amyloidosis diagnosis and classification in native kidney and renal transplants prof. dr. b. handan...

Amyloidosis Amyloidosis DDiagnosis and iagnosis and CClassification in lassification in NNative ative

KKidney and idney and RRenal enal transplants transplants

Prof. Dr. B. Handan Özdemir Prof. Dr. B. Handan Özdemir

Baskent UniversityBaskent University

Ankara-TurkeyAnkara-Turkey

Definition

Amyloidosis comprises a diverse Amyloidosis comprises a diverse group of systemic and local diseases group of systemic and local diseases

Characterized by organ involvement Characterized by organ involvement by the extracellular deposition of by the extracellular deposition of fibrils composed of subunits of a fibrils composed of subunits of a variety of normal serum proteinsvariety of normal serum proteins

Amyloid fibril protein occurs in tissue deposits as rigid, non-branching fibrils 7-to 10 nm in dm

When analysed by X-ray diffraction, the fibrils exhibit a characteristic cross Beta diffraction pattern

Physical NPhysical Natureature

Chemical NChemical Natureature

PPentagonal moleculeentagonal molecule

95% Protein Fibril 95% Protein Fibril

5% Glycoprotein P component 5% Glycoprotein P component

• Cofactors such as amyloid P component may have an important role in the tissue deposition and resorption resorption

• Promoting fibrillogenesis• Stabilization of the fibrils• Binding to matrix proteins• Affecting metabolism and proteolysis of formed

fibrils

Act by

Husby G. Clin Immunol Immunopathol 1994:70:2

Genetic factors may be involved in Genetic factors may be involved in predisposing to the development of predisposing to the development of

fibrillogenesis and amyloidosisfibrillogenesis and amyloidosis

What factors allow some proteins to What factors allow some proteins to aggregate into amyloid fibrils ?aggregate into amyloid fibrils ?

Patients with AA amyloidosis have levels ofPatients with AA amyloidosis have levels of

SAA protein no greater than those patientsSAA protein no greater than those patients

with inflammatory diseases who do not have amyloid with inflammatory diseases who do not have amyloid

Therefore some additional unknown Therefore some additional unknown stimulus is required for amyloid fibrils to stimulus is required for amyloid fibrils to form and precipateform and precipate

Cast nephropathy versus AmyloidosisCast nephropathy versus Amyloidosis

Certain light chains also may form high Certain light chains also may form high molecular weight aggregates in vitromolecular weight aggregates in vitro

In AL amyloidosis biochemical In AL amyloidosis biochemical characteristics of the light chain is characteristics of the light chain is important in determining amyloid important in determining amyloid

formation formation

The established amyloid fibril nomenclature is based on the chemical nature of the fibril protein

Which is designated protein A and followed by a suffix that is an abbreviated form of the parent or precursor protein name

For example, when amyloid fibrils are derived from immunoglobulin light chains, the amyloid fibril is AL and the disease is AL amyloidosis

Amyloid fibril protein nomenclature

Amyloid, 2010; 17(3–4): 101–104

At least 25 different precursor proteins are At least 25 different precursor proteins are known known

They They areare associated withassociated with variety of variety of

IInflammatorynflammatory

IImmunemmune

IInfectiousnfectious

Hereditary conditionsHereditary conditions

Amyloid fibril protein nomenclature

Typing of amyloid deposits isTyping of amyloid deposits is important important because of the difference in their treatment because of the difference in their treatment strategiesstrategies

Typing of the amyloid deposits can be Typing of the amyloid deposits can be performed withperformed with various techniquesvarious techniques

The most definitive method used is The most definitive method used is IF or IHC IF or IHC staining of tissue using antibodies that are staining of tissue using antibodies that are directeddirected against known amyloid proteins against known amyloid proteins

TYPING OF RENAL AMYLOIDOSIS

IIHCHC typing of amyloidosis poses several typing of amyloidosis poses several problems and requires experienceproblems and requires experience

WWide range of success in ide range of success in IHC IHC amyloid typing, amyloid typing, ranging from 38% to 87%ranging from 38% to 87% was reported was reported

IIHC HC diagnosis of AA type is relatively reliable,diagnosis of AA type is relatively reliable,

BBut there is a problem in the differentiation of ut there is a problem in the differentiation of AL andAL and hereditary amyloidoshereditary amyloidosisis

Kebbel A, Röcken C. Am J Surg Pathol. 2006;30:673

Amyloid Typing and Pitfalls

• Commercial antibodies are raised against the constant regions of the Ig light chains

