Different biochemical patterns in type II and type III mixed cryoglobulinemia in HCV positive patients

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ContentslistsavailableatScienceDirect

Digestive

and

Liver

Disease

j o u r n a l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / d l d

Liver,

Pancreas

and

Biliary

Tract

Different

biochemical

patterns

in

type

II

and

type

III

mixed

cryoglobulinemia

in

HCV

positive

patients

Umberto

Basile

a,1

_,

_Francesca

_Gulli

b,1

_,

_Laura

_Gragnani

c,∗

_,

_Krizia

_Pocino

a

_,

Cecilia

Napodano

a

_,

_Luca

_Miele

d

_,

_Stefano

_Angelo

_Santini

a

_,

_Mariapaola

_Marino

e

_,

Anna

Linda

Zignego

c

_,

_Gian

_Ludovico

_Rapaccini

d

a_Department_of_Diagnostic_Imaging_and_Laboratory_Medicine,_Fondazione_Policlinico_{Universitario}_Agostino_Gemelli_—_Università_Cattolica_del_Sacro_Cuore,

Rome,Italy

b_Department_of_Laboratory_Medicine_—_Madre_Giuseppina_Vannini_Hospital,_Rome,_Italy

c_Center_for_Systemic_{Manifestations}_of_Hepatitis_Viruses_(MaSVE),_Department_of_Experimental_and_Clinical_Medicine,_University_of_Florence,_Florence,_Italy d_Institute_of_Internal_Medicine,_Fondazione_Policlinico_{Universitario}_Agostino_Gemelli_—_Università_Cattolica_del_Sacro_Cuore,_Rome,_Italy

e_Institute_of_General_Pathology,_Fondazione_Policlinico_{Universitario}_Agostino_Gemelli_—_Università_Cattolica_del_Sacro_Cuore,_Rome,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29November2017

Receivedinrevisedform16March2018 Accepted19March2018

Availableonline4April2018 Keywords:

Antinuclearantibody(ANA) Cryoglobulins

HCV IgG3

Rheumatoidfactor

a

b

s

t

r

a

c

t

Background:Reversiblecryoprecipitabilityofproteinsisobservedasaconcomitantfeatureofimmune

complexformation.Mixedcryoglobulinemia(MC)issystemicvasculitis,associatedwithmixedIgMand

IgGcryoglobulins(CGs)showingrheumatoidfactor(RF)activity.Itisfrequentlyassociatedwithhepatitis

Cvirus(HCV).ThisstudyinvestigatesthepresenceofIgGRFandanti-nuclearantibodies(ANA)in

cryo-precipitatesofpatientswithtypeIIIandtypeIIMC,tounderstandthebiochemicalpatternsassociated

withdifferenttypesofMCtoagreaterdegree.

Methods:Serafrom70HCVuntreatedpatientswithtypeIIIortypeIIMCweretestedbyimmunoﬁxation

forIgG3andthroughELISAforIgGRF.CryoprecipitateswereanalysedforANAbyindirect

immunoﬂuo-rescencetoidentifyspeciﬁcpatterns.

Results:AfterstratiﬁcationaccordingtoMCtype,theANApatternsbetweentypeIIandtypeIIIMCwere

statisticallydifferent.IgG3levelsandIgG-RFpositivityweresigniﬁcantlyhigherintypeIIIcryoprecipitate.

WeobservedahigherpositivityofIgG3andasigniﬁcantdifferencebetweentheliverﬁbrosisstage,ANA

andIgG-RFinthecryoprecipitate.

Conclusion:Resultsshowacombinationofbiochemicalmarkersandautoantibodiesassociatedtomixed

cryoglobulinemia;theseﬁndingscouldbefurtherinvestigatedinordertoascertaintheirusefulnessin

assessingtheriskforthedevelopmentofmixedcryoglobulinemia.

