A SIMPLE PROCEDURE THAT INCREASES THE SPECIFICITY OF THE ACTIVATED PROTEIN C RESISTANCE TEST IN SAMPLES CONTAINING ANTIPHOSPHOLIPID ANTIBODIES

Thrombosis Research,Vol.86,No.5, pp.385-391,1997

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A SIMPLE PROCEDURE THAT INCREASES THE SPECIFICITY OF THE ACTIVATED PROTEI.N C RESISTANCE TEST IN SAMPLES CONTAINING ANTIPHOSPHOLIPID ANTIBODIES Eva MarieJacobseq FinnWislaff HaematologicalResearchLaboratory,Ullev?dUniversityHospital,Oslo,Norway (Received

17 March 1997by Editor H.C. Godal; revised/accepted 8 April 1997)

Abstract It iswellknownthatplasmaswithlupusanticoagulants(LA) maygivefalselow activated protein C (AX) ratios, and these false posit;vetests’are not necessarilycorrected by mixing with factor V deficientplasma (FVdef). In the present study, we show that 1+1withpoolednormalplasma@P) insteadof mixingwith repeatingthetest after-g FVdef confkn the presence of the Leiden mutation (FV-Leiden) in patients with antiphospholipidantibodies(APA). Samples from sixteen patients with a low APC-ratio were examined.Eight samples containedAPAs,includingfivesampleswith LA. Results: The APC-ratiosof elevensamples,includingfour with APAs, became normal when retested after mixingthe plasma1+1with NP. All of them were heterozygousfor FV-Leiden.One additionalpatient,who had partialcorrectionof the APC-ratio,proved to be homozygousforthe Leidenmutation.The remainingfour samples,all of whichhad high positivetests for APAs, remainedunchanged.One of these four was heterozygous for FV-Leide~ whilethe otherthree had normalFV. Conclusion:Normalisationof a low APC-ratiotier dilution1+1 with NP confirmsthe diagnosis of FV-Leiden.PCR analysiscould be reservedfor cases not tiected by this procedure. @1997 Elsevier Science Ud The conventionalmethod used to test for resistanceto APC has been to determinethe activated partialthromboplastin time(APTT)of a plasmaboth in the presenceand absenceof exogenousAPC andthencalculatetheratiobetweentheseclottingtimes(l). However,with this method,fhke normal Key words: Antiphospholipidantibodies,lupusanticoagulant,APC-resistance. Correspondingauthor:Dr Eva Marie Jacobsen.HaematologicalResearchLab. Ullev&lUniversity Hospital.0407 Oslo,Norway.Fax +47 22119181.

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ratiosoccurredwhentestingpatientstreated with oral anticoagulants,and a low ApC-l”aliodoes not alwayssigni~an inheritedmutationin factorV (2). For instance,manypatientswith LA havea low APC-ratio(3.4).Therehasbecusomecontroversywhetherthesepatientscarry the facto[-V mutation (5), but most reports suggestthat this is an acquiredAPC-resistance(6,7). These methodological problemsled severalinvestigatorsto concludethat plasmaswith a prolongedAPTT in the absence of APC could not be tested with this APC-resistancetest (2). The problemassociatedwith oral anticoagulantsseemsto havebeen solvedwith the modificationrecommendednow, i.e. mixingtest plasma 1+4withFVdefbeforetesting(8,9). However,the use of mixtureswithFVdefhas not been shownto eradicatethe problemof low APCI-ratiosin somepatientswith APAs. The aimof thisstudywasto examinewhethera simplemodificationof the APC-resistancetest could increasethe specificityof thetestin samplescontainingAPAs,Our hypothesiswas that mixingplasma from patientswith FV-Leiden 1+1 with NP insteadof mixingwith FVdef would provideenough normalFV to correct the low APC-ratio,whilean APC-resistanceduc to an inhibitorrather than a mutatedFV wouldnot beabolishedby a 1+1mixturewithNP. If so, a simplemodificationof the test could be used to vcrifi lhat an APC-resistanceis due to a defect FV. MATERJALANDMETHODS Patients 20 consecutivepatientswithLA weretestedfor APC-resistance using 1+4mixtureswith FVdef.Five

