a term that causes trouble to many clinicians, who then start complaining about having to work with BORDERLINE PATHOLOGISTs; The best verbal description I can give you is that the term refers to changes that look like rejection (in terms of the presence of active interstitial inflammation and tubulitis) but criteria for a higher grade are not satisfied. In terms of banff scores we can either have grade i1 inflammation with ANY grade of tubulitis OR we can have grade t1 tubulitis with ANY grade of inflam Most often there is grade t1 tubulitis and the inflammation score is i1 For biopsies with i2 or i3 I hesitate making a diagnosis of borderline changes suspicious for acute rejection; instead I carefully look for and usually find a higher grade of tubulitis that results in upgrading the rejection Theoretically lesions with the scores of i1t2t3 and i2i3t1 tubulitis are allowed in the borderline category, but in these situations I usually convey my concern to the clinician that I am worried about the case and they should consider treating it if warranted by the clinical circumstance (sometimes I call it borderline to mild tcmr, although this is not a term sanctioned by the banff panel))(in an older version of the schema it was acknowledged that i2t1 could be called mild acute rejection and treated as such if the clinical situation so dictated; of course the latter is true of i2t1 too and separate reference to i2t1 was deleted)— Of course these grey area cases may be patients who have evolving rejection or have already received treatment for previous biopsies with higher grades of rejection.---In interpretation these biopsies it is important remember that some cases of borderline change are evolving tcmr (as might be particularly seen in protocol biopsies) or already treatment tcmr

20 Here is an example of the most common biopsy picture I categorize as borderline changes suspicious for acute rejection; the inflammation grade is i1 (since % whole biopsy was occupied by infiltrates). The number of lymphocytes per tubular cross section did not exceed 2 o 3.

21 SHOULD BIOPSIES WITH BORDERLINE CHANGES BE TREATED AS REJECTION?
Scheweitzer et al. 58% CR, 30% PR Saad et al. 63% CR, 13% PR Dooper et al. 24% definite rejection Gaber et al. 8/8 (100%) CR Should ----- The literature is contoversial; for example here are 4 studies in which response rate to steroids varied between 24% and 100%

22 REASONS FOR HETEROGENEITY IN RESPONSE TO TREATMENT
Borderline changes SUSPICIOUS for AR Non responsive cases may be non-immune (dehydration, ATN, CNI, infection) AMR, underlying CR Responsive cases may be early stage TCMR Examples of sampling error where biopsy did not sample more significant i or t lesions The reasons for heterogeneity in response to treatment are multiple’ First of all let us remember that the EXACT BANFF WORDING that describes this condition. It says changes SUSPICIOUS for acute rej Non responsive cases may be due to non-immunologic conditions such as Of course non response can be due to immunologic conditions such as antibody mediated rejection or underlying chronic rejection. ON THE OTHER HAND responsive cases may be early stage TCMR where YOU WERE ABLE TO NIP THE ALLOIMMUNE RESPONSE IN THE BUD Or examples of sampling error Xxx I0,i1 t0t1 subjectivity in threshold: need not stress again as done elsewhere in the talk Protocol biopsy can not be cause for response or non-response (there is no rise of creatinine to assess this) Rejection treatment before biopsy can also not be called a problem if you review whole creatinine history

23 TYPE 1A Enough about borderline changes. Let us illustrate some higher grades of rejection. here is a tubule with t2 tubulitis with about 8 lymphocytes per cs; this would be type 1a tcmr PEAK T2 CONCEPT;

24 Type 1B A tubule with more than 10 lymphocytes per cross section, and example of type 1b tcmr

25 An example of tubular disruption, sometimes also referred to as the phenomenon of vanishing tubules. This tubule looks like it has been shattered by a laser beam; you can see the damaged by tubular epithelium admixed with activated lymphocytes. Lesions like this get the highest possible grade of tubulitis i.e. t3 and should be callled banff type 1b rejection. (two foci needed with inflammation at least i3) Unfortunately this lesion tends to be missed by pathologists who do not look regularly at transplant biopsies. Infact I have seen biopsies with extensive inflammation and tubular damage being called borderline or mild rej, because people get hung up on counting the number of lymphocytes in intact tubules, which may be hard to find in such cases

26 Another problem I regularly see in consult biopsies is under-calling of acute rejection in biopsies with interstitial fibrosis and tubular atrophy. Here is a biopsy with significant tubular atrophy which was simply scanned at low power and reported as chronic allograft nephropathy. On higher power I saw blastically transformed lymphocytes and tubular disruption that qualiifed for t3 tubulitis. I called a banff type 1b rejection and the patients responded well to steroids. Xxxx Yes some tcmr episodes may get missed if no non-atrophic tubules remain to be examined (all lost to inflammation) Hpf non-atrophic disruption

27 Intimal arteritis/ >25 luminal occl so type 2b

28 INTIMAL ARTERITIS NOT ALWAYS TCMR
56% had donor specific antibodies 33% had PTC C4d diffuse or focal Can be pure AMR, pure TCMR, or mixed AMR-TCMR Sis et al Am J Transplantation 10: 421 It is now being realized that--- In one study 56%

29 An e.g. type 3 amr with transmural inflamm and fibrinoid nec
This is a very serious lesion and can be viewed as a death sentence for the graft

30 Intimal arteritisis a lesion to be taken very seriously because it can rapidly lead to infarction

31 GRADING OF ACUTE REJECTION IS IMPORTANT FOR PROGNOSIS
Banff 1 = 93% CR, 5 yr GS 67% Banff 2 = 79% CR, 5 yr GS 56% Banff 3 = 47% CR, 5 yr GS 32% There is no question that--- cr means complete response to therapy, GS is graft survival Gaber’s kidney international dataBanff 1 = 93% CR, 5 yr GS 67% Banff 2 = 79% CR, 5 yr GS 56% Banff 3 = 47% CR, 5 yr GS 32%

32 HISTOLOGIC GRADING OF INDIVIDUAL LESIONS
Graft Loss t1 tubulitis 0/36 0% t2 tubulitis t3 tubulitis 10/36 28% v1 arteritis 11/36 31% v2 arteritis 11/18 61% v3 arteritis 11/11 100% Histologic grading of individual lesions is also important; here is some of our own data on graft loss (after 3 to five years follow up I think); Grafts which NEVER show tubulitis > grade t1 or t2 in their biopsies have no graft loss after 3 to 5 yrs of follow up ( I thinks, marida 2000) With t3 tubulitis graft loss is 28%, for v1 intimal arteritis its is 31%, for v2 61% and v3 100% (28 versus 31 not significant); Xxxxxxxxxxxx Yes you can find studies saying severity of tubulitis does not correlate with outcome; but actual numbers of t3 tubulitis are usually small (we pulled out 36 cases t3 tubulitis and did a head to head comparison with 36 cases intimal arteritis) Yes can score was higher in t3 than v1 but mean difference was only 1.0 (incidence OKT3 in v1 was only marginally higher, not significant) Not that v1 arteritis only 70% graft loss; most do ok

33 IMMUNOHISTOCHEMICAL STUDIES IN ACUTE REJECTION
Not necessary for Dx (except C4d) May occasionally help d/d PTLD vs AR Intraglomerular CD68 worse prognosis CD20 not correlation AMR or response CD4/CD8/CD68: stage of evolution, patient selection, immunosuppression. What about the role of immunohistochemistry in the evaluation of acute rejection These studies are not necessary routinely, except of course for C4d. Intraglom CD68 have been shown to have bad prognosis (independent of C4d status) by Magill’s laboratory, but these has not been validated by other centers (mean glomerular CD68 count > 1 per glomeruli confers hazard ratio of 3.89 for poor graft function at 2 yr= estimated gfr<30 ml/min) Of note presence of CD20 positive B-cells, which is a B-cells marker, does not corrrelate with amr or response to anti-rejection treatment (contoversial)t CD20 positive B-cells may reflect antigen presentation role for these cells Different investigators report varying proportions of CD4 positive T-cells, CD8 positive T-cells, and cd68 positive biopsies. These differences are likely related to the stage of evolution of the rejection, patient selection (eg early graft non-function) and type of immunosuppression (eg campath). XXXXXXXXXXMay provide dx useful information in selected cases (CD68 + cells glomerulitis, D/D ACR vs PTLD vs BKVAN L-activation markers potential future role in d/d NS infiltrates from those associated with early ACR (HLA-DR, ICAM, CD25)