1. Introduction

Cryoglobulinemiaisdeﬁnedasthepresenceof immunoglob-ulins(Igs)precipitatinginserumatatemperatureof<37◦Cand dissolvinguponreheatingofthespecimen.Reversible cryoprecip-itabilityofproteinsmaybeobservedasaconcomitantfeatureof immunecomplexformation.Therelevanceofthecryoprecipitation phenomenonbecameapparentwhenclinicalassociationswith

vas-∗ Correspondingauthorat:CenterforSystemicManifestationsofHepatitisViruses (MASVE),DepartmentofExperimentalandClinicalMedicine,UniversityofFlorence, LargoBrambilla3,50134Florence,Italy.

E-mailaddress:laura.gragnani@uniﬁ.it(L.Gragnani).

1 _Coauthor.

culitisandnephritis,resemblingthoseseeninexperimentalserum sickness,weredescribed[1].

Cryoglobulins (CGs) wereclassiﬁed byBrouet et al. [2] into threetypesaccordingtothecharacteristicsoftheconstitutingIg: typeIreferstothepresenceofasinglemonoclonalIg;typeIIis formedbytwoIgs,onemonoclonalandtheotherpolyclonal. Fre-quently,amonoclonalIgM-rheumatoidfactor(RF)complexedwith polyclonalIgG;typeIIIiscomposedofpolyclonalIgM-RFandthe correspondingantigen(usuallypolyclonalIgG)immunocomplex. Thelasttwotypesarereferredtoasmixedcryoglobulins(MCs). CGsinvivoprecipitateinthethinvesselsoftheextremitiesand theskin,wherethebloodtemperaturecandropwellbelow37◦C, aswellasinthekidney.TypeIIandtypeIIICGsweredeﬁnedas “essential”astheywerefoundinpatientswithoutanyclinically apparentdisease[3].

https://doi.org/10.1016/j.dld.2018.03.028

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Table1

Maincharacteristicsofthestudypopulation.

Typeofmixedcryoglobulinemia p

Total TypeII TypeIII

Total 70 30(43%) 40(578%)

Age(years)a ₆₄_±_11.97 _69.4_±₁₀ ₆₀_±_411.8

Gender(male/female) 24/46 October-20 14/26

Metavirscoreb F0 13 0 13 F1 20 17 3 F2 28 7 21 F3 4 1 3 F4 5 5 0 HCVgenotype 1 1(1.4%) 1(3.3%) – 1a 13(18.6%) 1(3.3%) 12(30%) 1b 34(48.6%) 16(53.3%) 18(45%) 2a 8(11.4%) – 8(20%) 2a/2c 5(17.1%) 5(16.7%) – 2a–3c 2(2.8%) – 2(5%) 3 1(1.4%) 1(3.3%) – 3a 2(2.8%) 2(6.6%) – MCsymptoms None 19(27.1%) – 19(47.5%) Neuropathy 25(35.7%) 18(60%) 7(17.5%) Meltzer’striad 30(42.8%) 20(66.7%) 10(25%) Renalinvolvement 5(7.1%) 3(10%) 2(5%) Raynaudphenomenon 12(17.1%) 11(36.5%) 1(2.5%) ALT(U/L)c 7–45U/L 13(19%) 13(43.3%) – p<0.001

>45U/L 57(81%) 17(56.6) 40(100%) TypeIIvs.typeIII

a_Data_is_expressed_as_mean_±_standard_deviation. b_Based_on_liver_stiffness_assessed_by_FibroScan. c _ALT_normal_range:_7–45_U/L.

MCsyndromeisasystemicvasculitis,affectingthesmallarteries andveins,associatedwiththepresenceoflargeamountsofmixed IgMand IgG cryoglobulins,showingRFactivity.It isfrequently associatedwithhepatitisCvirus(HCV)infection[4–6].

HCVchronicinfectionischaracterizedbythepossible devel-opmentofbothhepaticandextrahepaticmanifestations,anditis responsibleforpoly-oligoclonalB-lymphocyteexpansion,leading toseveralimmune-mediateddisorders.HCV-infectedpatients pre-dispositiontodevelopMCvasculitisremainsunclear,butthehost’s immuneresponsegenesmayplayarole[5].

In1992,Mussetetal.observedmicroheterogeneity character-izedbythepresenceoftwoormoremonoclonalIgMorIgG[7–9]

inpatientswithCGs.