of these(patients1-5,table I) showedAK-resistance and were includedin the studytogetherwith I I patients with low APC-ratio identifiedfrom our routine testing of outpatientsreferred for investigationofthrombophilia(patients6-16,tableI). Threeofthese 11patientshad low or moderate positivetests for anticephalinand/oranticardiolipinantibodies. Plasma samples All plasmasampleswerepreparedfrombloodcollectedin l/10thvolumeof 0.129 M bufferedsodium

citrate(Vaeutainer@, BectonDickinson,France).Afler centrifugationat 2000 g for 15minutes,the plasmawas stored in smallaliquotsat -70”C. Poolednormalplasma(NP) for LA testingas wellas plasmafi-ompatientswith APAs were filtered(Millex-GV0.22 ,umFilter Unit, Millipore,USA) beforefreezing. Poolednormalulasma(NP)forLAtestingwaspooledcitratedplasmafl-om20 healthyblooddonors and was filteredbefore storageat -70°C. A specialbatch of NP was preparedfor APC-resistance testingfrom 36 blood donors,each with normalAPC-resistancetest. Assays

APC-resistancewasdeterminedwiththe commercialkit Coatest@APCTM-Resistance (Chromogenic AB, Sweden).Thekitisbasedon a modifiedAPTT performedin the presenceand absenceof APC, respectively.Theratiobetweenthe clottingtimesis then calculated.The APC-resistancetestingwas performedon 1+4mixtureswith FVdef(ChromogenixAB). In addition,all plasmaswere retested using 1+1mixtureswithNP insteadof mixtureswith FVdef. APC-ratioswereexpressedas normalizedratios(nAPC-ratio)by dividingby the correspondingratio for NP definedin eachtest cycle.Theuseof nAPC-ratioshasbeenshownto givegood discrimination betweennormal,heterozygousandhomozygousvalues,respectively(2). DeRonde and Bertinafound thatpatientswithnormalFV-Leidenhada nAPC-ratioabove0.84 and patientsthat are heterozygous