34 MOLECULAR DIAGNOSIS TCMR
MDx potentially valuable non-invasive tool Dx TCMR:sensitivity & specificities of 70-90% Disagreements in an average of 20% Reflect sampling issues, arbitrary grading thresholds & low frequency diagnoses Provides information that is complementary ..In the past decade there has been a lot of interest in– ..Mdx is potentially non-invasive tool for screening and avoiding unnecessary biopsies ..Current ..Disagreements between molecular and histologic methods occur in about 20% of cases. ..These reflect sampling issues and overlap with low frequency diagnoses like pyeloneph and BKVN (sampling illustrated by only a small terminal part of biopsy being taken for microarrays)(thresholds of inflammation & tubulitis used for unequivocal diagnosis of (mild) acute rejection are arbitrary)(thresolds for overexpression and underexpression of specific genes in microarray analysis are also arbitrary—2 fold over controls, positive results with 9 or more probe sets out of 15 chosen for a given gene) and are difficult to resolve; both metho ..I believe mdx methods can provide information that is complementary to & not necessarily a substitute for routine histology (if a biopsy reading is borderline changes suspicious for acute rejection and microarrays or urine cytokine analysis show marked t-cell activation we should take that seriously and keep the patient under observation)(on the other hand the biopsy shows grade 2A rejection and the small piece taken for microarrays is WNL, this should not dissuade us from treating the patient aggressively) Nonetheless

35 At this time the bx remains the gold std for dx of both T-cell mediated rejection and amr
All new molecular markers are currently all validated against this yardstick; this may change in years to come as mdx assays become less expensive and more widely available;

36 ACUTE TUBULAR NECROSIS ACUTE TUBULAR INJURY ACUTE KIDNEY INJURY
. ACUTE TUBULAR NECROSIS ACUTE TUBULAR INJURY ACUTE KIDNEY INJURY Let me now take up a pattern of injury which has conventionally been referred to as acute tubular necrosis. Since overt necrosis of cells was is not often seen in these cases the term ATI more appropriate. Clinicians tend to use the word AKI if they can not tell if the injury is restricted to the tubular compartment or whether there is any tubular injury at all (so called pre-renal acute tubular necrosis or volume responsive AKI to encompass early acute tubular injury that responds to treatment) CJASN Renal biopsy in the very elderly (D’Agati) offers these DEFINITIONS OF AKI Doubling of serum creatinine if prior renal function normal OR rise >50% in patients with prior chronic kidney injury OR creatinine > 2mg/dl if baseline renal function unknown 1

37 The most common biopsy finding in acute tubular injury its that of tubular dilatation and intraluminal proteinaceous material. A PAS stain shows focal loss of the brush border. Note that the tubular basement membranes, which can be looked upon as the cytoskletal scaffolding of this anatomic compartment remain well preserved. Hence once the noxious influence that caused AKI has been removed, remnant epithelium can proliferate and completely restore the original architecture of the tubules

38 More pronounced tubular injury results in pigmented casts
More pronounced tubular injury results in pigmented casts. Most often this is lipofuscin, but the possibility of myoglobin should be kept in mind, if there is history of trauma to the skeletal muscles; less commonly we can get hemoglobin casts if there is intravascular hemolysis, or even bile casts under conditions of marked hyperbilirubinemia) D.D unusual casts includes light chain disease and rapamycin toxicity

39 True coagulative necrosis is only seen in the more advanced cases

40 The glomeruli are of course intact in ATI; if they are necrotic the lesion really becomes an acute infarct.

41 An interesting finding seen in some patients with with significant aki, is extramedullary hematopoiesis in the IST compartment; these immature myeloid, erythroid, and lymphoid cells are presumably derived from some stem cells either residing in the kidney itself or migrating into it from the bone marrow

42 CALCINEURIN INHIBITOR TOXICITY
Cyclosporine Tacrolimus Moving on to calcineurin inhibitor toxicity. Both cyclosporine and tacrolimus produce almost identical lesions 1

43 CALCINEURIN INHIBITOR CAN CAUSE FUNCTIONAL TOXICITY
Elevation in serum creatinine Blood levels Tac/Csa may be elevated Biopsy shows no specific pathology Reduction of dosage restores serum creat Graft dysfunction attributed to vasospasm ONE important concept THAT I want to emphasize in this context is that — The typical clinical history is an elevation in serum creat 1

44 CALCINEURIN INHIBITORS CAN CONTRIBUTE TO ATI
CNI induced AKI confirmed in animals Clinically, prolonged cases of DGF can recover once calcineurin inhibitors switched Likely mechanism is reversal of drug induced vasoconstriction I also want to add that--- Calcineurin inhibitor induced- Prolonged swithed to MMF or rapamycin 1

45 The most widely recognized lesion associated with acute calcineurin inhibitor toxicity, is however, the so called isometric vacuolization of the tubular epith

46 TUBULAR VACUOLIZATION IS NOT SPECIFIC FOR CNI
Use of plasma expanders (dextran, mannitol), radiocontrast media, IVIG preps containing sucrose as a stabilizer, hyperosmolar sucrose infusions Frequently seen in context of ACR Occurs in donor biopsies Patients maintained on Azathioprine Attributed to CNI by exclusion It should be kept in mind that— Use Frequenly Occurs Patients IT SHOULD ONLY BE ATTRIBUTED TO CALCINEURIN INHIBITOR TOXICITY BY A PROCESS OF EXCLUSION; THAT IS A GENERAL PRINCIPLE TO KEEP IN MIND FOR MAKING A DIAGNOSIS OF DRUG TOXICITY IN ANY ORGAN SYSTEM XXXXXXXXXXXXXXXXXXX Saad: 70% responded to steroids (all cases) and increased tacrolimus (2/3 cases); double diagnosis can not exclude in non-responders,although complicating factors like acute tubular necrosis and chronic allograft nephropathy were present in some 1

47 In severe cases the small isometric vacuoles can fuse and form larger more coarse vacuolization.
Coarse vacuolization is generally considered to be a manifestation of ischemic injury to the tubules but experimental models confirm that pronounced calcineurin inhibitor induced injury can produce an overlapping picture with an admixture of both fine and coarse vacuoles XXXXX Biopsies with clinical ATN show histologic overlap with those corresponding to nephrotoxicity Experimental models of calcineurin inhibitors toxicity show mixed vacuolization

48 Just like we get tubular vacuoles in calcineurin inhibitor toxicity we can also get myocyte vacuolization in small arterioles. Indeed arteriolar vasospasm is an important mechanism in the pathogenesis of acute calcineurin inhibitor toxicity.

49 OTHER CAUSES OF MYOCYTE VACUOLIZATION
Amphotericin B therapy Use of vasopressors Injury secondary to cholesterol emboli Acute cellular rejection with intimal arteritis Myocyte vacuolization is not a specific lesion for calcineurin inhibitor toxicity either; Other causes of myocyte vacuolization include 1

50 A lesion sometimes mentioned in the context of acute calcineurin inhibitor toxicity is giant mitochondria; it is usually seen as A FEW SCATTERED EOSINOPHILIC GLOBULES OF VARIABLE SIZE located in the tubular epithelium; Large numbers of small and uniformly shaped mitochondria are of course seen in normal proximal tubules

51 GIANT MITOCHONDRIA Association with CNI confirmed in rats
Lesion of limited diagnostic utility, since it is uncommon, not readily recognized, & may needs EM to exclude lysosomes Not specific: described in azathioprine Rx, glomerulonephritis, acute tubular necrosis Association of calcineurin inhibitor with Nonetheless, it is a lesion It is also not specific

52 THROMBOTIC MICROANGIOPATHY
. THROMBOTIC MICROANGIOPATHY Moving on to--

53 Here is florid example: fibrin thrombi can be seen plugging virtually every capillary loop in this glomerulus

54 A bx artifact that is sometimes confused with thrombi is reflux of exfoliated tubular epithelium into the glomeruli capillary lumina; I have seen this phenomenon dignified by the term tubular epithelium emboli. To recognize this artifact one simply has to note that the material inside the capillary loops looks identical to the cytoplasm of cells that are clearly renal tubular epithelium cells

55 A second case where thrombi were seen in the afft arterioles but not the glomeruli

56 A third case where a small interlobular arteriole shows only subtle damage in the form of edema and a few strands of eosinophilic material; there is no definite thrombosis.

