Laboratory Testing used Laboratory Testing used in diagnosis and in diagnosis and l i f V li il i f V li ievaluation of Vasculitisevaluation of Vasculitis

EisaEisa SalehiSalehi PhDPhDEisaEisa SalehiSalehi PhDPhDAssistant ProfessorAssistant Professor

Tehran University of Medical SciencesTehran University of Medical Sciences

Classification of vasculitisClassification of vasculitisClassification of vasculitisClassification of vasculitis

Rationales of laboratory testing in Rationales of laboratory testing in vasculitis:vasculitis:

Check the general inflammationCheck the general inflammationDifferential diagnosisDifferential diagnosisggCheck the disease activityCheck the disease activityCheck the affected organs for the extent of injuryCheck the affected organs for the extent of injuryCheck the affected organs for the extent of injuryCheck the affected organs for the extent of injuryTreatment follow upTreatment follow up

Lab testing in Lab testing in small vessels small vessels vasculitisvasculitis

BUN/BUN/CreCreANCAANCAANCAANCAESRESRCRPCRPCBCCBC--DDCBCCBC DDUrinalysis, Urinalysis, UprotUprot//crcrWAWAvWAgvWAg

ANCAANCAAnti-Neutrophil Cytoplasmic Antibodies

ANCA i i ll d ib d i D i t l i 1982 i t lANCAs were originally described in Davies et al. in 1982 in segmental necrotising glomerulonephritis,[14] and by van der Woude et al.in 1985 in Wegener's.[15]

The Second International ANCA Workshop, held in The Netherlands in May 1989, fixed the nomenclature on perinuclear vs. cytoplasmic patterns, and the antigens MPO and PR3 were discovered in 1988 and 1989,and the antigens MPO and PR3 were discovered in 1988 and 1989, respectively.[16]

International ANCA Workshops have been carried out every two years.

PathogeneticPathogenetic role role ofof ANCAANCAClinical evidenceClinical evidenceCorrelation between ANCA values and activity of Correlation between ANCA values and activity of vasculitis, as well as relapsesvasculitis, as well as relapsesDrugDrug--induced ANCA vasculitisinduced ANCA vasculitisIn vitroIn vitro studiesstudiesActivation of neutrophils by binding of ANCAsActivation of neutrophils by binding of ANCAs →→Activation of neutrophils by binding of ANCAs Activation of neutrophils by binding of ANCAs →→release of destructive enzymes and toxic reactive oxygen release of destructive enzymes and toxic reactive oxygen radicals, as well as neutrophil extracellular traps;radicals, as well as neutrophil extracellular traps;factors released by activated neutrophils activate thefactors released by activated neutrophils activate thefactors released by activated neutrophils activate the factors released by activated neutrophils activate the alternative complement pathway;alternative complement pathway;ANCAs bind also to ANCA antigens adsorbed to anionic ANCAs bind also to ANCA antigens adsorbed to anionic endothelium and GBM, enhancing complement dependentendothelium and GBM, enhancing complement dependentendothelium and GBM, enhancing complement dependent endothelium and GBM, enhancing complement dependent cytotoxicity; cytotoxicity; ANCAs disregulate neutrophil apoptosis ANCAs disregulate neutrophil apoptosis →→ necrosisnecrosis; ; specific T lymphocytes for PRspecific T lymphocytes for PR33specific T lymphocytes for PRspecific T lymphocytes for PR33Several animal models of ANCA diseaseSeveral animal models of ANCA diseaseMPOMPO--ANCA, PRANCA, PR33--ANCA, antiANCA, anti--LAMPLAMP--2 2 antibodiesantibodies

ANCA

hυ

hυ’

c-ANCA:

CCytoplasmatisch

Diffuus, granulair, cytoplasmatisch patroon.

p-ANCA:

PPerinucleair

Perinucleaire tot nucleaireaankleuring.

ANCA can be divided into

c-ANCA-------------------PR3 c-ANCA (atypical)------------ PR3 + BPIPerinuclear ANCA (p-ANCA)(p )

classical p-ANCA--------------MPOp-ANCA without nuclear extension ----p ANCA without nuclear extension BPI,cathepsin G, elastase, lactoferrin and lysozymegranulocyte specific-antinuclear antibody (GS-ANA).g y p y ( )

atypical ANCA (a-ANCA or x-ANCA).