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for FV-Leidenhave a ratio between 0.45 and 0.70. Homozygouspatientshavea ratio below 0.45. To be sure to identi~ patientswith abnormalAPC-ratios,we includedpatientswith a nAPC-ratio below0,80, DNA analvsis.DNA was preparedilom EDTA wholebloodusingDynabeads@DNA DirectlMkit (DyrAAS, OS1O, Norway)accordingto the manufactuds instructions.PCR analysiswas performed essentiallyas describedby Bertinriet al. (10). A 220 bp fragmentof exon 10/intron10 of the factor V gene was amplifiedby PCRbeforetreatmentwith restrictionenzymeMrd 1. LutMsanticoamdants.Two semi-automated,integratedprocedureswere used as describedearlier (11).Theproceduresare APTT-basedand RVVT-based,respectively.In each procedureplasmais mixed1+1withNP and clottingtimeswith two differentphospholipidconcentrationsare recorded. Crudecephalinhorn bovinebrain(a gift fromNycomedPbarma,Oslo,Norway) are used as source of phospholipid.A computerprogramcalculatesthe ratio betweenthese clottingtimesobtainedwith low and high concentrationsof cephalin. The ratio is then normalizedby dividingwith the correspondingratioforNP. Theresuhingratio is definedas the lupusratio (LR) of that plasma.The 97.5percentileof theLR in a normalpopulationhasbeen calculated(10) and definedas one LA-unit (LA-U).Usingdilutionsof a strongLA-positiveplasmaas a standard,the LR is transformedto LAunits. A resultbetweenone and two LA-U is considereda low positiveLA betweentwo and four LA-U is a moderatepositiveresultand more than four LA-U signifiesa highpositiveLA. Anticenhalin antibodies {Aceuha),An enzyme-linkedimmunosorbentassay (ELISA test) for quantitation of anticephalinantibodieswas performed as describedearlier (12). This assay has previouslyshowna highconcordancewith a diluteAPTT screeningtest for LA. The resultswere divergentin somecases,though,and the ELISA test may havehighersensitivity(12). Anticardioliuinantibodies {ACAl The patient samples were analysed for ACA essentiallyas describedby Gharaviet al (13). The standardsdefinedby Harriset al. (14) were used. The results of both ELISA tests are presentedas low (+), medium(+-+)or high(+++) positive. RESULTS Theplasmaswerelint assayedafter mixing1+4with FVdef.Fiveof 20 (25VO)consecutivepatients withLA hadAPC-resistancedefied as a nAPC-ratiobelow0.80(patients1-5,table I). PCR analysis confirmedheterozygosityforFV-Leidenintwo ofthesepatients(1OYO of the patientswith LA), while three had normalallelesfor FV. TableI showsthe resultsof the testingof allthe 16 patientswith APC-resistance,i.e.the fivepatientswithLA andthe 11patientswith APC-resistanceidentifiedtlom the routinetestingof outpatients. Theplasmawasthentestedtier mixing1+1withIV. Withthis procedure,normalAPC-ratioswere obtainedin 11of the 16 patients,includingone patientwith LA and the three patientswith positive tests for AcephaandlorACAbut no LA. PCR analysisconfirmedheterozygosityfor FV-Leidenin all. One firther patient(patientno. 10)had a substantialincreaseof the ratio withoutreachingthe reference range; this patientturned out to be homozygousfor FV-Leiden.In the remainingfour patients, the APC-ratio remainedlow after 1+1 mixingwith NP. All these were stronglyAPA positive,and they were positivefor both m Acephaand ACA PCR analysisshowedthat one of themwas heterozygousfor the FV-Leidenmutatio~ whilethe rernainingthree had normalFV.

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TABLE I Effect on Low NormalizedAPC-Ratios(nAPC-ratio)of Mixing 1+1with Normal Plasma.RatiosThat BecameNormal Are Shownin Bold Face. patient no, 1 2

3 4 5 f> 7

8 9 10 II 12 12

14 15

16

nAPC-ratio 0.76 0.69 (i.71 0.58 0.67 0.65 057 ().63 0.64

0.47 0.62 ().65 [}.57 0.62 0.63 0.65

1+1 NP 0.58

0.97 0.62 (1.(,4 (t64 0.99 0.84 0.92 1.19 ().77 1.06 0.90 1.08 1.01 0.ss 0.89

LA-IJ* 1.9/1.6 1.314.2 4. 1/>6 4.O/~6 4,5/6,0 neg. neg. neg. ncg, neg. ne~. Nl) neg ncg ncg, neg.

ACA*+*

Acepha** rgw Igw Iq-i F++ lgM+-kI Ig[* neg. ncg, neg. TIGj+ Ilcg. neg. neg. neg. TgG+t IgM+t Ighf+

1gG+t+r’M+

Igtwt Igw-H

lgCW+/M+ IgC;W+/M+ neg. mY rwg ncg, neg. neg. nefi Iwg. I@++

ncg, ne~.

PCR normal hctcrozygous normal hetcrmy~ows nomwl hctcroqgous hetcrozygous heternzygotls Iwtermygous hnmur.ygous hetcrozygous heterozygous heterozygous helermygous helw(nqgous hctcmzygms