• A subset of AL, in which amyloid fibrils are derived from a truncated light chain

“containing only variable regions”

will be nonreactive with commercial antibodies

• Therefore, negativeTherefore, negative light chain staining does light chain staining does notnot rule out AL amyloidosisrule out AL amyloidosis

Amyloid Typing and Pitfalls

• The typical antibody panel should include The typical antibody panel should include • Amyloid P componentAmyloid P component• Kappa & lambda Ig light chainsKappa & lambda Ig light chains• Amyloid A proteinAmyloid A protein• TransthyretinTransthyretin• FibrinogenFibrinogen• Beta-2 microglobulinBeta-2 microglobulin

• This panel allowed definitive This panel allowed definitive

typing of amyloid in 90% of kidney biopsiestyping of amyloid in 90% of kidney biopsies

Immunopathology in Renal Amyloidosis

Systemic Systemic AAmyloidosismyloidosis

Primary Primary AAmyloidosismyloidosis

Secondary Secondary AAmyloidosimyloidosiss

Classification of AmyloidosisClassification of Amyloidosis

Localized amyloidosisLocalized amyloidosis

Senile cerebralSenile cerebral

Senile cardiacSenile cardiac

Type 2 diabetesType 2 diabetes

Primary Primary SSystemic ystemic AAmyloidosismyloidosis

Disease nameDisease name Type of amyloidType of amyloid PrecursorPrecursor

MM..MyelomaMyeloma ALAL Light Light chainschains

PrimaryPrimary ALAL Light Light chainschains

SecondarySecondary SSystemic ystemic AAmyloidosismyloidosis

Disease nameDisease name Type of amyloidType of amyloid PrecursorPrecursor

Chronic Chronic inflammatinflammatory ory diseasedisease

AAAA SAASAA

Hemodialysis Hemodialysis associateassociate

AAββ2- 2- micro micro globulinglobulin

ββ2- 2- micro globulinmicro globulin

The most common type of amyloidosis depends on The most common type of amyloidosis depends on the population studiedthe population studied

In the USA and the Western World AL amyloidosis In the USA and the Western World AL amyloidosis is the most prevelant, followed by AA amyloidosisis the most prevelant, followed by AA amyloidosis

In Turkey AA amyloidosis with an underlying In Turkey AA amyloidosis with an underlying disease of FMF is the most common typedisease of FMF is the most common type

In developing countries AA amyloidosis is far more In developing countries AA amyloidosis is far more common than AL amyloidosis (Tbc !)common than AL amyloidosis (Tbc !)

Ozdemir AI. Am J Gastroenterol 1969; 51:311

DDerived from the immunoglobulinerived from the immunoglobulin light chain light chain

Can occur alone or in association with

M. Myeloma

Malignant lymphomas Malignant lymphomas

MacroglobulinemiaMacroglobulinemia

ALAL Amyloidosis Amyloidosis

Kyle RA et al N Engl J Med. 2006;354:1362

Light chain deposition disease has a similar pathogenesis with AL amyloidosis

Primary difference is that

Deposited light chain fragments do not form fibrils and do not engender deposition of amyloid cofactors


• Wide spectrum of organ system involvementWide spectrum of organ system involvement

• The kidneys are commonly affectedThe kidneys are commonly affected

• ProteinuriaProteinuria

• Edema and hypoalbuminemiaEdema and hypoalbuminemia

• Mild renal dysfunction is frequentMild renal dysfunction is frequent

• Rapidly progressing renal failure is rareRapidly progressing renal failure is rare


Waxy appearance of intradermal amyloid deposition around the eye

Macroglossia showing teeth indentations

Peri-orbital haemorrhage (raccoon or panda eyes)

Gross lymphadenopathy

The AA amyloid proteins resultThe AA amyloid proteins result from a from a proteolytic cleavage proteolytic cleavage SAASAA protein protein

SAA is an SAA is an acute-phase reactant acute-phase reactant produced by liverproduced by liver

Sustained high concentration of SAA Sustained high concentration of SAA prerequisite for AA amyloidosisprerequisite for AA amyloidosis

AAAA Amyloidosis Amyloidosis

Benditt EP et al. Ann N Y Acad Sci. 1982;389:183

Acquired and hereditaryAcquired and hereditary diseases can lead diseases can lead to AA amyloidosisto AA amyloidosis


Chronic inflammatory diseases

Rheumatoid arthritis

Inflammatory bowel disease

FMF

Bronchiectasis,

Tuberculosis

• KKidney is the most frequently affected target idney is the most frequently affected target organorgan

• The primary clinical manifestation is The primary clinical manifestation is proteinuriaproteinuria

• The underlying disease usually is The underlying disease usually is longstandinglongstanding

• AActive inflammation typically is presentctive inflammation typically is present when when

amyloidosis becomes evidentamyloidosis becomes evident


CCaused by deposition of geneticallyaused by deposition of genetically variant variant proteins proteins

AAssociated with mutationsssociated with mutations in the genes for in the genes for

TransthyretinTransthyretin AApolipoprotein AI,polipoprotein AI, Apolipoprotein AIIApolipoprotein AII LysozymeLysozyme Fibrinogen A.Fibrinogen A.