TypeIIIpatternCGissuggestedtobeatransitionalstatethat evolvesfromapolyclonalpopulationofB-cellstoanoligoclonal, andﬁnallytoamonoclonaltypeIICG[9].

PersistentHCVinfectionassociatedwithMCshowsserological features of an autoimmunity process. Presence of non-organ-speciﬁc autoantibodies including antinuclear antibodies (ANA) is wellestablished, and is a peculiar characteristic of Systemic Autoimmune-RheumaticDiseases,althoughtheirpathogenicrole hasnotbeenuncoveredyet[10].Asserologicbiomarkers,ANAsare usefulfordiagnosingpatientswithautoimmuneorconnective tis-suediseases,butarealsoassociatedwithHCV-inducedresponses

[11,12].

HCVinfectionmaybeconsideredasamultiplestagediseaseand itshowsacorrelationbetweenIgGsubclassesandthepresenceof IgG-RF,andANAinHCVwithtypeIIICGs[13].IgG3presencein cryoprecipitatestypeIIIinHCV-andANA-positivepatientswould constituteadecisivefactorforthepossibleactivationof autoim-munemechanismsinthelongterm[13].

IgG3areknowntobeautoreactiveclonesandtheirabilityto activateseveralcellclonesisconﬁrmedbymanyclinicalstudies

[14].

Consideringtheincreasedriskofimmunologicaldisordersin patientswithHCV,itisnecessarytodeterminesensitiveand spe-ciﬁcsetsofbiomarkers.Thisstudyaimstoinvestigatethepresence ofIgGRFandANAsincryoprecipitatesoftypeIIIandtypeIICGs, aswellastosearchforarelationshipbetweentheiroccurrencein HCV-positivepatientsandHCV-relatedmixedcryoglobulinemia.

2. Materialsandmethods

2.1. Patients

CGssampleswerecollectedfrom70patientswithHCVinfection. PatientswereenrolledattwoItaliancenters(CenterforSystemic ManifestationsofHepatitisViruses(MaSVE),Departmentof Experi-mentalandClinicalMedicine,UniversityofFlorence,Florence,Italy andFondazionePoliclinicoAgostinoGemelliUniversitàCattolica DelSacroCuore,Rome,Italy).

Subjectswereincludedinthestudyaccordingtothefollowing criteria:presenceofHCV-RNAintheserum,absenceofantiviral treatmentand/orimmunosuppressivetherapy,andthepresence ofmixedcryoglobulinemiasymptoms(Table1).

MCwasdiagnosedwhenthepresenceofserumCGswasfound inatleasttwometachronoustests.ALTvalueswereavailablefor allthepatientsandtheMETAVIRscore,wasassessedbytransient elastography(Fibroscan).Exclusioncriteriawas:thepresenceof co-infections(HIVandHBV),ongoingdrugtreatmentandthe pres-enceofotherautoimmunedisorders.Written informedconsent wasobtainedfromallindividualsandthestudywasconducted inaccordancewiththeDeclarationofHelsinki.

2.2. Clinicalandlaboratoryassessment

Twentymlofperipheralbloodwascollectedusingtubes with-outanticoagulant,andkeptat37◦Cbothbeforeandaftersample collectionforatleast30minuntilcompleteclotting[15,16].

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Fig.1.DifferentbiochemicalpatternsinthestudypopulationstratiﬁedaccordingtoMCtype.PanelA:resultsofANAdetection;panelB:IgG3;panelC:IgGRF positiv-ity/negativity;panelD:ALTnormallevel(7–45U/L)andabnormallevel(>45U/L)distribution.

Thisimpliesasimplebiochemicalquantiﬁcation,which never-thelessrequiresstrictpre-analyticalprotocoladhesioninorderto maintainthesampleatastabletemperatureof37◦C, especially throughouttheinitialstepsofthetest[17].