*LA-U = The resultsof the APTT-basedand the RVVT-basedtests for LA (see material and methods).**ELISAtest ~’oranticephalinantibodies.*** ELISA test ~’or anticardiolipin antibodies.ND = not done. Plasma from two patientswho retained low APC-ratiosafter mixingwith NP was retested after progressivedilutionsinFVdefandin NP, respectively(1+1, 1+4, 1+9and 1+19mixtures)(tableII). Oneofthesepatientshad normalFV (patientno. 1) and the other was heterozygousfor FV-Leiden (patientno. 4). The APC-ratioin the patientwho was heterozygousfor FV-Leidenbecamenormal afler 1+4mixingwithNP, but remainedlow in alldilutionswith FVdef The patientwith normalFV had similarAPC-ratioswith FVdefand NP at each dilutionstep and the ratios eventuallybecame normalwith both FVdefand NP when diluting1+19. TABLE II NormalizedAPC-Ratios(rtAPC-ratio)in Two PatientsWho RetainedLow APC-Ratios after MixingPlasma 1+1withNP. nAPC-RatiosAbove0.80 Are Shownin Bold Face. 1+1 mixtures

1+4 mixtures

1+9 mixtures

1+19 mixtures

plasma mixed with FVdef

0.45

0.76

0.74

0.83

plasma mixed with IW

0.58

0.66

0.71

0.85

plasma mixed with FVdef

0.49

0.58

0,64

0.66

plasma mixed with NP

0.64

1.03

1.09

1.14

patientno. 1 (normal

PCR)

patientno. 4 (heterozygous)

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DISCUSSION Theprincipalaimof thisstudywasto examinewhetherrepeatingtheAPC-resistancetest after mixing 1+1whhNP couldaid in the differentiationbetweenlow ratiosdue to FV-Leiden~d those due to the influenceof APAs. Whenthe low APC-ratiois due to a defectFV, mixingwith FV-deficient plasmacan not correctthe defect.I! however,the plasmais mixedwith NJ?,one would expectthe normalFV intheNP to causea partialor totalcorrectionof the APC-ratiodependingon whetherthe patientis homo-or heterozygousfortheFV-Leidenmutation.The use of 1+1mixtureswith NP was chosenbecausethis degree of dilutionin NP is the mixtureused when testingfor LA to prove the inhibitoryeffect.A higherdilutioncouldperhapsneutralizea weak LA. As shownintable1,allpatientswhoseplasmasampleshad normalAPC-ratioswhen mixingwith NP wereheteroqgousforFV-Leiden.Furthermore,thisgroupincludedallheterozygouspatientsexcept onewho in additionhad strongAPAs.Oneadditionalplasmahad a partialcorrection(patientno. 10) and this patientturned out to be homozygousfor FV-Leiden.If a patientis homozygousfor FVLeidq 1+1mixingwithNP wouldgivea plasmawith the sameamountof normalFV as in a plasma ikoma heterozygouspatient.Evenifthiswouldnot correct the AK-ratio, the rise in ratio shouldbe evidentas seen here. The four remainingplasmas,where mixing1+1with NP fhiledto correct the ratio, were strongly positivefor APAsandallwerepositiveforLA. Thiscouldbe explainedby an inhibitor,as mixing1+1 withNP wouldnot be suilkientto neutralizethe inhibito~effectof an APA. In accordancewith this, PCRanalysisshowedthat three of the four patientshad normalFV. The last one was heterozygous forFV-Leidenandthe APC-resistanceofthispatientmaybe dueto the combinedeffectsof a mutated FV andan inhibitor.It is not possiblefrom our resultsto concludewhichAPAsthat are responsible for this inhibito~ effect as all four had strong positive ELISA tests for anticephalinand/or anticardiolipinantibodiesas well as positivetests for LA. Theoccurrenceof lowAPC-ratioswithoutthe presenceof FV-Leideninpatientswith APAs has been relatedto the prolongationof APTT often seenin plasmawith LA (4). Becausethe APC-resistance test is based on the APTT test, one mighthave suspecteda correlationbetweenthe strengthof the APTT-basedLA test and the influenceof antibodieson the APC-resistancetest. We couldnot find sucha linkbetweenthese tests. Of the three patientswith low APC-ratiosand normalFV, one had onlya weakpositiveAPTT-basedLA test. Also,onlythree out of ninepatientswith strongpositive APTT-basedLA tests(datanot shown)hadlowAPC-ratios,oneof whomwas heterozygousfor FVLeiden.Oneexplanationfor this couldbe that the importanceof differentphospholipidsin each test varies,eventhoughboththe APC-resistancetest and the LA-test is based on the APTT. In an APTT withoutAPC, the crucialstepsare the activationof factor X and the conversionof prothrombinto thrombin.Themostimportantphospholipidinthesereactionsisthoughtto be phosphatidylserine(15) and a LA with stronginhibitionof these reactionswillbe expectedto givea highpositiveresultin an APTT-based LA test. On the other hand, if one measuresthe ratio between the APTT in the presenceand absenceof APC, respectively,the crucialstepwillbe the inactivationof FV by APC. Therehavebeenseveralreportson the inhibitionby LAs on both the activationof proteinC and the degradationofFV byAPC(16-18).As shownby SmirnovandEsmonjtheinactivationof FV by APC is stronglyenhancedby phosphatidylethanolrunine, whilephosphatidylethanolamine had very little influence on prothrombinactivation(19). The same group has shownthat the inhibitionof APC activityby LA is enhancedby phosphatidylethanokunine and, in somepatientswith LL APC activity is inhibitedmore stronglythan is prothrombinactivation(20).