HereditaryHereditary Amyloidosis Amyloidosis

Typically they associated with Typically they associated with polyneuropathy and cardiac involvement polyneuropathy and cardiac involvement but but can affect kidneyscan affect kidneys

RRenal deposits may be clinically silentenal deposits may be clinically silent

Isolated glomerular involvement with no Isolated glomerular involvement with no amyloidamyloid in the tubules, interstitium, or in the tubules, interstitium, or vessels has beenvessels has been found to be characteristic found to be characteristic of fibrinogen Aof fibrinogen A

Renal failure develops rapidlyRenal failure develops rapidly

HereditaryHereditary Amyloidosis Amyloidosis

Blood. 1997;90:799–805

Clinically evident renal involvement occurs Clinically evident renal involvement occurs mainly inmainly in AA and AL amyloidosis AA and AL amyloidosis

The deposition of AThe deposition of Abetabeta22 M occursM occurs in patients in patients on prolonged dialysison prolonged dialysis

But But diagnosis on the kidney biopsy is diagnosis on the kidney biopsy is unexpectedunexpected

Eight precursor fibrils are particularly Eight precursor fibrils are particularly importantimportant

for kidney for kidney

RENAL AMYLOIDOSIS

The incidence The incidence of amyloid in patients with of amyloid in patients with nephrotic syndromenephrotic syndrome or proteinuria was or proteinuria was found in 2% to 12% of native renalfound in 2% to 12% of native renal biopsies biopsies

RENAL AMYLOIDOSIS

Arch Pathol Lab Med. 2007;131:917–922

Amyloidosis without therapy usually progresses to endstage kidney disease

Deposits may also regress

N Engl J Med. 2003;349:583–596.

Ozdemir BH. Transplant Proc. 2006;38:432–434.

Diagnosis of Diagnosis of AAmyloidosismyloidosis

• Can be very difficultCan be very difficult

• No blood test can diagnose or exclude No blood test can diagnose or exclude amyloidosisamyloidosis

• Usually relies on clinical suspicionUsually relies on clinical suspicion

• Possibility supported byPossibility supported by• Underlying chronic inflammatory state – AAUnderlying chronic inflammatory state – AA• Underlying plasma cell dyscrasia – ALUnderlying plasma cell dyscrasia – AL• Family history - hereditaryFamily history - hereditary• Evidence of Evidence of renal renal dysfunctiondysfunction

In In H&EH&E stained sections, amyloidstained sections, amyloid is is recognized as amorphous hyaline and recognized as amorphous hyaline and eosinophilic eosinophilic materialmaterial

Weakly PAS positiveWeakly PAS positive

Detection of Renal Amyloid

Detection of Amyloid

• AAmyloid do not stain by silver stainingmyloid do not stain by silver staining

• Occasionally Occasionally may stain with silver stains andmay stain with silver stains and

show spicules (Jones silver)show spicules (Jones silver)


• Congo red is the gold standard is the gold standard

• Slides must be examined under polarized light

• Apple-green birefringent deposits is diagnostic

Caution is advised regarding ‘‘overinterpreting’’ collagen as amyloid

Because deposits of amyloid are frequently very focal and irregularly distributed in tissue sections

Multiple and thicker (5–10 m) sections may need to be examined

Picken MM. Curr Opinion Nephrol Hypertens. 2007;16:196


Other stains, or techniques

Fluorescence Thioflavin S and T Methyl violet Sulphonated Alcian blue They are less specific

Curr Opinion Nephrol Hypertens. 2007;16:196


Sen S. Arch Pathol Lab Med 2010: 134: 532

• Characterized by randomly disposed, rigid, Characterized by randomly disposed, rigid, nonbranching, variably long, 7-to 10nm-dm nonbranching, variably long, 7-to 10nm-dm fibrilsfibrils