57 LABORATORY ABNORMALITIES
Thrombocytopenia Schistocytes on PBF Elevated serum bilirubin Low serum haptoglobin Presence of free Hb in plasma In cases with subtle and inclusive findings of the type I just showed you, laboratory abn may be helpful in establishing a confident diagnosis of thrombotic microangiopathy. These findings include-- 1

58 COMMON DRUGS ASSOCIATED WITH THROMBOTIC MICROANGIOPATHY
Cyclosporine Tacrolimus Sirolimus? The most common drugs associated with thrombotic microangiopathy are Cyclosporine and thrombotic microangiopathy A few cases of sirolimus have also been reported, but the evidence is not as strong (need more patients with careful exclusion of other etiologies) 1

59 LESS COMMONLY USED DRUGS
Vasopressors: dopamine Antimicrobials: penicillin, metronidazole Anti-inflammatory: penicillamine Anti-epileptic: phenytoin Chemotherapeutic agents: mitomycin Less commonly used drugs in the transplant setting are: --- just list them xxxxxx Vasopressors: esp dopamine which causes a unique lesion called segmental arterial mediolysis----(actually ischemic injury to endothelium may itself be a cause of thrombosis) We have antimicrobial compounds such as— Anti-inflammation Anti-epilep Chemo And cyclosporine, tacrolimus, and possibly sirolimus; although I have listed these drugs last, they are probably the commonest cause TMP in the transplanted pts 1

60 THROMBOTIC MICROANGIOPATHY IS MAY BE OF IMMUNE ORIGIN
Antibody mediated rejection T-cell mediated ac rej with intimal arteritis. Ac rejection treated with OKT3 Autoimmune disorders with circulating autoab (ACA, APA, ANCA) In the differential diagnosis of drug induced thrombotic microangiopathy, is imp to remember that this condition may be of immune origin. Examples include: & xxxxxx ACA anti-cardiolipin (hepatitis C, autoimmune hepatitis, primary biliary cirrhosis, esld); APA antiphospholipid is SLE, drugs I hydrlazine, procainamide, Mdopa), collagen vascular disease, old age, idiopathic); cANCA wegner is really interlobular and muscular arteries; 1

61 INFECTIONS ARE IN IMPORTANT CAUSE OF TMP
Gastroenteritis (E.coli, Shigella) CMV infection (damage to endothelium) HCV (anti-cardiolipins) Parvovirus infection (targets endothelium) HIV infection (TTP syndrome) Infections are anther important— Examples include --- xxxxxxxxxxx GE due to e. coli or shigella WHICH is THE COMMONEST CAUSE OF SPONTANEOUSLY RESOLVING hus in clinical practice. Other possibilities are: CMV infection (where the virus directly damages the endothelial) Hepatitis C virus which is occasionally associated with anti-cardiolipin antibodies Parvovirus infection (where pure red cell aplasia will be a useful clue) And HIV infection (where some patients are known to develop ttp= thrombotic thrombocytopenic purpura) In one study from Boston, Hepatitis C virus serology was + in 30% of patients with de novo thrombotic microangiopathy in kid transplant pts 1

62 Recurrent rate depends on mutation
RECURRENT GENETIC HUS Recurrent rate depends on mutation 50% for CFH, CFI (inhibitors complement) -liver-kid transplant may be curative Not expected MCP/CD46 (present in donor kidney, will catalyse breakdown of C3b, C4b normally) -extrarenal activation of complement -chimerism in glomerular capillaries - -Thrombotic microangiopathy in an allograft can also be manifestation of Recurrent genetic (atypical) -Recurrence rates depends on the exact mutation. T -It is about 50% for atypical hus associated with the deficiency of COMPLEMENT factor H or I, proteins produced primarily by liv -Theoretically, recurrence is not expected in membrane cofactor protein or cd46 deficiency -since this protein is produced by most organs, and the donor kidney is likely not to have the same mutation -nevertheless at least one case of recurrence has been reported, and has been speculated to be the result of EXTRA-RENAL activation of complement by (extra-renal) mutant protein or chimerism in glomerular capillary endothlium (localised production of mutant protein), Xxxxxxxxxxxxxxxxxxxxxx Recurrent genetic (atypical) hus (30-50% of atypical HUS have genetic mutations (complement factor mutations make you more prone to activation of alternate or classic complement pathway which in turn can activate coagulation pathway) Theoretically, recurrence is not expected in membrane cofactor protein or cd46 deficiency (in which there is impaired breakdown of complement proteins c3b and c4b), -since this protein is produced by most organs, and the donor kidney is likely not to have the same mutation nevertheless at least one case of recurrence has been reported, and has been speculated to be the result of EXTRA-RENAL mutant protein or microchimerism, i.e. the partial replacement of the allograft parenchyma by recipient derived (CAPILLARY ENDOTHELIAL mononuclear or epithelial ?stem) cells-----localization but is widespread including both epithelial and mononuclear cells since cd46 can act as a receptor for both measles virus (neurons) and hhv6 (cd46 also present in sweat glands, basal and squamous cancers, cutaneous nevi, spermatozoa) 1

63 OTHER BIOCHEMICAL DEFECTS ASSOCIATED TMP
Deficiency of anti-thrombin III Protein C Protein S Factor V mutation (resistance to Protein C) Homocysteinemia (B6, B12 deficiency) Less common biochemical abnormailities associated thrombotic microangiopathy include -- -- And Homocysteinemia which may have an underlying CORRECTABLE deficiency of vitamin B6 or B12 xxxxxxxxxxx (vitamins must be involved in normal metabolism of homocys, which is toxic to endothelial cells when present in high concentrations in the blood) coag abn listed here actually cause VENOUS thrombosis more often; factor V mutation is interfering with normal ANTI-coag function of protein c) 1

64 CNI ASSOCIATED ACUTE ARTERIOLOPATHY
Intimal edema and fibrin Necrosis of myocytes leaves behind empty basement membranes bags Filled up by knot like protein deposits Knots fuse to form ring of pearls IN addtion to thrombotic microangiopathy calcineurin inhibitor have been associated with acute— It is characterized by intimal Medial Knot

65 An example of acute cyclosporine associated arteriolopathy (from a textbook probably brenner’s); severe endothelial injury has led to marked intimal edema and fibrin leakage. CERTAINLY THESE CASES CAN PROGRESS TO THROMBOTIC MICROANGIOPATHY & EVEN PRESENT AS HUS

66 Fibrinoid change in arteriolar wall Collapse of distal glomeruli
ARTERIOLONECROSIS Fibrinoid change in arteriolar wall Collapse of distal glomeruli Healing by onion skin change The most severe from of arteriolopathy is --— It is characterized by fibrinoid--- Collapse of Healing by myofibroblast proliferation results in onion skinning

67 Examples of arterioles with fibrinoid necrosis.
Personally I have never seen cases of calcineurin inhibitor toxicity this severe, although they have described in cyclosporine literature from the 80’s. This photograph is from a native kidney with malig hypertension

68 Healing of arterolar necrosis is effected by concentric prolf of fblasts that leads to an onion skinned appearance of the affected small artery

69 CHRONIC GRAFT DYSFUNCTION
Evolves slowly Usually begins > 3m post-tx Typically progressive Rate of decline, may be punctuated by periods of stability UPTO NOW my talk focused on ACUTE causes of graft dysfunction; I will now shift gears and talk about CGD; Cgd IS A graft dysfunctiion that evolves slowly--