Followingcentrifugation,thesupernatantserumsampleswere transferredintoWintrobetubesandincubatedat4◦C.The precipi-tationprocessmanifestsitselfinavarietyofwaysamongsamples, dependingontheconcentrationofCGspresent,andmayrequire eitherafew hours(whenCGs-oftentypeI-becomeinsolubleat roomtemperature)or longerperiodsof time (7days), particu-larlyintheeventoflowconcentrationsoftypeIIICGs.Cryocrit percentagewasassessed,thesupernatantwasremoved,andthe remainingcryoprecipitatewasrecoveredandwashed3times[16]. Washingwascarriedoutbyusinga4%polyethyleneglycol6000 (PEG6000)solutionobtainedbymixing4gofPEG6000in100ml ofphosphatebuffersolution(PBS)(4%p/v).2mlof4%PEG solu-tionwasaddedtoeachcryoprecipitatesample,whichwasthen centrifugedat3000×gfor5minat37◦C.Cryoprecipitatewas re-dissolvedintheappropriatevolumeof a3%PEG6000solution, andre-solubilizedfor30minat37◦C.Igsweretypedand charac-terizedbyimmunoﬁxationelectrophoresis(IFE)ontheG26Fully automatedsystem(Interlab,Italy),accordingtothemanufacturer’s instructions.IFEwasperformedusingantiseraagainst␥,␣,␮,␬, and␭.IgG3subclasseswerecharacterizedbymeansofantisera (BindingSite,UK).AstainedbandintheIFErepresentsareaction betweenanantiserumandaspeciﬁcproteinonthespecimen.IgG RFwastestedusingELISAkitsforquantitativemeasurementofIgG RF(INOVA,USA).Analiquotofthesamplewaspreviouslyremoved

forELISAtesting,andassayedat37◦C.Allsampleswereanalysed atthesametimefollowing themanufacturer’sinstructions,and plateswereimmediatelyreadonaplatereaderasindicatedbythe manufacturer.FollowingClinicalandLaboratoryStandards Insti-tute(CLSI)guidelines[18],wetested20healthydonorsfromthe localpopulationtoverify adherencetoCLSI EP28A3C[18] and accordingtothemanufacturer,IgGRF≥9wastobeusedascut-off positive.

ANA determination was carried out by means of indirect immunofluorescence(IIF)onHEp-2cells,AmericanTypeCulture Collection(ATCC)(INOVA,USA),whichenablestheexpressionof antigensthroughoutall stagesof thecellcycle[19].Cellswere cultured onglass slides in multiplewellplates and the appro-priate titer of cryoprecipitate was added to cells. Slides were incubated for 30min and then washed with PBS in order to removeanynon-specificbindingorexcess.Asecondarypolyclonal antibody (anti-human IgG polyclonal sheep antiserum, FITC-conjugatedanti-IgGanti-serum,Invitrogen,LifeTechnologies)was subsequentlyadded.Excessconjugatedsecondaryantiserumwas removedby washingwith PBSsolution.Slides were fixedwith glycerolandmountedformicroscopy.Cryoprecipitatedilutionfor ANAdetection wasin PBS1:5.Thisiscurrentlytheone indica-tionconcerningadequatetiterstobeusedforANAdetectionin cryoprecipitates[13].

QuantitativeHCV-RNAdetectionincryoprecipitatewas deter-minedbyaroutinemethodandavirusgenotypewasdetermined for each sample (Siemens Healthcare, Germany). Presence and quantiﬁcation of HCV-RNAwere determined bymeansof

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real-Fig.2.DifferentbiochemicalpatternsinthestudypopulationstratiﬁedaccordingtoMETAVIRscore.PanelA:resultsofANAdetection;panelB:IgGRFpositivity/negativity; panelC:MCtypedistribution;panelD:ALTnormallevel(7–45U/L)andabnormallevel(>45U/L)distribution.

time polymerase chain reaction (PCR), transcription-mediated ampliﬁcation(TMA),andmulti-probereversehybridizationofthe 5-untranslatedregion(5-UTR)oftheHCVgenome.TestingforHCV genotypeandsubtypewasperformedthroughreverse transcrip-tionandPCRandreversehybridizationoftheHCVgenome.