• Ultrastructural immunogold labelling can Ultrastructural immunogold labelling can depict the precursor protein in amyloif fibrilsdepict the precursor protein in amyloif fibrils

Electron Microscopy

• Rare cases exhibit massive aggregates of Rare cases exhibit massive aggregates of amyloid fibrils in subendothelium amyloid fibrils in subendothelium

• They arranged in thightly packed, electron-They arranged in thightly packed, electron-dense structures and can be easily confused dense structures and can be easily confused with with

• MPGNMPGN• Cryoglobulinemic glomerulopathyCryoglobulinemic glomerulopathy

• These cases are usually associated with These cases are usually associated with monoclonal kappa light cahinsmonoclonal kappa light cahins

Electron Microscopy

• Enlarged kidneysEnlarged kidneys• Pale, waxy appearing cut Pale, waxy appearing cut

surfacessurfaces• Increase in the weight of kidneyIncrease in the weight of kidney

Gross Pathology of Renal Amyloidosis

Weight of the kidney did not correlate Weight of the kidney did not correlate with with

Renal function Renal function

The site of renal amyloid deposition The site of renal amyloid deposition

Inversely proportional to the amount of Inversely proportional to the amount of amyloidamyloid

• Amyloid can be found in any of Amyloid can be found in any of the renal compartmentthe renal compartment

• TThe most common andhe most common and the the earliest site of amyloid earliest site of amyloid deposition in the kidneydeposition in the kidney is the is the glomeruliglomeruli

• Glomerular amyloid formations Glomerular amyloid formations begins in the mesangiumbegins in the mesangium

• Extends into capillary wallsExtends into capillary walls

Microscopy of Renal Amyloidosis

• Amyloid deposition in glomeruli may Amyloid deposition in glomeruli may occuroccur

• SegmentalSegmental

• Diffuse mesangialDiffuse mesangial

• NodularNodular

• Pure basement membrane patternsPure basement membrane patterns


Renal amyloidosis was divided into 6 classesSimilar to the classification of SLE

A proposed histopathologic classification

Sen S. Arch Pathol Lab Med 2010: 134: 532

• Early segmentalEarly segmental deposits are small and confined deposits are small and confined to mesangium without creating nodularity to mesangium without creating nodularity

• It is very easy to miss this early form It is very easy to miss this early form


• In the In the diffuse formdiffuse form

• The mesangium is uniformly expanded by The mesangium is uniformly expanded by depositdeposit


• In the In the nodular formnodular form • Mesangium is asymmetrically expanded by Mesangium is asymmetrically expanded by

amyloidamyloid

• Distinguish from diabetic nephropathyDistinguish from diabetic nephropathy• Other forms of nodular glomerulosclerosisOther forms of nodular glomerulosclerosis


• Subepithelial amyloid depositionSubepithelial amyloid deposition

Associated with spikes Associated with spikes

Can be seen at the periphery of mesangial areasCan be seen at the periphery of mesangial areas


• Rarely cresents can be seenRarely cresents can be seen

Highlighting the fact that capillary wall Highlighting the fact that capillary wall rupture has occuredrupture has occured


• Interstitial amyloid are seen in 50% casesInterstitial amyloid are seen in 50% cases

• Generally begins in areas adjacent to blood Generally begins in areas adjacent to blood vesselsvessels

• Medullary amyloid deposits are more frequent Medullary amyloid deposits are more frequent


• AA amyloidosisAA amyloidosis• Certain mutants of transthyretinCertain mutants of transthyretin

• May show amyloid deposition limited to the May show amyloid deposition limited to the interstitium and medullainterstitium and medulla

• Such patients present with renal failure not Such patients present with renal failure not associated with proteinuriaassociated with proteinuria


• Renal vessels are often involvedRenal vessels are often involved

• Arteriolar deposits being most frequent Arteriolar deposits being most frequent

• Followed by deposits in arteries, PTCs and Followed by deposits in arteries, PTCs and veinsveins


• Therapies are not successful in patients diagnosed at advanced stages of amyloidosis

• Supportive therapy is essential

• There are two choices for the therapy

• Hemodialysis

• Transplantation

RENAL AMYLOIDOSIS

• The survival of patients begining dialysis is poor

• Mean survival is only 33 months

• Worse than patients with other renal diseases

Hemodialysis in Renal Amyloidosis

• The European experience showed a 76% survival at 2 years for all patients compared with 53% among amyloidosis patients

• Similarly in our center the 2-year survival was 50% among patients with amyloidosis