70 MAGNITUDE OF PROBLEM This graph gives you an example of the Magnitude of the problem. (x-axis) If we look at patients 8 yrs out, (y-axis) approximately 50% of the grafts are functioning; and this projected graft half life has not changed over the years, although the rate of acr has fallen from 80% in the initial days of transplantation to <10% under modern immsup regimens J. Actual numbers Varies of course, other centers or even 60. this data is from a national database published by Meier 2004:4:378 )(SRTR ; scientific registry of transplant recipients) J. Acute cellular rejection incidence improved from 80% to less than 10% nationwide 1 yr survival increased 40% to 95% Improvements in half life seen primarily in high risk patient with greatly improved management

71 CAUSES OF CHRONIC GRAFT DYSFUNCTION
REJECTION RELATED CATEGORIES -Chronic active T-cell mediated rejection -Cronic active antibody mediated rejection NON-REJECTION RELATED CATEGORIES -Calcineurin inhibitor toxicity -Glomerulonephritis -Recurrence of original disease -Donor disease (age, hypertension) -RA stenosis, reflux nephropathy, recurrent UTI/BKN -Technical & vascular complications Of the various causes of cgd, I will cover one rejection related category, namely, catcmr, since camr already covered bob colvin In the non rejection category I will cover--- There will be no time to talk about --- h

72 CHRONIC REJECTION (BANFF 1991)
No distinction made between chronic active TCMR or chronic AMR Chronic Transplant Glomerulopathy New onset arteriosclerosis (not pre-existing donor disease) Let us begin with chronic rejection as defined in the banff 1991 conf; At that time no distinction--- There were two criteria laid down for diagnosis, namely xxxxxxxxxxxxx Subsequently, in the late 90’s--, an additional criterion—peritubular capillary reduplication by electron microscopy- was added (cannot really see it officially in any report, before the 2005 report that is still being drafted), and most recently in 2003, a chronic antibody mediated rejection category was added, which I will discuss next. (original formulation of chronic rejection, there was no subdivision into the chronic active cellular and chronic active antibody mediated categories) In the 2005 conf, the official proceedings of which were published in 2007, both transplant gmp and peritubular capillaries duplic are mentioned in the chronic active antibody mediated rejection group, ALTHOUGH it is recognized that a currently undefined percentage of these patients have no c4d deposits in the tissue, and no circulating donor specific antibody, so that these might be the end result of cellular rather than antibody mediated rejection J. txgm: number of glomeruli to be examined not standardized; ivanyi at least capillary loops should have 2 fold or more thickening

73 CHRONIC TRANSPLANT GLOMERULOPATHY
Reduplication of GBM Subendothelial translucent material Mesangial interposition (tram track MST) Suggested increase in capillary wall thickness of >2 fold, at least 3 capillary loops Ivanyi 2001; Mod Pathol 14:1200 Chronic transplant glomerulopathy is by --- list 3 bullet points It has been suggested that the diagnosis be based on an increase in capillary thickness that is at least 2 fold ABOVE NORMAL, and AFFECTS AT LEAST 3

74 LM of a biopsy with chronic transplant glomerulopathy: glomerular basement membranes thickened, but cannot really see the duplication needed to make a firm dx

75 This is an illustration of the chronic phase of thrombotic microangiopathy. There is reduplication of glomerular basement membranes. Mesangial cells insinuate between the epithelial and endothelial layers of the basement membranes and produce a lesion that mimics chronic transplant glomerulopathy

76 . On em electron micrographs show the presence of g’ar transluc material s/e; no icds seen as one might expect in mpgn

77 This micrograph shows mes interpos---i. e
This micrograph shows mes interpos---i.e. an insinuation of mes cell cytoplasmic processes between the two layers of gbm

78 DIAGNOSTIC CRITERIA FOR CHRONIC REJECTION (BANFF 1991)
Chronic Transplant Glomerulopathy New onset arteriosclerosis (exclude pre-existing donor disease) - intimal fibrosis without elastosis - fibroelastosis may develop after prolonged graft dysfunction The SECOND diagnosis criterion for chronic rejection in the banff 91 conference is new onset arteriosclerosis; by new onset we mean that we must exclude pre-existing donor disease, an increasingly important consideration in an era, where donors above the age of 50 or even 60 are being accepted for transplantation; The lesion is typically described as intimal fibrosis— However fibroelastosis may develop -- ---elastic stain said to be necessary to distinguish fibroelastosis of hypertension (mihatsch); he also says it is pseudoelastin= actually collagen despite staining characteristics xxxxxx iagnosis criteria for chronic active cellular rejection; In the First Banff Conference, two criteria were recognized— Subsequently, in the late 90’s--, an additional criterion—peritubular capillary reduplication by electron microscopy- was added (cannot really see it officially in any report, before the 2005 report that is still being drafted), and most recently in 2003, a chronic antibody mediated rejection category was added, which I will discuss next. (original formulation of chronic rejection, there was no subdivision into the chronic active cellular and chronic active antibody mediated categories) In the 2005 conf, the official proceedings of which were published in 2007, both transplant gmp and peritubular capillaries duplic are mentioned in the chronic active antibody mediated rejection group, ALTHOUGH it is recognized that a currently undefined percentage of these patients have no c4d deposits in the tissue, and no circulating donor specific antibody, so that these might be the end result of cellular rather than antibody mediated rejection J. txgm: number of glomeruli to be examined not standardized; ivanyi at least capillary loops should have 2 fold or more thickening

79 An example of intimal fibrosis affecting inv of a larger interlobar artery running through the medulla; similar lesions may be seen in the main hilar arteries as well as smaller interlobular arteries that feed the glomeruli

80 R. Colvin Dr Robert Colvin, ATC 2006
An elastic tissue stain showing absence of absence of elastosis in allograft arteosclerosis (to the left, single layer), in contrast to native kidney arteriosclerosis attributed to hypertension, where we see marked duplication and Multilayering of the elastica (This slide is from one of Bob Colvin’s talks at ATC 2006_ R. Colvin Dr Robert Colvin, ATC 2006

81 DIFFERENCES BETWEEN GRAFT AND ENDEMIC ATHEROSCLEROSIS
Extent of involvement (diffuse vs proximal) Type of luminal compromise distinctive Calcification is late & less common in GAS. Differences in biochemical composition (Apo-A,E, biglycans vs Apo-B, decorin) Differences are relative & not absolute There are ALSO OTHER differences— The extent of The type of ----concentric vs eccentric Differences in ---- but these may reflect AGE of the lesion or the RATE of development and not true differences in the biology of these two lesions We should keep in mind that these differences are relative and not absolute; for example, if we make an early diagnosis by cardiac gax by intravascular cardiac ultrasound, we will find lesion that are neither diffuse nor concentric

82 . An example of the concentric nature of graft arteriosclerosis; THIS IS EXPECTED since endothelial cells all around the circmuf bear the same HLA-antigens and are equally likely to be targeted by alloimmune mechanisms

83 WHO: Eccenteric arteriosclerosis

84 At autopsy or allograft nephrectomy, vessels from organs with chronic rejection can show visible atheromatous plaques similar to those seen in patients with myocardial infarction secondary to coronary artery disease

85 Angiograms show multiple areas of kinking and deformity can be used to make a diagnosis of chronic rejection. This is particularly imp in the heart and liver, where allograft biopsies seldom sample arteries showing the diagnostic lesions; For ht transplant recipients some centers periodically monitor their patients with coronary angiography for development of chronic rej

