Parmjeet Randhawa

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

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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

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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

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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

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ACUTE REJECTION

• Acute T-cell mediated rejection
• Acute cellular rejection

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ACUTE T-CELL MEDIATED REJECTION
IL-2
Th
M?
TNFa
IFNg
IL-4 IL-10
anti-HLA Ab
B
Dr. David Rush
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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

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HISTOLOGIC CRITERIA FOR DIAGNOSIS OF ACUTE T-CELL
MEDIATED REJECTION

• Predominantly mononuclear infiltrate
• Presence of parenchymal damage
• Occurrence of subendothelial inflammation in
  venules, arteries

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GRADING OF ACUTE TCMR-1

• Severity of inflammation
• Intensity of tubulitis
• Disruption of tubular architecture
• Presence of arteritis

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GRADING OF INTERSTITIAL INFLAMMATION

• Limit assessment to cortex unless medulla alone
  sampled
• Ignore areas in continuity with capsule
• lt10 area is assigned grade 0
• Grades 1, 2, 3 are 10-25, 26-50, gt50
• Area is evaluated not the intensity

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INFLAMMATION IN AREAS OF SCARRING
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IMPLICATIONS OF LESION SCORING IN AREAS WITH
FIBROSIS

• DeKAF study 265 bx from 7 centers (7.5/-6.1 y)
• 72 biopsies had tatr scoresgt0(conventional t
  0)
• 50 had iatr scores gt0 (conventional i 0)
• Inclusion of modified scores in data analyses
  yielded prognostic information
• Matas et al. Am J Transplant 2010 10 315

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GRADING OF TUBULITIS

• Define area of maximal involvement
• Avoid tubules cut tangentially
• Grades 0-3 (0, 1-4, 5-10, gt10)
• Look for disrupted tubules (2 foci, i2, i3)
• Atrophic tubules historically ignored
• Do not overlook non-atrophic areas or lymphocytes
  with blast transformation

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GRADING OF INTIMAL ARTERITIS

• Look closely if interstitial hemorrhage,
  glomerulitis, or PTC inflammation
• Caveat added to report if lt 4 arteries
• Grade v1 (lt25) (mild to moderate)
• Grade v2 (gt25) (severe)
• Grade v3 fibrinoid change, necrosis, transmural i
  (transmural intimal arteritis)

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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
  
  

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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

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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

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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

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TYPE 1A
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Type 1B
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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

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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

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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
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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.

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MOLECULAR DIAGNOSIS TCMR

• MDx potentially valuable non-invasive tool
• Dx TCMRsensitivity specificities of 70-90
• Disagreements in an average of 20
• Reflect sampling issues, arbitrary grading
  thresholds low frequency diagnoses
• Provides information that is complementary

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• ACUTE TUBULAR NECROSIS
• ACUTE TUBULAR INJURY
• ACUTE KIDNEY INJURY

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CALCINEURIN INHIBITOR TOXICITY

1. Cyclosporine
2. Tacrolimus

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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
  
  
  
  

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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

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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

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OTHER CAUSES OF MYOCYTE VACUOLIZATION

• Amphotericin B therapy
• Use of vasopressors
• Injury secondary to cholesterol emboli
• Acute cellular rejection with intimal arteritis

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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

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• THROMBOTIC MICROANGIOPATHY

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LABORATORY ABNORMALITIES

• Thrombocytopenia
• Schistocytes on PBF
• Elevated serum bilirubin
• Low serum haptoglobin
• Presence of free Hb in plasma

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COMMON DRUGS ASSOCIATED WITH THROMBOTIC
MICROANGIOPATHY

• Cyclosporine
• Tacrolimus
• Sirolimus?