• The mean survival of our patients on hemodialysis was 33 months


• Common complications of amyloidosis are

• Extrarenal progression of amyloidosis

• Infections

• Major causes of death


• It has been reported that 21 of the 31 deaths in the group that underwent dialysis were due to extrarenal progression of amyloidosis, namely to cardiac amyloidosis

• Similarly, we have observed that infection and extrarenal progressive amyloid deposition was the cause of death in 15 of our 30 patients who died approximately 9 months after starting hemodialysis


Ozdemir BH et al. Transplant Int 2004: 17: 241–246

Other Choice of Therapy is TransplantationOther Choice of Therapy is Transplantation

• The outcomes of renal tx in patients with amyloidosis is still controversial

• Pasternack• 3-year survival • Patients with amyloidosis 51%• Patients with glomerulonephritis 79%

Transplantation in Renal Amyloidosis

Pasternack A et al. Transplantation 1986; 42:598

• Pras noted similar graft and patient survival rates among patients with amyloidosis and GN

• Similarly our 5-year survival rates for amyloidosis patients (78%) were equal to the survival rates of patients with GN

Transplantation in Renal Amyloidosis

Pras M et al. Adv Nephrol Necker Hosp 1984; 13:261

Ozdemir BH et al. Transplant Proc 2006: 38, 432

Ozdemir BH et al. Transplant Int 2004: 17: 241–246

The rates ofThe rates of recurrent amyloidosis vary from recurrent amyloidosis vary from

centcenter er to to centcenter er ((13 to 50%13 to 50%) )

ThisThis is mainly because only a small number ofis mainly because only a small number of patients have been studied in single-centpatients have been studied in single-centerer series series

Recurrence of Amyloidosis

The rates of recurrence is dependent on the indication of graft biopsy

Since early amyloidosis could exist without urinary abnormalities or impairment of renal function

In In our centerour center we observed higher rates we observed higher rates of of amyloid recurrence amyloid recurrence than have been reportedthan have been reported previouslypreviously

Recurrent Recurrent amyloidosis was diagnosedamyloidosis was diagnosed by by renal allograft biopsy in 20renal allograft biopsy in 20 of our 30 of our 30 cases cases

Of 20 grafts, 18 were from livOf 20 grafts, 18 were from livinging and 2 were and 2 were fromfrom cadaveric donors (P<0.01)cadaveric donors (P<0.01)

Four of the five patientsFour of the five patients (80%) with HLA-(80%) with HLA-identical LRDs showed identical LRDs showed amyloid amyloid recurrencerecurrence


Ozdemir BH et al. Transplant Int (2004) 17: 241–246

PreviousPrevious reports document amyloid recurrence in reports document amyloid recurrence in renal grafts renal grafts atat 6 months to 4 years after t6 months to 4 years after txx


Transplantation 1981; 32:6

In our patients, amyloidosis developed in the In our patients, amyloidosis developed in the graftsgrafts 18 months to 10 years after 18 months to 10 years after txtx

This is a very wideThis is a very wide range, emphasising that range, emphasising that amyloid recurrence can developamyloid recurrence can develop at any time at any time after after txtx

Arch Intern Med 1979; 139:1135


RRecipients with LRD showed shorter times to ecipients with LRD showed shorter times to recurrence (33.6±5.2 months)recurrence (33.6±5.2 months) than for those than for those with cadaveric graftswith cadaveric grafts ((78±24 months; 78±24 months; P<0.01)P<0.01)

The overall 3-, 5- and 10-year graft survival The overall 3-, 5- and 10-year graft survival rates forrates for the recipients with amyloidosis the recipients with amyloidosis recurrence were 77%,recurrence were 77%, 70%, and 36%, 70%, and 36%, respectivelyrespectively

The corresponding patientThe corresponding patient survival rates survival rates were 93%, 78%, and 35%were 93%, 78%, and 35%



WWhy sohy so many patients showed such a high many patients showed such a high incidence of amyloidosisincidence of amyloidosis recurrence, even recurrence, even

though they were taking colchicinethough they were taking colchicine ? ?

Part of the reason for this may be Part of the reason for this may be poorpoor D Drugrug compliance compliance

Doubting the need for medication

Preference for self-care measures other than medication

Convenience, side effects and lack of demonstrated benefit

• Amyloidosis patients maintained on chronic dialysis have high mortality rates

• Better survival was noted among renal transplant patients even with a recurrence

• These results encourage transplantation for renal end-stage disease due to amyloidosis

CONCLUSION

amyloidosis diagnosis and classification in native kidney and renal transplants prof. dr. b. handan...

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