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The principleof our mixingprocedurewas confirmedby progressivelydilutingtwo APA positive plasmaswith low APC-ratiosin FVdef and in NP (tableII). The plasmafromthe patientwho was heterozygousfor the FV-Leidenmutationhad, as expected,low APC-ratioswith allmixtureswith FVdef.WithNP, however,the ratiowasnormalwith allmixturesexceptthe first one (1+1 mixture). Theseresultscouldbe explainedby the presenceof FV-Leidencombinedwith a weak inhibitionby an antibody.The patientwithoutFV-Leiden(patientno. 1) had parallelresultswhen mixingwith FVdefrmdwithNP. ThenAPC-ratiowas lowwith 1+1,1+4and 1+9mixturesbut eventuallybecame normal (i.e. above 0.80) when testing 1+19mixtures.This suggeststhat in this plasmathere is a stronginhibitionof the APCanticoagulantsystem.Interestingly,this patienthas onlya weak positive APTT-basedtest for LA. In conclusio~ifthe reasonforthe APC-resistanceis a defectFV with no influencefrom an inhibitor, the APC-ratio will become normal (heterozygouspatient) or substantiallyhigher (homozygous patient)whenmixing1+1withNp. p(ll Aysis maynot be necessaryinthesepatients.If the APCratiois influencedby an AP& however,the 1+1mixingwith NP insteadof 1+4with FVdefwillnot givethe expectedrise of the APC-ratio.To revealthe presenceof the FV-Leidenmutationbesides the effectof an AP~ PCR testingmustbe performed. REFERENCES 1. DAHLBACK, B., CARLSSON, M. and SVENSSON, P.J. Familialtrombophiliadue to a previouslyunrecognizedmechanismcharacterizedby pooranticoagulantresponseto activatedprotein C: Predictionof a cofactorto activatedproteinC. Proc Natl Acad Sci90, 1004-1008,1993. 2. DE RONDE, H. and BERTIN& R.M. Laboratory diagnosisof APC-resistance:A critical evaluationof the test and the developmentof diagnosticcriteria.ThrombHaemost 72, 880-886, 1994. 3. BOKAREW& M.I., BLOMBACK, M., EGBERG, N. and ROSEN, S. A new variant of interactionbetweenphospholipidantibodiesandtheproteinC system.Blood Coag Fibrinol5, 37-41, 1994. 4. HALBMAYE~ W.M., HAUSHOFEK A., SCHON,R. and FISCHER M. Intluenceof lupus anticoagulanton a commerciallyavailablekit for APC-resistance.ThrombHaemost 72, 645-646, 1994. 5. BOKAREW4 M.I., BREMME, K., FALK, G., STEN-LINDERj M., EGBERG, N. and BLOMBAC&M. Studieson phospholipidantibodies,APC-resistanceand associatedmutationin the coagulationfactor V gene. ThrombRes 78, 193-200,1995. 6. EHRENFOR~ S.,RADTKE,K.P. and SCHARRE~ I. AcquiredactivatedproteinC-resistance in patientswith lupusanticoagulants.ThrombHaemost 74, 797-798,1995. 7. VILLA P., AZNN J., JORQUEIQ J.I. and CAS~~ P. Laborato~ Diagnosisof APCresistancein patientswith lupusanticoagulant.ThrombHaemost 74, 1606-1607,1995. 8. JORQUEw J.I., MONTORO, J.M., FERNkWIEZ, M.A., AZN& J.A. and AZNN J. Modifiedtest for activatedproteinC resistance.Lancet 344, 1162-1163,1994. 9. TROSSfiRT, M., CONARD, J., HORELLOU, M.H., SAMAM-%M.M., IRELAND, H., BAYSTON, T.A. and LANE, D.A. Modified APC resistance assay for patients on oral anticoagulants.Lancet 344, 1709, 1994. 10.BERTIN~ R.M., KOELEMAN,B.P.C., KOSTE~ T., ROSENDAAL,F.R., DIRVEN, R.J., DE RONDE, H., VAN DER VELDEN,P.A. and [email protected]. Mutationin blood coagulation factor V associatedwith resistanceto activatedproteinC. Nature 369, 64-67, 1994. 11. SCHJETLEIN,R., SLETNES, K.E. and WISLOFF, F. A quantitative,semi-automatedand