86 CLASSIFICATION OF CHRONIC REJECTION (BANFF 2005)
Chronic active T-cell mediated rejection - chronic graft arteriopathy (intimal thickening +/- inflamm+/- neointima) - absence of C4d and DSA Chronic active antibody mediated rejection -chronic tissue injury (cg, ci, ptcdd) - with C4d and DSA Due to work done in the late 90’s and the early part of this decade it became clear that some cases of chronic rejection were antibody mediated; Hence in the 2005 banff conference chronic rejection was offcially subdivided into catcmr & camr. A diagnosis of ctcmr requires demonst of chronic graft arteriopathy defined as intimal thickening w/wo inflammation w/wo neointma It is important to show absence of C4d and donor specific antibodies since this lesion has clearly been shown to occur in amr too (proof esp convincing in mice ht transplant) Chronic active amr is defined by a lesions of chronic tissue injury such as – with -- XXXXXXXXXXXX Chronic graft arteriopathy is really graft arteriosclerosis ----(what % of these are tcmr and what camr is currently undefined; donor specific antibodies probably found in 20-60% of different studies; on the other hand it is my experience that a similar range of biopsies have tcells demonstrable in the intima) Neointimal is second layer intima formed by myofibroblasts lining up between internal elastic lamina and endothelium

87 CHRONIC TXGM IS FRQUENTLY DUE TO CHRONIC AMR
30-70% biopsies DSA (mostly class II) 20-40% have C4d in peritubular capillaries ENDAT seen on microarray analyses Significance of C4d –ve cg+ biopsies? AMR, TCMR, TMP, MPGN (as pointed out by Bob before me) It has NOW being increasingly recognized that ---- 30-70, Endothelium associated transcripts are seen on microarray analysis of these biopsies and the pattern of expression overlaps that seen in classic amr The significance of is presetly unknown;--- some regard these as evidence of past antibody damage (so called hit and run antibody injury) while others consider it to be an antibody independent (possibly cell mediated) mechanism of glomerular basement membrane damage; A minority of cases represent healed thrombotic microangiopathy or an immune complex disorder such as mpgn. ENDAT seen on microarray analyses in many biopsies (limited studies, but not surprising in a capillary damage disease) The significant of C4d negative cases has become a debatable issue: AMRloving/philiac pathologists think all cases of cg represent amr whether or not antibodies are demonstrable at the time of diagnosis; I think that is going too far; there is really no reason why T-cells mediated immune injury can not damage glomerular capillary endothelium and result in basement membranes duplication-----Xxxx cell mediated immunity contribution could vary with immunosuppression protocol; Monkeys correln is with amr and not tcmr

88 REDUPLICATION OF PERITUBULAR CAPILLARY BASEMENT MEMBRANES
Needs EM: single PTC with 7 layers 3 PTC’s with 5-6 layers Duplication >60-75% of circumference Thus defined, lesion will only rarely occur in other diseases: TMP, reflux, Phenacetin kidney (repeated endothelial injury) Another defining lesion for chronic rejection is ----(amr, if c4dDSA+ & presumed tcmr if negative) Demonstration of this lesion requires electron microscopy and we can either have a single PTC— Or we can have 3 ptcs --- The duplication must affect --- of the capillary loop examined Thus defined— xxxxxxxxxxx Mihatsch says phenacetin kidney the correct term (not analgesic nephropathy); ist change is capillary thickening (capillary sclerosis) in peritubular capillaries and mucosal capillaries in descending urinary tract Xxxxxxxxxxxxxxxxxxx Again per cent capillaries actually C4d positive is not well defined; the investigators who described this lesion originally did no j. Number of capillaries to be studied not yet standardizes; ivanyi studied all available; gough minimum of 10.

89 . Here is a p/t cap where the endoth bm is throw into multiple layers; you can definitely count more than 7 in this area;

90 CHRONIC CALCINEURIN INHIBITOR TOXICITY
Cyclosporine Tacrolimus The next clinical entity to be discussed is --- > The remarks that I make/lesions that I will illustrate, will be applicable to both cyclosporine and tacrolimus nephrotoxicity.

91 PATTERNS OF TISSUE INJURY IN CHRONIC CNI TOXICITY
Arteriolar lesions Striped Interstitial fibrosis/tubular atrophy Chronic phase thrombotic microangiopathy Ischemic glomerulopathy Focal segmental glomerulosclerosis Juxtaglomerular hyperplasia Tubular vacuolization and calcification Five patterns of tissue injury have been recognized in chronic calcineurin inhibitor toxicity. These are ---; I will have time to discuss only the first two lesions, which are the most common ones we see in practice.

92 No complete internal elastic lamina
ARTERIOLAR LESIONS 1-3 layers smooth muscle No complete internal elastic lamina Diameter < 1/3 rd of a glomerulus Let us begin with arteriolar lesions. These affect small vessels that have xxxxxxxxxxxxxxxxxxxx By definition ARTERIOLAR HYALINOSIS is a lesion that affects arterioles; Arterioles should be distinguished from small arteries and one reasonably workable definition proposes that An arteriole is a vessel with 1-3 layers of smooth muscle, no COMPLETE internal elastic lamina, and a diameter that perhaps is less than that of a glomerulus sectioned approximately through its center. The SIZE criterion is arbitrary but I find it useful because sometimes vessels are just at first glance too large to be called arterioles but their muscular wall may be atrophied &/or the elastic lamina not well visualized)

93 ARTERIOLAR HYALINOSIS
Deposition of glassy material Initially in endothelial layer Followed by circumferential extension Medial involvement The commonest arteriolar lesion of chronic calcineurin inhibitor toxicity is arteriolar hyalinosis. It is characterized by ---- xxxxxxxxxxxx By definition ARTERIOLAR HYALINOSIS is a lesion that affects arterioles; Arterioles should be distinguished from small arteries and one reasonably workable definition proposes that An arteriole is a vessel with 1-3 layers of smooth muscle, no COMPLETE internal elastic lamina, and a diameter that perhaps is less than that of a glomerulus sectioned approximately through its center. The SIZE criterion is arbitrary but I find it useful because sometimes vessels are just at first glance too large to be called arterioles but their muscular wall may be atrophied &/or the elastic lamina not well visualized)

94 Here is an eg of arteriolar hyaline and eosinophilic material that permeates the vascular wall and has become focally transmural.

95 ARTERIOLAR HYALINOSIS
Donor disease Diabetes mellitus Hypertension PSS Chronic thrombotic microangiopathy Radiation Ah is of course not a lesion that is specific for calcineurin inhibitor toxicity. I can be seen in a host of other conditions, viz---

96 On immunofluorescence examination affected arterioles may stain for IgM, c3, because the hyaline material is derived from insudation of plasma proteins Note medial and adventitial involvement

97 On electron microscopy hyalin material is variably electron dense; It can form Knot like deposits if it fills up empty basement membranes bags left behind by necrotic myocytes On electron microscopy, granular appearance, the ELECTRON DENSITY density of which can vary considerably. A NODULAR configuration of these deposits AND DEGENERATIVE or even necrotizing changes in the smooth muscle are said to be characteristic of calcineurin inhibitor toxicity. However, in my experience nodular hyalin change can occur with aging and prolonged hypertension and diabetes mellitus

98 INTERSTITIAL FIBROSIS IN CALCINEURIN INHIBITOR TOXICITY
. INTERSTITIAL FIBROSIS IN CALCINEURIN INHIBITOR TOXICITY I will now discuss

99 PATTERNS OF FIBROSIS Striped fibrosis: alternating areas of atrophic AND normal or even hypertrophic renal parenchyma Diffuse interstitial fibrosis: more extensive and uniformly distributed collagen deposition There are two patterns of fibrosis reported with the use of calcineurin inhibitor. One is the so called striped fibrosis which is comprised of ----- The second is a diffuse ---- (may be a later stage of the striped pattern of injury) Diffuse fibrosis to cyclosporine/tacrolimus: contributions from isch and immune injury are difficult to exclude in the reported cases 1

100 An example of the so called
There is a linear band of tubular atrophy and ci bounded on either side by This lesion is probably secondary to the arteriolar and interlobular (calcineurin inhibitor hypertension) lesions I have just described. Let us imagine there is one interlobular artery running here (gesture it) and another parallel one running here. If both these vessels become narrowed due to intimal sclerosis, we would expect a narrow area of renal parenchyma in the watershed zone to become atrophic, while the tissue in immediate proximity to the vessels will retain a reasonable blood flow; this would resut in a striped pattern of fibrosis xxxxxxxxxxx SECOND EXPLANATION: area most prone to injury and hence fibrosis (high metabolic demand, low pO2, due to its being farthest available from arterial supply