Other less common antigens incl de:include:

HMG1 (p ANCA pattern)HMG1 (p-ANCA pattern),HMG2 (p-ANCA pattern),alpha enolase (p and c-ANCA pattern),catalase (p and c ANCA pattern)catalase (p and c-ANCA pattern),beta glucuronidase (p-ANCA pattern),azurocidin (p and c-ANCA pattern),actin (p and a ANCA)actin (p and a-ANCA)h-lamp-2 (c-ANCA)

8585 toto 9090% of WG patients are positive for% of WG patients are positive for85 85 to to 9090% of WG patients are positive for % of WG patients are positive for ANCA, among them ANCA, among them 8080% are % are cANCAcANCA positive positive andand 2020% are% are pANCApANCA positivepositiveand and 2020% are % are pANCApANCA positivepositiveIn MPA In MPA 8080% among them % among them 8080% are % are pANCApANCApositive andpositive and 2020% are% are cANCAcANCA positivepositivepositive and positive and 2020% are % are cANCAcANCA positivepositiveCSS CSS 4040% positive mostly anti MPO% positive mostly anti MPO

Sensitivity and specificity of ANCA (Sensitivity and specificity of ANCA (IIF IIF ++ ELISA for PRELISA for PR33, , MPOMPO) from different studies in the literature) from different studies in the literature

Disease Sensitivity of ANCADisease Sensitivity of ANCA___________________________________________________

Limited Wegener's granulomatosis 50-66 %G li d W ' l t i 80 98 %Generalized Wegener's granulomatosis 80-98 %Microscopic polyangiitis 82-90 %Pauci-immune necrotizing extracap GN 90-95 %g pChurg-Strauss syndrome 60-70 %

Control group Specificity of ANCAControl group Specificity of ANCA____________________________________________________

Patients with various other diseases 76-91 %H lth bj t 94 99 %Healthy subjects 94-99 %

Inflammatory bowel diseaseInflammatory bowel diseaseInflammatory bowel diseaseInflammatory bowel disease

HenochHenoch--SchonleinSchonlein PurpuraPurpuraL b Fi diL b Fi diLaboratory FindingsLaboratory Findings

There is NO definitive diagnostic test.There is NO definitive diagnostic test.ggIgA levelsIgA levels may be elevated in may be elevated in 5050--7070% of patients.% of patients.Pl t l t t dPl t l t t d t di h ld b lt di h ld b lPlatelet counts and Platelet counts and coagcoag studies should be normal.studies should be normal.Inflammatory markers may be elevated.Inflammatory markers may be elevated.UrinalysisUrinalysis

Red cells, white cells, casts, proteinuriaRed cells, white cells, casts, proteinuriappMay not be present until later in the courseMay not be present until later in the courseRemember to continue UA screenings after the acute Remember to continue UA screenings after the acute ggphase.phase.

Negative RF and ANA.Negative RF and ANA.

Cryoglobulinemia

Type Composition Percent

Type I isolatedmonoclonalimmunoglobulins 10–15% of the total cases

Type II immunocomplexesformed 50–60% of reported casesType II bymonoclonal IgM 50 60% of reported cases

Type III immunocomplexesformed bypolyclonal IgM

25–30% of the reported cases

Cryoglobulinemic vasculitis

25%

Cryoglobulin precipitate in a cryocrit tube Serum protein electrophoresis

Kawasaki disease Kawasaki disease Laboratory EvaluationLaboratory Evaluation

Markers of systemic inflammationMarkers of systemic inflammationMarkers of systemic inflammationMarkers of systemic inflammationElevated CRP, ESR, Elevated CRP, ESR, leukocytosisleukocytosis with left shift, with left shift, reactive reactive thrombocytosisthrombocytosis (up to (up to 11 million)million)yy ( p( p ))

Anemia (Anemia (normocyticnormocytic, , normochromicnormochromic))SterileSterile pyuriapyuria (urethral origin don’t do a(urethral origin don’t do a cathcath))Sterile Sterile pyuriapyuria (urethral origin, don t do a (urethral origin, don t do a cathcath))TransaminaseTransaminase elevation (mild to moderate)elevation (mild to moderate)CSF fi diCSF fi diCSF findingsCSF findings

Mononuclear Mononuclear pleocytosispleocytosis, , hypoglycorrhachiahypoglycorrhachia, elevated protein, elevated protein