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computer-assistedtest for lupusanticoagulant.ThrombRes 69, 239-250, 1993. 12.SLETNES,K.E., KEIRUNG,G. and WISLOFF,F. Quantitationof anticephalinantibodiesin a computer-assistedenzyme-linkedimrnunosorbentassay(ELISA): relationto lupus anticoagulant. ThrombRes 57, 235-246, 1990. 13. GHARAVI,A.E., HARRIS, E.N., ASHERSON,R.A. and HUGHES, G.R.V. Anticardiolipin antibodies:isotypedistributionand phospholipidspecificity. AnnRheumDis 46, I-6, 1987. 14.HARRIS, E.N., GHARAVI,A.E., PATEL, S.P. and HUGHES, G.R.V. Evaluationof the anticardiolipinantibodytest: report of an internationalworkshopheld4 april 1986.ClinExp Immunol 68,215-222, 1987. 15.ROSING,J, VANRUN,J.L.M.L.,BEVERS,E.M., VAN DIEIJEN, G., COMFURKJS,P. and ZWA4L, R.F.A Theroleof activatedhumanplateletsinprothrombinand factor X activation.Blood 6S, 319-332, 1985. 16. CARIOU, R., TOBELEMjG,, BELLUCCI, S., SORI~ J., SORIA C., MACLOUF,J. and CAEN, J. Effect of lupus anticoagulanton antithrombogenicproperties of endothelirdcells Inhibitionof thrombomodulin-dependent proteinC activation.ThrombHaemost60, 54-58, 1988. 17,MARCINIA&E. andROMOND,E.H. Impairedcatalyticfimctionof activatedproteinC: a new in vitro manifestationof lupusanticoagulant.Blood 74,2426-2432,1989. 18.LO, S.C.L., SALEM H.H.,HOWARD,M,A., OLDMEADOW,M.J. and FIRKIN,B.G. Studies of natural anticoagulant proteins and anticardiolipinantibodies in patients with the lupus anticoagulant.Br J Haematol76,380-386, 1990. 19. SMIRNOV, M.D. and ESMON, C.T. Phosphatidylethanolarnine incorporationinto vesicles selectivelyenhancesfiwtorVainactivationby activatedproteinC. J BiolChem269, 816-819, 1994. 20. SMIRNOV,M.D., TRIPLETT,D.T., COMP,P.C., ESMON,N.L. and ESMON, C.T. On the roleof phosphatidylethanohunine in the inhibitionof activatedproteinC activityby antiphospholipid antibodies.J ClinInvest95, 309-316,1995.