101 GLOMERULONEPHRITIS IN THE ALLOGRAFT KIDNEY
Donor transmitted glomerulonephritis De-novo glomerulonephritis Recurrence of original disease Morphologic spectrum similar to native kidneys Regarding glomerulonephritis in the allograft kidneys; we should try to classify them into one of 3 categories---- The morphologic spectrum of glomerulonephritis is similar to that seen in native kidneys; It has been said that the transplant kidney is after all a kidney (hunsicker/halloran) XXXXXXXXX – de nov0 Occasional---- IgA in east and even cases of lupus (which resolved in new recipient) By far the most common --- see next slides---

102 DONOR TRANSMITTED GLOMERULONEPHRITIS
IgA nephropathy SLE MPGN Examples of donor transmitted glomerulonephritis documented in the literature include--- Interestingly immune complex deposits associated with these diseases gradually resolve after transplantation; unless the kidney is by sheer chance given to a recipient who has circulating immune complexes similar to those present in the organ donor xxxxx Donor biopsy scenario; go by chronicity; wont’ progress MPGN may not be seen on frozen donor; no catastrophe; will likely improve– review old and new heptinstall

103 DE-NOVO GLOMERULONEPHRITIS IN THE ALLOGRAFT KIDNEY
Work up infection, neoplasms, autoimmune Review medication (captopril, hydralazine) Elicit past h/o horse ATG for TCMR Recently C4d + cases MGN recognized Most frequently, however, no cause found The term denovo glomerulonephritis refers to glomerulonephritis occurrin in the allograft kidney of a patient who had some non-glomerular cause of end stage kidney disease When this happens, it is important to work up the patient for---- We should review the medication as some drugs like captopril and hydralazine can result in immune complex deposits in the kidney One should elcit past history of administering horse Atg as these animal proteins can also occasionally deposit in the glomeruli Recently C4d -- Frequently no cause of glomerulonephritis is identified; these cases are probably alloimmune in origin Xxx Of course we are refering to c4 in peritubular capillaries of biopsies with mgn C4d + cases have been recognized recently suggesting that some cases are really manifestations of amr xxxxxxx Hepatitis C virus, hepatitis B, mucin producing adenocarcinoma pancrease and colon/lung statistically Autoimmune syndromes systemic lupus erythematosus and mctd Gold salts, captopril membranous nephropathy, The most common drugs that cause drug-induced lupus erythematosus are hydralazine, procainamide, quinidine, isoniazid, diltiazem, and minocycline procainamide lupus like syndroms

104 DE-NOVO ANTI-GLOMERULAR BASEMENT MEMBRANE DISEASE
Seen in patients with Alport’s (<15%) Exposure to Goodpasture’s antigen Often subclinical but may cause graft loss Histology: proliferative or crescentic GN ELISA, IF confirmation antiGBMab In passing I would like to mention that It is due to exposure of the recipient to GPantigen for the first time after transplantation. The disease is often subclinical-- xxxx

105 RECURRENT GLOMERULONEPHRITIS
3rd most frequent cause of graft loss at 10y (1=chr rej, 2=death with functional graft) Accounts for 8.4% of all lost grafts overall Range %, highest if male sex, primary disease FSGS or MGN High pra was listed as increasing graft loss rate along with male sex and h/o fsgs/mgn; Tx not contraindicated (overall graft loss at 10 yr is 45.4% vs 45.8% Briganti et al 2002; NEJM 347: 103

106 RECURRENT GLOMERULAR DISEASE
Focal segmental glomerulosclerosis 3.4y Membranoproliferative GN 3.6y Membranous nephropathy 3.3y IgA nephropathy 4.4y Life of avg CRTX 13y, LRD 21y Once recurrence has occurred there is a significant effect on graft longevity; the projected half life for a graft with recurrent fsgs is -, for recurrent mpgn it is, recurrent mgn-,IgA, 4.4, recurrent diabetes mellitus, recurrent hus— By comparison the LIFE (not half life) of an average cadaveric--, living related --- Hariharan et al 1999; Transplantation 68:635

107 Hariharan et al 1999; Transplantation 68:635
RATES OF GRAFT FAILURE Focal segmental glomerulosclerosis 65% Membranoproliferative GN 66% Membranous nephropathy 44% IgA nephropathy 41% Diabetes mellitus 53% HUS/TTP 63% Data from the same study, expressed as rates of graft loss; for recurrent fsgs the rate of graft loss is --, recurrent mgn--, recurrent IgA— Of course graft failure in most of these cases will be multifactorial xxxxxxxxx Probably a 10 year data; all causes of graft failure lumped together presumably pooled (UNOS database); united network of organ sharing Hariharan et al 1999; Transplantation 68:635

108 I CAN not possibly cover the whole spectrum of recurrent glomerulonephritis in the allograft. But I will give you a couple of examples. Here is a trichrome stain showing recurrent focal segmental sclerosis

109 On immunofluorescence exam segmental sclerosis lesions show ns trapping IgM and c3 confined to the segmentally sclerotic area of the glomerulus

110 The earliest stages of recurrent disease can only be diagnosed by electron microscopy, and the most imp finding here is the extensive fusion of podocyte foot processes. Hence if a patient transplanted for focal segmental glomerulosclerosis devs puria early after transplantation it is important to save tissue for electron microscopy; light microscopy may not show any segmental sclerotic lesions since these take several weeks to develop. xxxxxxxxxxxxxxxxxxx Here is a glom capillary loop (6 o’clock); this is the endothelial cell; here is a podocyte on the epith aspect with some remnant foot processes. However, most of the glomerular capillary loops have no visible foot processes remaining; you can only see this cytoplasmic band corresponding to

111 MANAGEMENT OF PATIENTS TRANSPLANTED FOR FSGS
Early dx & intervention best prognosis Ur.prot/creat ratio OD to disch, wx4, mx12, Ratio > 0.5 consider 24 hr measurement Puria >2g/day consider biopsy confirmation Plasmapheresis removes permeability factor I would like to make a few remarks on Early Ur If the ratio If Puria Plasmaph Xxxxxx Heavy native proteinuria may confound some cases (consider pre-transplant medical nephrectomy with NSAID or A-IIR blockade

112 DE NOVO FOCAL SEGMENTAL GLOMERULOSCLEROSIS
FSGS in patient tx for another cause Common finding in ISTA Most cases months to years after Transplant -Ischemic glomerulopathy secondary to ah -Hyperfiltration injury, a compensatory response to loss renal mass Denovo fsgs refers to the occurrence of segmental lesions in a patient who was transplanted for some cause other than focal segmental glomerulosclerosis. It is a common Most— The pathogenesis is related to two factors, namely, ischemic

113 Let me quickly run through another example of reference glomerulonephritis, namely IgA nephropathy: On lm, the commonest finding is--

114 By immunofluorescence we see IgA dominant or co-dominant deposits in a primarily mesangial locations

115 Electron microscopy confirms the primarily mesangial and parames location of the edds

116 RECURRENT METABOLIC DISEASES
Diabetes mellitus Amyloidosis LCDD Lipoprotein glomerulopathy Fabry’s disease Metabolic diseases also recur after transplantation; this is expected since the transplantation procedure itself does not correct for the metabolic defect; Examples include---- light chain deposit disease and Fabry’s disease xxxxxxxxxxxxxxxxxxxxxxxx Primary hyper-oxaluria type I (deficiency of alanine/glyoxalate aminotransferase) and type II (deficiency of glyoxalate reductase/hydroxypyruvate) frequently recur in the allograft kidney, unless prior or concomitant liver transplantation is performed to correct the underlying enzyme deficiency ( ). Type II enzyme is not deficient in the liver, and it is not clear if liver transplantation can be recommended in type II hyperoxaluria (198). Post-transplant stone formation can be prevented by aggressive hydration and serial measurements of oxalate crystal volume in the urine (199). In amyloidosis, the presence of systemic disease makes the post-operative management clinically difficult ( ). Patients who do survive a year or more post-transplant show recurrent amyloid in the graft in about 20% of the cases. Amyloid deposits progress slowly and do not directly influence graft survival. Dialysis related amyloid regresses after transplantation (230). Fabry’s disease, an X-linked deficiency of alpha-galactosidase A with systemic accumulation of glycosphingolipids, has been noted to recur in the allograft despite the fact that the donated organ would be expected to have normal enzyme activity. Histologic recurrence does not impact clinical outcome if patients are screened for cardiovascular complications before being accepted for transplantation (231). Indeed, heterozygous female carriers with very mild renal lesions have been successfully used as organ donors (232). Graft thrombosis has been described in some patients who have associated protein C deficiency (233). Lipoprotein glomerulopathy can recur in the transplanted kidney ( ). Biopsies show lipoprotein thrombi in the glomerular capillaries. Biochemical investigations show abnormalities in apolipoprotein E. Recurrence is also documented for another hereditary disorder, lectithin cholesterol acyl transferase deficiency, characterized by foam cell accumulation in the mesangium, interstitium and tubules (239, 240). Finally, it deserves mention that renal transplantation has been successfully performed in patients with renal cell carcinoma and underlying Von Hippel-Lindau disease without tumor recurrence (241).

