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Parmjeet RandhawaProfessor, Division of Transplantation Department of PathologyUniversity of Pittsburgh
ROLE OF ALLOGRAFT BIOPSY IN THE MANAGEMENT OF TRANSPLANTED PATIENT
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
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
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
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
ACUTE REJECTION
• Acute T-cell mediated rejection
• Acute cellular rejection
ACUTE T-CELL MEDIATED REJECTIONACUTE T-CELL MEDIATED REJECTION
ThThTh
IL-2IL-2
CTL
Granzyme BGranzyme BPerforinPerforinGMP-17 (TIA-1)GMP-17 (TIA-1)
Fas LigandFas LigandCD95CD95
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MTNFTNFIFNIFN
B antiantiHLA AbHLA Ab
IL-4IL-4IL-10IL-10
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IL-2IL-2
AllorecognitionAllorecognition• DirectDirect• IndirectIndirect
IL-15IL-15
Dr. David Rush
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
HISTOLOGIC CRITERIA FOR DIAGNOSIS OF ACUTE T-CELL
MEDIATED REJECTION
• Predominantly mononuclear infiltrate
• Presence of parenchymal damage
• Occurrence of subendothelial inflammation in venules, arteries
GRADING OF ACUTE TCMR-1
• Severity of inflammation
• Intensity of tubulitis
• Disruption of tubular architecture
• Presence of arteritis
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
INFLAMMATION IN AREAS OF SCARRING
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
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
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)
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
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
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
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
TYPE 1A
Type 1B
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 2010. Am J Transplantation 10: 421
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%
HISTOLOGIC GRADING OF INDIVIDUAL LESIONS
Graft Loss
t1 tubulitis 0/36 0%
t2 tubulitis 0/36 0%
t3 tubulitis 10/36 28%
v1 arteritis 11/36 31%
v2 arteritis 11/18 61%
v3 arteritis 11/11 100%
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.
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
.
• ACUTE TUBULAR NECROSIS
• ACUTE TUBULAR INJURY
• ACUTE KIDNEY INJURY
CALCINEURIN INHIBITOR TOXICITY
1. Cyclosporine
2. Tacrolimus
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
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
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
OTHER CAUSES OF MYOCYTE VACUOLIZATION
• Amphotericin B therapy• Use of vasopressors• Injury secondary to cholesterol emboli• Acute cellular rejection with intimal arteritis
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
.
THROMBOTIC MICROANGIOPATHY
LABORATORY ABNORMALITIES
• Thrombocytopenia
• Schistocytes on PBF
• Elevated serum bilirubin
• Low serum haptoglobin
• Presence of free Hb in plasma
COMMON DRUGS ASSOCIATED WITH
THROMBOTIC MICROANGIOPATHY
• Cyclosporine
• Tacrolimus
• Sirolimus?
LESS COMMONLY USED DRUGS
• Vasopressors: dopamine
• Antimicrobials: penicillin, metronidazole
• Anti-inflammatory: penicillamine
• Anti-epileptic: phenytoin
• Chemotherapeutic agents: mitomycin
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)
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)
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-
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)
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
ARTERIOLONECROSIS
• Fibrinoid change in arteriolar wall
• Collapse of distal glomeruli
• Healing by onion skin change
CHRONIC GRAFT DYSFUNCTION
• Evolves slowly
• Usually begins > 3m post-tx
• Typically progressive
• Rate of decline, may be punctuated by periods of stability
MAGNITUDE OF PROBLEM
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
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)
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
.
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
Dr Robert Colvin, ATC 2006R. Colvin
DIFFERENCES BETWEEN GRAFT AND ENDEMIC ATHEROSCLEROSIS
1. Extent of involvement (diffuse vs proximal)
2. Type of luminal compromise distinctive
3. Calcification is late & less common in GAS.
4. Differences in biochemical composition (Apo-A,E, biglycans vs Apo-B, decorin)
Differences are relative & not absolute
.
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
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
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)
.
CHRONIC CALCINEURIN INHIBITOR TOXICITY
• Cyclosporine
• Tacrolimus
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
ARTERIOLAR LESIONS
• 1-3 layers smooth muscle
• No complete internal elastic lamina
• Diameter < 1/3 rd of a glomerulus
ARTERIOLAR HYALINOSIS
• Deposition of glassy material
• Initially in endothelial layer
• Followed by circumferential extension
• Medial involvement
ARTERIOLAR HYALINOSIS
• Donor disease • Diabetes mellitus • Hypertension• PSS• Chronic thrombotic microangiopathy • Radiation
.
INTERSTITIAL FIBROSIS IN CALCINEURIN INHIBITOR
TOXICITY
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
GLOMERULONEPHRITIS IN THE ALLOGRAFT KIDNEY
• Donor transmitted glomerulonephritis
• De-novo glomerulonephritis
• Recurrence of original disease
Morphologic spectrum similar to native kidneys
DONOR TRANSMITTED GLOMERULONEPHRITIS
• IgA nephropathy
• SLE
• MPGN
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
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
Briganti et al 2002; NEJM 347: 103
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 5.9-12.0 %, highest if male sex, primary disease FSGS or MGN
Hariharan et al 1999; Transplantation 68:635
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
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%
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
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
RECURRENT METABOLIC DISEASES
• Diabetes mellitus
• Amyloidosis
• LCDD
• Lipoprotein glomerulopathy
• Fabry’s disease
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%
RELATIVE IMPORTANCE OF VARIOUS RELATIVE IMPORTANCE OF VARIOUS CAUSES OF CHRONIC GRAFT DYSFUNCTIONCAUSES OF CHRONIC GRAFT DYSFUNCTION
Non-ImmuneCauses
80%
ChronicRejection
20%
1/3-1/2 C4d+
NON-IMMUNE FACTORSNON-IMMUNE FACTORS
Unclassified
37%
Drug Toxicity
14%
Recurrent disease
16%
De novo GN
13%
2% if EMDNA-MA
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
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)
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)
PROGNOSTIC IMPORTANCE OF HISTOLOGIC GRADING
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
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
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
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
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
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