2.3. Statisticalanalysis

StatisticalanalysiswasperformedusingtheStatisticalPackage forSocialScience(SPSS)version15.0(Chicago,Illinois). Distribu-tionofthedatainthegroupswaspreliminarilyevaluatedviathe Kolmogorov–Smirnovtest.Continuousvariableswerepresentedas mean±SD.Categoricalvariablesweredescribedasnumbersin per-centageandcomparedusingthe␹2_test._The_differences_in_molecule

levelsbetweengroupswereanalyzed usingtheKruskal–Wallis, Mann–WhitneyTest,andmultiplecomparisonsposthoctestsas appropriate.Atwo-sidedpvalueoflessthan0.05wasconsidered, associatedwithstatisticalsigniﬁcance.

3. Results

Atotalof70patients,46femaleand24male,meanage64years (range35–79)wereretrospectivelyenrolled.IgG-RFwaspresent in42.9%(N=30)ofpatientcryoprecipitates.TheIgG3subclasswas negativein37/70(53%)patients.

ANAdeterminationassaywasnegativein32/70subjects(46%) andpositiveintheremaining38/70(54%);ALTlevelswere nor-mal(mean29.9±9.8U/L)in13/70patients(19%)andincreased

(mean106.1±52.7U/L)in57/70(81%),normalvaluesrangefrom 7to45U/L.

Results of cryoglobulin type assessment showed that 30/70 patients(43%)hadtypeIIand40hadtypeIIIMC(57%).Stratifying resultsaccordingtoMCtype,theANApatternswerestatistically differentbetweentypeIIandtypeIIIMC(p<0.001)(Fig.1,panel A);IgG3levelsandIgG-RFpositivitywerehigherintypeIII cryopre-cipitate(p<0.004,Fig.1,panelBandp<0.001panelCrespectively); ALTlevelswerealsosignificantlydifferentbetweentypeIIandtype IIIMC(p<0.001):inthetypeIIMCgroup13/30patients(43%)had normalALT(mean29.9±9.8U/L)and17/30(57%)hadan abnor-malALTlevel(mean84.3±43.4U/L);alltypeIIIMCpatientshad anabnormalALTvalue(mean115.4±53.9U/L)(Fig.1,panelD). Dividingthetotalpopulationintotwogroups,accordingtoALT lev-els(normalandabnormal),wedidnotfindasignificantcorrelation withIgG3positivity(p=0.056;datanotshown).

Stratifyingfortheliverfibrosisstage,accordingtotheMETAVIR scoringsystem,weobservedahigherpositivityofIgG3and,a sta-tisticallysignificantdifferencebetweenthestageofliverfibrosis andANA(p<0.001,Fig.2,panelA),IgG-RFinthecryoprecipitate (p=0.027;Fig.2,panelB)andalsoadifferenceinMCtypefrequency (p<0.001)(Fig.2,panelC).ALTlevelsstratifiedaccordingtoliver fibrosisstagesareshowninFig.2,panelD.

4. Discussion

The pathogenesis of HCV-related MC is still not completely known[20].Keyfactorsareprobablychronicstimulationofthe

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immune systemthroughHCV infection,and thedysfunction of thereticuloendothelial system[21],therebyleading tothe pro-ductionandpersistenceofantibodiesthatareabletoprecipitate belowbodytemperature[22].ThereisahigherprevalenceofIgG3 responsestoHCVantigensinHCV-relatedMCpatientsthaninHCV patientswithoutMC[23].

IgG3ﬁxescomplementmoreefﬁcientlythanothersubclasses, therebyleadingtotheactivationoftheclassicalpathway[13,24]. CGsareheterogeneousimmune-complexes,anddonotalways cor-relatewiththeseverityofsymptoms,[25].

HCVpatientsexpressagreatvarietyofautoantibodiesusually atalowtiter[26],andtheymaybeassociatedwithunderlying autoimmunedisordersorliverinﬂammationinHCVinfection.A possiblereasonforantibodyproductionisoveractivationand pro-liferationofBlymphocytes,viainteractionwiththeHCVsurface protein[27].

DatashowsadifferentpresenceofautoantibodiesandIgG3in typeIIandtypeIIICRGsthatprobablyrepresentsamodulationof theimmunesystemtocontinuousantigenicstimulation.