117 RECURRENT DIABETIC NEPHROPATHY
Electron microscopy thickening 6 months Arteriolar hyalinosis 2 years Nodular glomerulosclerosis 18% by 13 years Rate of progression slow 10 year graft survival 32% vs 52% in one study Graft loss attributable entirely to DM<10% One third or more of all transplants are done for diabetes mellitus; therefore let me briefly discuss recurrent nephropathy. Although not clinically significant recurrent diabetic injury starts occurring immediately after transplantation; Electron microscopy can demonstrate— xxxxxxxxxxxxxx 1

118 A trichrome stain showing a so-called capsular drop lesion of recurrent dm; this is basically hyaline material that has extended distally from the afft arteriole and deposited just underneath the parietal layer of the bce.

119 Later stages of diabetic nephropathy show these mesangial nodules, so called Kimmelstein Wilson lesions, which tend to be associated with nephrotic syndrome xxxxxxx Review collagen type---iv (reticulin) and v , laminin, fibronectin (not type III which is blue on collagen)

120 RELATIVE IMPORTANCE OF VARIOUS CAUSES OF CHRONIC GRAFT DYSFUNCTION
Rejection 20% Non-Immune Causes 80% I will now discuss the RELATIVE importance of the various causes of cgd that I have illustrated in the past several slides It has been historically believed that only 20% of cgd is due to immune causes, and that the remaining 80% if accounted for by non-immune factors. However, increasing use of C4d stain to kidney biopsies has shown that perhaps a third of the cases that were labeled as non-immune have amr as AT LEAST a PARTIAL contributing factor xxxxxxxxxxxxx (ENDAT transcripts 64% of all tissues obtained from patients with graft loss at Edmonton– see caveats already discussed in mdx of amr ) (molecular transcripts chronic amr, only 40% C4d positive) 1/3-1/2 C4d+

121 NON-IMMUNE FACTORS 2% if EM DNA-MA Drug Toxicity 14% Unclassified
Recurrent disease 37% 16% 2% if EM DNA-MA -Within the non-immune category of cgd (even after C4d cases excluded) drug toxicity accounted for 14% of cases in one large series from Boston, recurrent dis 16% and denovo glomerulonephritis was - -- -37% remained unclassified in this cohort of patients. PURPORTEDLY This number has been brought down to as little as 2% in medical centers that make intensive use of ancillary techniques such as electron microscopy for diagnosis recurrent glomerulonephritis and DNA microarray analyses to diagnosis amr (showing endothelium associated transcripts EDAT of amr)series XXXXXXXXXXXX number can vary from center to center, and may be lower if diseases like glomerulonephritis are an important cause of kidney transplant in your practice; cosio in mayo 36% have glomerular Pathology of which 18% is cg; if arteriolar hyalinosis attributed simplistically to calcineurin inhibitor 98% have (at least one) identifiable cause (but cannot be the sole cause since donor age, hypertension, diabetes mellitus likely common causes of multifactorial injury) Also, DNA microarray studes at University Alberta indicate that perhaps a 64% of unclassifed cases have the molecular profile of chronic amr De novo GN 13%

122 ASSIGNING PRIMARY CAUSE OF CGD HAS LIMITATONS
ah often simplistically taken as CNIT - virtually all patients hypertensive - 1/3 rd are diabetic - variable proportion are ECD of age >50 C4d +ve CGD cases are classified as CAMR -many have had prior episodes of TCMR -most will have hypertension &/or diabetes However, it is important to remember that the process of assigning the primary cause of cgd in any given case has inherent limitations For example the presence of arteriolar hyalinosis is often simplistically taken as evidence of calcineurin inhibitor toxicity, but -----, & these concurrent conditions must be contributing to the devtOFah Likewise, C4d Positive cases of cgd can be labeled as having camr, but many patients have had prior episodes of tcmr, and most will have hypertension &/or diabetes mellitus Xxxxxxxxxxxxxx ICD on EM used to establish a dx of GN -concurrent subendothelial translucent material

123 CHRONIC ALLOGRAFT NEPHROPATHY
Coined at 1991 Banff Meeting - to recognize multifactorial nature CGD - inability to determine etiology in all cases Widely & variably used to denote a process assumed to have no prev/therapeutic measures Banff 2005 proposed elimination & suggested ISTA-NOS (no evidence of specific pathology) No lecture on renal transplant Pathology will be complete without mentioning the word chronic allograft nephropathy. The term WAS first coined by pathologists at the Banff meeting in 1991(published in KI93 and defined by the diagnostic triad of ci, ct, cv); The intent was to recognize the multifactorial nature of cgd Unfortunately---it became widely AND VARIABLY used: some used it in a generic way,while others would exclude cases with an immune contribution to graft damage. In some publications you can see it defined on clinical criteria using renal function alone Another problem was that it had a rather catchy and intellectual sounding name and came to be regarded as a specific disease entity For these reasons, banff 2005 conference proposed elimination of this term. The suggested replacement was the intentionally boring/ MUNDANE term ISTA not specified xxxxxxxxxxxxxxxxxxx 2003– new term; nonetheless the term is deeply entrenched in the literature & is unlikely to go away soon, In this talk I am going to REFER TO IT REPEATEDLY BY THE ACRONYM CAN-SLASH-ISIT j. ---PERSONALLY, nothng objectionalbe in using it as long as we recognize the limitations of the term. Etiologically non committal term encompassing varying combinations of the following lesions ci ct cv

124 GRADING OF CAN/ISTA NOS
Interstitial fibrosis (ci 0-3) (<5, 25, 50, >50) Tubular atrophy (ct 0-3) (0, <25, 50, >50) Arteriosclerosis (cv 0-3) (% luminal occl) Arteriolar hyalinosis (ah 0-3) (#, severity) Tx glomerulopathy (cg 0-3)(<10,25,50,>50) WHETHER YOU CALL IT CHRONIC ALLOGRAFT NEPHROPATHY OR ISTA-NOT SPECIFIED there is no denying the fact that grading of this lesion is important in every renal allograft biopsy; Ci and Ct can be graded from 0-3 depending on the % area of the biopsy involved by the process CV is graded by the % luminal compromise of the most affected area Arteriolar hyalinosis by the number of arterioles involved as well as the severity of inv And Finally txgm is graded by the percentage of glomerular CAPILLARY LOOP showing duplic IN THE MOST AFFECTED GLOMERULUSi xxxxxxxxxxxxxxxxxxxxxxxxxxx It is not enough to simply state that chronic allograft nephropathy/ista is present or absent. A comment on the severity of the process expressed as mild,mod or severe, is very helpful to the treating physician; This grade, along with clinical parameters of graft function, can help decide when it would be appropriate to list a patient for re-transplantation. Without going into (boring you with) the details (of interest only to pathologists) let me just say that Grades from 0-3 can be assigned to xxxxxx(review new ah scoring, cg, mm and also cadi scoring; put it all here instead of on a printed out page which needs copying everytime)-----Acknowledge focalilty and problems in individual cases; yet there is good correlation in population based studies; see curves ahead; yes useful taken in conjunction with clinical parameters

125 PROGNOSTIC IMPORTANCE OF HISTOLOGIC GRADING
Grading can or ista has prognostic implications Here is data from Jeremy Chapman’s group showing that if the interstitial fibrosis score of a PROTOCOL BIOPSY AT 3 MONTHS IS 0, the 15 yr graft survival exceeds 90%; for interstitial fibrosis=1 it is about 80%, whereas for scores of 2 and above it is down to about 60% Xxxxx Statisticians can debate on whether or not it is mathematically correct to combine mutually interdependent entities like ci and ct into a composite score. The fact remains that histologic grades of individual lesions, as well as sum scores of all lesions combined, help to predict graft prognosis (in studies including sufficient number of patients spanning the entire spectrum of lesions from mild to severe CANistaNOS.