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LESS COMMONLY USED DRUGS

• Vasopressors dopamine
• Antimicrobials penicillin, metronidazole
• Anti-inflammatory penicillamine
• Anti-epileptic phenytoin
• Chemotherapeutic agents mitomycin

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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)

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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)

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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
• -

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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)

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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

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ARTERIOLONECROSIS

• Fibrinoid change in arteriolar wall
• Collapse of distal glomeruli
• Healing by onion skin change

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CHRONIC GRAFT DYSFUNCTION

• Evolves slowly
• Usually begins gt 3m post-tx
• Typically progressive
• Rate of decline, may be punctuated by periods of
  stability

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MAGNITUDE OF PROBLEM
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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

h
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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)

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CHRONIC TRANSPLANT GLOMERULOPATHY

• Reduplication of GBM
• Subendothelial translucent material
• Mesangial interposition (tram track MST)
• Suggested increase in capillary wall thickness of
  gt2 fold, at least 3 capillary loops
• Ivanyi 2001 Mod Pathol 141200

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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

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R. Colvin
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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

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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

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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

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REDUPLICATION OF PERITUBULAR CAPILLARY BASEMENT
MEMBRANES

• Needs EM single PTC with 7 layers
• 3 PTCs with 5-6 layers
• Duplication gt60-75 of circumference
• Thus defined, lesion will only rarely occur in
  other diseases TMP, reflux, Phenacetin kidney
  (repeated endothelial injury)

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CHRONIC CALCINEURIN INHIBITOR TOXICITY

• Cyclosporine
• Tacrolimus

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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

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ARTERIOLAR LESIONS

• 1-3 layers smooth muscle
• No complete internal elastic lamina
• Diameter lt 1/3 rd of a glomerulus

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ARTERIOLAR HYALINOSIS

• Deposition of glassy material
• Initially in endothelial layer
• Followed by circumferential extension
• Medial involvement

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ARTERIOLAR HYALINOSIS

• Donor disease
• Diabetes mellitus
• Hypertension
• PSS
• Chronic thrombotic microangiopathy
• Radiation

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• INTERSTITIAL FIBROSIS IN CALCINEURIN INHIBITOR
  TOXICITY

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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

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GLOMERULONEPHRITIS IN THE ALLOGRAFT KIDNEY

• Donor transmitted glomerulonephritis
• De-novo glomerulonephritis
• Recurrence of original disease
• Morphologic spectrum similar to native kidneys

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DONOR TRANSMITTED GLOMERULONEPHRITIS

• IgA nephropathy
• SLE
• MPGN

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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

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DE-NOVO ANTI-GLOMERULAR BASEMENT MEMBRANE DISEASE

• Seen in patients with Alports (lt15)
• Exposure to Goodpastures antigen
• Often subclinical but may cause graft loss
• Histology proliferative or crescentic GN
• ELISA, IF confirmation antiGBMab

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RECURRENT GLOMERULONEPHRITIS

• 3rd most frequent cause of graft loss at 10y
• (1chr rej, 2death 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

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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

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RATES OF GRAFT FAILURE

• Focal segmental glomerulosclerosis 65
• Membranoproliferative GN 66
• Membranous nephropathy 44
• IgA nephropathy 41
• Diabetes mellitus 53
• HUS/TTP 63

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MANAGEMENT OF PATIENTS TRANSPLANTED FOR FSGS

• Early dx intervention best prognosis
• Ur.prot/creat ratio OD to disch, wx4, mx12,
• Ratio gt 0.5 consider 24 hr measurement
• Puria gt2g/day consider biopsy confirmation
• Plasmapheresis removes permeability factor

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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

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RECURRENT METABOLIC DISEASES

• Diabetes mellitus
• Amyloidosis
• LCDD
• Lipoprotein glomerulopathy
• Fabrys disease

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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 DMlt10

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RELATIVE IMPORTANCE OF VARIOUS CAUSES OF CHRONIC
GRAFT DYSFUNCTION
Chronic Rejection
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Non-Immune Causes
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1/3-1/2 C4d
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NON-IMMUNE FACTORS
Drug Toxicity
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Unclassified
Recurrent disease
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2 if EM DNA-MA
De novo GN
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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 gt50
• C4d ve CGD cases are classified as CAMR
• -many have had prior episodes of TCMR
• -most will have hypertension /or diabetes

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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)

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GRADING OF CAN/ISTA NOS

• Interstitial fibrosis (ci 0-3) (lt5, 25, 50, gt50)
• Tubular atrophy (ct 0-3) (0, lt25, 50, gt50)
• Arteriosclerosis (cv 0-3) ( luminal occl)
• Arteriolar hyalinosis (ah 0-3) (, severity)
• Tx glomerulopathy (cg 0-3)(lt10,25,50,gt50)

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PROGNOSTIC IMPORTANCE OF HISTOLOGIC GRADING
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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

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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

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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

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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

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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

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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

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