Inourcohort,weanalysediftherewasanycorrelationbetween typeIIand typeIIIMC withthepresence ofANA, IgG-RF,IgG3 subclassandALTelevation(Fig.1).

Thedifferencebetweenthetwogroupsisprobablyduetothe existenceofa progressiveevolutionof CGs.ElevatedALTlevels intypeIIIareprobablyduetoabroaderimmune responsethat involvesbothIgG1,whichtakepartintherecognitionofthevirus, andthepresenceofIgG3thattriggerssomeautoimmuneresponses aswellasIgG-RFthatparticipatesintheformationoftheimmune complex[15].ItisalsoconceivablethatthepresenceofANAand thenautoreactive clones representsthetriggerfor extrahepatic autoimmunemanifestations.

TypeIICGsarisebya later clonalselectioncharacterizedby thepresenceofamonoclonal␬−IgM responsiblefortypicalMC symptoms.LowtemperaturesandIgG3seemtotriggerareversible cryoprecipitation,possiblybyinducingstericmodificationsofIg molecules[28].ThebiologicalactivitiesofeachsubclassofIgGare notfullyknown.IgGreceptorsarestrikinglynumerousinhumans. Theyare comprised of high-affinityand low-affinity. The pres-enceofIgG3incryoprecipitatesofHCV-andANA-positivepatients constitutesadecisivefactorforthepossibleactivationof autoim-munemechanismsinthelongterm[15].Inaddition,IgG3positive patientsarealso positive for IgG-RF, knowntobe autoreactive clones,andtheircapacitytoactivateseveralcellclones.Thus,the presenceofIgG3incryoprecipitatesmaysuggest amorehighly activatedimmune system, which is then more exposed tothe mechanismsofautoimmunediseases[15].Thepossiblepresenceof IgG-RForANAmaybetheresultofselectedclonesofautoreactive IgGinHCVpatients.

Hepaticﬁbrosisstimulatestheproductionandaccumulationof collagenandextracellularmatrixproteins.Moleculesderivedfrom bothpathogenandhostcaninduceinﬂammationinarigidly con-trolledprocess,thatengagesimmunecellsandparenchymalcells viadifferentsignaltransductionpathways[29].

InthepresentstudypatientswithMetavirscoreF2,hadatypeIII CGpattern.AllpatientswithatypeIIICGpatternhadelevatedALT. TheelevatedALTalsocorrelatedwiththepresenceofIgG-RFinthe cryoprecipitateandparticularlyfinespeckledANA(Fig.2panelD). Thesefindingswereunexpected,andfurtherstudieswillbeuseful inconfirmingifamildfibrosisstagewithhighALTlevelsis cor-relatedwitha“secretory”behavior,highlevelsofcryoprecipitate, andANApositivity.

Resultsshowthatacombinationofbiochemicalmarkerscould have predictive value for the diagnosis of clinically signiﬁcant extrahepaticmanifestationsinpatientswithCG,anddetectionof autoantibodieshasasigniﬁcantroleinthemosaicofautoimmunity asanimportantriskfactorforthedevelopmentofautoimmune

disease[30].Consideringthedouble-edgednatureofantibodies, unveilingthe origin and pathological implications of infection-drivenautoantibodiescouldbeamilestoneinthedevelopmentof effectivetherapeuticstrategiesagainstbothchronicinfectionsand autoantibody-mediatedsystemicdisorders.Moreover,the hypoth-esisthatIgG3couldplayanimportantrolebothintheinduction andpersistenceofcryoglobulinemia,aswellastheassumptionof anevolutionofsuchaconditionfromtypeIIICRGstotypeIICRGs, havebeenconﬁrmedbyotherauthorsaswell[28].