126 GROUPING OF LESIONS INTO CLUSTERS
Tubulo-interstitial inflammation (i, t) Microcirculation lesions (g, v, ptc, cg) Scarring hyalinosis lesions (ci, ct, cv, ah) Based on 234 unselected biopsies graded for acute and chronic Banff lesions Sis et al. Am J Transplant 2010: 10: 421 Mathematical analyses indicate that the lesions we tend to score individually in biopsies with ISTA NOS or CAN are often related to each other and should probably get a group score. ONE Study suggested that the three lesion clusters for which a composite score needs to be developed are ---- (ptcd occupied ambiguous position between microcirculation and scarring clusters). Such composite scores will better reflect the biology of disease and may give more prognostic information than sum scores; and also are reasonable groups for the purposes of conducting therapeutic trials These reommendations were based on an evaluation of 234 unselected— xxxxx (patients with inflamm may need trial with anti-inflammatory therapies; fibrosis alone with fibroblast directed therapies, cg for antibody pathway therapies)(place in routine day to day diagnosis is less clear)

127 UTILITY OF HISTOLOGIC CLUSTERS IN DeKAF STUDY
Two different analyses performed Only g, i, mm, ct, cv, ah scores used in 1 analysis All scores including i-atr,t-atr used in 2nd analysis Six clusters difft prognosis 18 m GS 96% best cluster  75% worst cluster Matas et al. Am J Transplant 2010: 10: 315 The utility of histolgic clusters has also been shown by the so-called DeKaf study I mentioned earlier Two different – G, I, All banf scores including scores for inflammation and tubulitis in atrophic areas Using these scores they were able to classify their cases into six clusters— 18 month graft survival was 96% xxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxx (Late onset)Delayed Kidney Allograft Function Study 265 New Exclude defined entities such as bkn, glomerulonephritis)-creatinine rise >25% or new onset proteinuria defined as alb/creatinine ration >0.2 or prot/creatinine ratio >0.5) G, i (other scores excluded because they were tightly correlated with the scores listed here)(i&t ci&ct had r=0.8) xxxx\ Miscellaneous: tubulitis-atrophy score almost identical to total i-score in 3 clusters and gave same prognostic information on univariate analysis Local pathology reading said not to differentiate clusters based on the argument that all had similar % of biopsies with diagnosis of chronic allograft nephropathy However review of data indicates % acute rejection clearly different; and % transplant glomerulopathy and calcineurin inhibitor toxicity diagnoses also seem to differ although no statistic analysis is even attempted (cluster 1 definitely less acrCGcni; others less different but then their % graft survival difference is also less pronounced) (this same group states in another paper that CAN in LATE POST-TRANSPLANT BIOPSIES 7.5+/-6.1 Years provides no prognostic information!– obviously it would if there were enough cases with >70% scarring) C4d and DSA similar proportion in 5 clusters with varying pathology (was low only in cluster 1 with mild ct)

128 The chronicity scoreS in an allog bx ARE somewhat like the odometer of a car
Tells you how much mileage remains in the kid, so to speak, and can be used as a guide to relist patients for a second tx

129 PATHOGENESIS OF CAN/ISTA NOS
Multifactorial parenchymal INJURY Exhausted REPARATIVE ABILITY due to impaired tub regen or vascular remodeling Persistence of injury inflammation, progressive fibrosis, and GS Self-perpetuating hyperfiltration injury culminating as graft failure Before I conclude, I want to make a couple of remarks on -- It should be apparent from what we have discussed that the pathogenesis of CAN/ISIT would involve a Multifactorial parenchymal injury. However, an EQUALLY important element may be the – ; This would lead to ---; Once A CRITICAL MASS of glomeruli have become sclerotic, a self-perpetuating --- is initiated. For example, if 40% of the glomeruli have become sclerotic, the remaining 60% have to bear the whole burden of maintaining an adequate GLOMERULAR FILTRATION RATE. This puts these glomeruli at abnormal stress. Increased capillary pressures lead to fsgs and a VIcIOUS CYCLE of FURTHER GS AND HYPERFILTRATION, CULMINATING IN graft failure Exhaustion ‘senescence’ J.—p16 and p27 cyclin dependent kinase inhibitor genes (Am J Kid Dis 2003;41: ); do upregulate in aging kidney and yes upregulation found in chronic allograft nephropathy (implying accelerated senescence); in this study chronologic age of graft was defined as DONOR age+time since transplant, and this age was always less than predicted age using p16 p27 staining (age vs p27 curves obtained from non-transplant ageing humans).

130 CAUSES OF RENAL ALLOGRAFT DYSFUNCTION
REJECTION RELATED CATEGORIES -Acute & chronic Ab mediated rejection -Acute & chronic T-cell mediated rejection NON-REJECTION RELATED CATEGORIES -ATN, CNIT, Infections, GN, Recurrent native disease, donor derived pathology Let me now BEGIN TO conclude my talk by saying that the causes Rejection Non-rejection d/d --- AND REQUIRES A BX 1

131 RENAL BIOPSY REPORTS SHOULD USE STANDARDIZED TERMINOLOGY
Example of non-standardized report: Allograft kidney, needle biopsy:   Interstitial inflammation and tubulitis c/w ac acute rejection (?TCMR, AMR, ?grade) Patchy interstitial fibrosis & tubular atrophy Renal biopsy reports should use standardized terminology: TO CONVEY THE Pathology findings CONCISELY AND CLEARLY in a manner that can be UNDERSTOOD BY transplant physicians WORLDWIDE Here is a real life NON-STANDARDIZED pathology report that accompanied a biopsy that was sent to me for consultation from a small community hospital: The first diagnostic line read: Interstitial inflammation and tubulitis consistent with acute rejection; DID not say whether the rejection was T-cell or antibody mediated or what its severity was The second diagnostic line read: Patchy interstitial fibrosis with occasional sclerotic glomeruli, again without mentioning how bad the changes really were; Chs ; A real report with minor edits to make some important points

132 A STANDARDIZED BIOPSY REPORT
Diagnosis: acute T-cell mediated rejection Grade severity: Banff grade 1 (g0,i2,t3,v0) Worse tubulitis compared to prior biopsy Chronicity: severe interstitial fibrosis (cg2, ci3, ct3, cv2) We use standardized Banff scoring templates -saves time & facilitates large databases In contrast, THIS IS THE STANDARDIZE REPROT that I issued on this case: I mentioned the mention the dx of . I graded the severity of the process, stating that it was a Banff grade 1a rejection, and add the banff scores for the defining lesions. With experience these scores can be assigned in less than 5 minutes, and they can actually replace a lengthy microscopic description using non-standardized terminology I compared the biopsy with the previous one pointing out that the tubulitis had worsened in the interim interval Finally, I quantified the chronicity in the biopsy by noting that there was severe interstitial fibrosis, thereby communicating to the clinicians that they should consider re-listing the patient for a second transplant. I also gave scores for cg, ci, ct and CV lesions in the biopsy We use a standardized banff scoring templates in ALL of our Pathology reports, because it saves time & allows construction of large databases that are very useful in clinical research; If any one in the audience would like copy of this templates we use in our day to day practice, send me an , or talk to me after the conference, or at the banquet tonight.

133 Time for questions

134


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