S i l fl id i fl iS i l fl id i fl iSynovial fluid inflammationSynovial fluid inflammation

Urgently Urgently need good need good biomarkers biomarkers

To distinguish active disease from damage or infection To distinguish active disease from damage or infection To predict relapse treatment response and prognosisTo predict relapse treatment response and prognosisTo predict relapse, treatment response, and prognosis. To predict relapse, treatment response, and prognosis. In the prototypic smallIn the prototypic small--vessel vasculitis, the autoantibodies vessel vasculitis, the autoantibodies for which it is named are unsuitable as biomarkers of diseasefor which it is named are unsuitable as biomarkers of diseasefor which it is named are unsuitable as biomarkers of disease for which it is named are unsuitable as biomarkers of disease activity. activity. A recent metaA recent meta--analysis concluded that the data is insufficient analysis concluded that the data is insufficient yyto support using persistently positive or rising ANCA titer to support using persistently positive or rising ANCA titer alone to guide treatment decisions.alone to guide treatment decisions.Also, traditional acuteAlso, traditional acute--phase reactants such as erythrocyte phase reactants such as erythrocyte sedimentation rate (ESR) or Csedimentation rate (ESR) or C--reactive protein (CRP) lack reactive protein (CRP) lack sufficient sensitivity and specificity to predict relapse in a sufficient sensitivity and specificity to predict relapse in a clinically significant manner.clinically significant manner.

New biomarkers not ready for New biomarkers not ready for primetimeprimetimeprimetimeprimetime

Subsets of B and T cells

AA i d (CD19 /CD38 ) B ll CD25 lAActivated (CD19+/CD38+) B cells vs CD25+ regulatory B cells

FF i ki i i b h bi i f B llFFor some patients taking rituximab the combination of B cell reconstitution and PR3 level can serve as a biomarker for relapsing diseaserelapsing disease.Activated T cells are detectable in the sera of AAV patients with both active and quiescent disease, but transcriptomewith both active and quiescent disease, but transcriptomeanalysis has identified CD8+ T cell expression profiles that divided patients into two distinct subgroups differing only in their risk of relapse.

New biomarkers not ready for New biomarkers not ready for primetimeprimetime

Activation of the alternative complement pathway is implicated in AAV pathogenesis.p y p p gPatients with active renal AAV show higher circulating plasma levels of C5a a common pathwaycirculating plasma levels of C5a, a common pathway component, and of fragment Bb, 3 proteins CXCL13 (BCA 1) (MMP 3) and tissue3 proteins CXCL13 (BCA-1), (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) that may distinguish active AAV from remission bettermay distinguish active AAV from remission better than ESR and CRP.

large vessel vasculitides, Giant Cell Arteritis (GCA) d T k A i i (TA)(GCA) and Takayasu Arteritis (TA),

Elevations of traditional inflammatory markers areElevations of traditional inflammatory markers are not specific for active vasculitis (and may be affected by therapy)by therapy).IL-6 is elevated in the arterial lesions and the peripheral circulation in GCA and TAperipheral circulation in GCA and TA. IL-6 levels in the serum correlate with disease

i i 13 Thi b i i f hactivity.13 This observation may inform therapy:A large study using IL-6 blockade with tocilizumab is under way for the treatment of new and relapsing GCA.14

Levels of another pro-inflammatory cytokine IL-Levels of another pro inflammatory cytokine, IL17, are also increased in the inflammatory lesions in active GCA They may predict response toactive GCA. They may predict response to glucocorticoid treatment.Certain circulating proteins including several matrixCertain circulating proteins including several matrix metalloproteinases have been suggested to be useful biomarkers for TA disease activityuseful biomarkers for TA disease activity.

Very recent biomarkers:Very recent biomarkers:

Pentraxin-3 in giant cell arteritis and Takayasu'sarteritis;arteritis; von Willebrand factor antigen in childhood central nervous system vasculitis;nervous system vasculitis; eotaxin-3 and other markers related to eosinophils

Th2 i i i hilior Th2 immune responses in eosinophilicgranulomatosis with polyangiitis (Churg-Strauss

d )syndrome);

Curr Opin Rheumatol. 2014 Jan;26(1):24-30. doi: 10.1097/BOR.0000000000000009.

Thanks for attentionThanks for attention