ThepresenceofautoantibodiesinthecourseofchronicHCV infection(in particular ANA) is a phenomenon that couldbe a virus-inducedautoimmunity,ofwhichcryoglobulinemiacouldbe apossiblemodel.Thedetectionofanautoantibodyisnot neces-sarilyrelatedtoafrankautoimmunedisease,butthepresenceof ANAcouldbeakeyfactorinautoimmunemechanismsactivation and,theirdetectioncouldbeascreeningtest,andabasictoolinthe diagnosisandmanagementofpatientswithautoimmunedisease. Inconclusion,HCVdiseaseistheresultofamultifactorialand multistepbiochemicalandpathogeneticprocess.Ourresults,along withlongitudinalstudies,demonstratethatthediscussedsetof biomarkerscanbeusedasapredictivetoolforvariationsin clini-calmanifestations.Someextrahepaticmanifestationsareimmune mediatedwhileothersseemtobedrivenbychronicinﬂammation. Also,theavailabilityofthesenon-invasivetestscouldbevaluablein ordertoassesstheeffectivecontrolofHCV-related lymphoprolifer-ationsorautoimmunediseases,afterviraleradication,highlighting anareaofinterestthatmaywarrantfurtherinvestigation,inorder todeterminevariationsincryoprecipitatesassociatedwith differ-entoutcomesinHCVpatients.

Conﬂictofinterest

Nonedeclared.

Guarantor

UB.

References

[1]FerriC,ZignegoAL,PileriSA.Cryoglobulins.JClinPathol2002;55(1):4–13.

[2]BrouetJC,ClauvelJP,DanonF,KleinM,SeligmannM.Biologicandclinical signiﬁcanceofcryoglobulins.Areportof86cases.AMJMed1974;57:775–88.

[3]MeltzerM,FranklinEC.Cryoglobulinemia:astudyof29patients.AmJMed 1966;40:828–36.

[4]FerriC,SebastianiM,GiuggioliD,ColaciM,FallahiP,PilusoA,etal.Hepatitis Cvirussyndrome:aconstellationoforgan-andnon-organspeciﬁc autoim-munedisorders,B-cellnon-Hodgkin’slymphoma,andcancer.WorldJHepatol 2015;7(March(3)):327–43.

[5]CacoubP,GragnaniL,ComarmondC,ZignegoAL.Extrahepaticmanifestations ofchronichepatitisCvirusinfection.DigLiverDis2014;46S5:S165–73.

[6]ZignegoAL,GragnaniL,PilusoA,SebastianiM,GiuggioliD,FallahiP,etal. Virus-drivenautoimmunityandlymphoproliferation:theexampleofHCVinfection. ExpertRevClinImmunol2015;11(1):15–31.

[7]MussetL,DiemertMC,TaibiF,ThiHuongDuL,CacoubP,LegerJM,etal. Charac-terizationofcryoglobulinsbyimmunoblotting.ClinChem1992;38:798–802.

[8]TissotJD,PietrograndeM,TestoniL,InvernizziF.Clinicalimplicationsofthe typesofcryoglobulinsdeterminedbytwodimensionalpolyacrylamidegel electrophoresis.Haematologica1998;83:693–700.

[9]PontenF,HalimiC,BrocardA,DelacourT.BiclonalimmunoglobulinM dysglob-ulinaemia:evolvingaspectsinacaseofprimarySjögren’ssyndrome.EurJClin ChemClinBiochem1997;35:287–90.

[10]Agmon-LevinN,Damoiseaux J,Kallenberg C,SackU, WitteT,Herold M, etal.Internationalrecommendationsfortheassessmentofautoantibodies tocellularantigensreferredtoasanti-nuclearantibodies.AnnRheumDis 2014;73:17–23.

[11]Zusinaite E,Metskula K,SalupereR. AutoantibodiesandhepatitisCvirus genotypesinchronichepatitisCpatientsinEstonia.WorldJGastroenterol 2005;11:488–91.

[12]ChrétienP,ChoustermanM,AbdAlsamadI,OzenneV,RosaI,BarraultC,etal. Non-organ-speciﬁcautoantibodiesinchronichepatitisCpatients:association withhistologicalactivityandﬁbrosis.JAutoimmun2009;32:201–5.

[13]BasileU,GulliF,TortiE,DeMatthaeisN,ColaciccoL,CattaniP,etal. Anti-nuclearantibodydetectionincryoprecipitates:distinctivepatternsinhepatitis Cvirus-infectedpatients.DigLiverDis2015;47:50–6.

(6)

[14]IzuiS,BerneyT,ShibataT,FulpiusT.IgG3cryoglobulinsinautoimmune MRL-lpr/lprmice:immunopathogenesis,therapeuticapproachesandrelevanceto similarhumandiseases.AnnRheumDis1993;52(Suppl1):S48–54.

[15]BakkerAJ,SlompJ,deVriesT,BoymansDA,VeldhuisB,HalmaK,etal.Adequate samplingincryoglobulinaemia:recommendedwarmly.ClinChemLabMed 2003;41:85–9.

[16]KallemuchikkalU,GorevicPD.Evaluationofcryoglobulins.ArchPatholLabMed 1999;123:119–25.

[17]BasileU,TortiE,Dell’AbateMT,ColaciccoL,GulliF,ZuppiC,etal.Pre-analytical phaseincryoglobulin(CRG)detection:analternativemethodforsample trans-port.ClinChemLabMed2016;54(4):e123–6.

[18]ClinicalandLaboratoryStandardsInstitute.EP05-A3evaluationofprecisionof quantitativemeasurementprocedures:approvedguideline.3rded.Wayne,PA 19087USA:ClinicalLaboratoryStandardsInstitute;2014.

[19]MeroniPL,SchurPH.ANAscreening:anoldtestwithnewrecommendations. AnnRheumDis2010;69:1420–2.

[20]Zignego AL,Ramos-CasalsM,Ferri C,SaadounD, ArcainiL, RoccatelloD, etal.InternationaltherapeuticguidelinesforpatientswithHCV-related extra-hepatic disorders. Amultidisciplinaryexpert statement.Autoimmun Rev 2017;16:523–41.

[21]Minopetrou M, Hadziyannis E, Deutsch M, Tampaki M, Georgiadou A, DimopoulouE,etal.HepatitisCvirus(HCV)-relatedcryoglobulinemia: cryo-globulin type and anti-HCV proﬁle. Clin VaccineImmunol 2013;20(May (5)):698–703.

[22]GulliF,BasileU,GragnaniL,FognaniE,NapodanoC,ColaciccoL,etal. Autoim-munityandlymphoproliferationmarkersinnaïveHCV-RNApositivepatients withoutclinicalevidenceofautoimmune/lymphoproliferativedisorders.Dig LiverDis2016;48(August(8)):927–33.

[23]OtaniM,KurokiA,KikuchiS,KiharaM,NakataJ,ItoK,etal.Sialylation determinesthenephritogenicityofIgG3cryoglobulins. JAmSocNephrol 2012;23(November(11)):1869–78.

[24]DispenzieriA,GorevicPD.Cryoglobulinemia.HematolOncolClinNorthAm 1999;13:1315–49.

[25]ChristodoulouDK,DalekosGN,MerkouropoulosMH,KistisKG,GeorgitsiG, ZervouE,etal.Cryoglobulinemiaisnotamajorprobleminclinicalpractice duetochronicviralhepatitisinfections.EurJInternMed2001;12:435–41.

[26]LunelF.HepatitisCvirusandautoimmunity:fortuitousassociationorreality? Gastroenterology1994;107:1550–5.

[27]Yang DH, Ho LJ, Lai JH. Useful biomarkers for assessment of hepatitis Cvirusinfection-associated autoimmunedisorders.WorldJGastroenterol 2014;20(11):2962–70.

[28]BasileU,GulliF,GragnaniL,FognaniE,NapodanoC,PocinoK,etal.IgG3 sub-class:ApossibletriggerofmixedcryoglobulincascadeinhepatitisCvirus chronicinfection.DigLiverDis2017;49:1233–9.

[29]SzaboG,PetrasekJ.Inﬂammasomeactivationandfunctioninliverdisease.Nat RevGastroenterolHepatol2015;12(July(7)):387–400.

[30]ShoenfeldY,BlankM,Abu-ShakraM,AmitalH,BarzilaiO,BerkunY,etal.The mosaicofautoimmunity:prediction,autoantibodies,andtherapyin autoim-munediseases—2008.IsrMedAssocJ2008;10:13–9.