DISTR. : LIMITEDDISTR. : LlMITEE

BS/87.1561

ENGLISH ONLY

WORLD HEALTH ORGANIZATION

ORGANISA TION MONDIALE DE LA SANTE

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva 1- 9 December 1987

A CDUAOORAT IVE sruDY TO mrABLISH AN INrERNAT I~ sr~ FOR PRJrEIN C IN ~

A.R. Hubmrd and D.P. TlvmasDivision of HaernatolCX3Y

WHO International L3.00ratory for Biological StandardsNational Institute for Biological Standards and Control

Potters Bar , Herts. EN6 JOG, U.K.

INrOODucrlrn

Human Protein C is a vitamin K dependent serine protease ~ich, upon activation, dis-plays antiooagulant properties, ~ inactivation of blood ooagulation Factors V arrl VIII. ThefXlysiolCXJical iDlfX)rtance of Protein C has teen deI[K)nstrated in nUIllerous families where a con-.genital deficiency of Protein C has been linked with recurrent thraribotic disease.

The requirement for an International Standard for Protein C in plasma has been discussedin the Protein C Su1:x..'Ommittee of the International COImnittee on ThranOOsis and HaeIrkJStasis,and it was concluded that an International Standard ~uld be preferable to the present situ-ation where many laOOratories use frozen local pooled plasmas as reference material.

The objectives of the present study were:

1. To calibrate a freeze-dried p:X>led oormal plasma for Protein C ~ assay of b:>th acti-vity and antigen aga1nst fresh, p:X>led l1Ormal plasma which, ~ definition, containsone unit of Protein C per ml.

2. To oompare local standards currently used to measure Protein c.

3. To compare intra-laboratory assay var~ation.

4. To compare the measurement of Protein C ~ di fferent met11cxis

PARI'ICIPANI'S

A total of 22 1a1:x:>ratories frcm 12 different oountries agreed to particip3.te in thestudy. This report has been co~iled from the results which have been received from 17 ofthe 22 la1:x:>ratories. Partic1p3.ting Laboratories were assigned code numbers to retain con-

fidentiality.

~

I. The prqJ()Sed Il"lternational Standard (cx:xied 86/622} oonsists of a p::JOl of nonnalplasma oollected from 15 donors in the United Kingdom J.n August 1986. Each OOnation wastested for the presence of Hepatitis B surface antigen and anti-HIV antib:>dies and was foundto 00 negative. The donations were centrifuged twice to renove cells. The p::JOled plasma wasfilled into approximately 4,000 glass ampoules, each oontaining 1 ml plasma, freeze-dried,secondary dessicated and finally sealed under nitrogen.

2. Fresh fXX)led normal plasma was D:)llected and prepared b:l the particifeting laOOra-tories, as described in AppendlX 1.

3. Participants' local standards were also lncluded in the assays. Of the 17 labora-

Les opinions exprimees dans les documents par des auteurscites nommement n'engagent que lesdits auteurs.

The views expressed in documents by named authors aresolely the responsibility of those authors.

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of WHO.

Ce document n'est pas destine a etre distribue au grand publicet tous les droits v afferents sont reserves par l'Organisationmondiale de la Sante (OMS) .II ne peut etrecommente, resume,cite, reproduit ou traduit, partiellement ou en totalite, sansune autorisation prealable ecrite de \'OMS. Aucune partiene doit etre chargee dans un svsteme de recherche documen-taire ou diffusee sous quelque forme ou par quelque movenque ce soit .electronique, mecanique. ou autre -sans une auto.risation prealable ecrite de j'OMS.

BS/87.1561 Page 2

tories ~ich have returned results, 13 used a frozen p:>oled mrmal plasma and four used afreeze-dried IXX>led plasma as local standard.

These materials are referred to henceforth according to the £ollowing code:

Ccxie M3.terial

pNL

proposed lst International Standard Protein C plasma ( 86/6~2 )Fresh fX)Oled normal plasma

Participant's local standard

ASSAY l'ErH()DS

Proteill C acti vi ty

The many different IOOthcxis currently used for lOOasuring Protein C activity were dividedinto four groups, depending on the methcxi of activatirl(,j Protein C (either thranbin or snakevenom) and the methcrl of detecting the activated Protein C (either chrCX0CX3enic or clotting) .

a) Thrombin activation/clotting assays The methcxis used by the three laOOratories werevery similar to that descrired ~ Francis and Patch ( I) except that one la]::x)ratory adopted acolorimetric prr using the Thranbcx.Juant reagent instead 0! a conventioml clotting end-fx:>int .A second la1:x>ratory included throm1:x>mcxiulin, \Oihich is an optional cofactor in the activationof Protein C ~ thrombin in vitro.

b} Thrombln activation/chrOlrKX:Jenic assays Five laOOratories used the ~th:xi of Bertinaet al. (2} , t\\Q of which used this in the form of the Coa-Set Protein C kit (KabiVitrum,Sweden} , and two included throm}:x)nOOulin in the activation step. One additional laOOratory-used the ~thod of Sala et al. ( 3} .

c) Snake verX)m activation/clottJ.ng assays Five out of the seven 1.aOOratories usedcommercial kits for this assay. One lab::>ratory perfonned a colorJ.metric prr usJ.ng theThromb::>quant reagent instead of a conventional clotting tire.

d) Snake venom activation/chr~enic assays Three out of eight laOOratories used can-mercial ki ts for this assay.

Protein C antigen

participants used either the Laurell I rocket I

tori es ) or an ELISA rnethcx:l ( 5 laOOrator les ) .electro-imm~say tedhnique ( 9 labora-

r-t:Ist participants carried out assays using one activity and one antigen method.laOOratorles used Ilk:>re thall one activity ~thcrl.

Scme

DESIGN

Sufficient anIfX)ules of t:.e prq:JOSed standard (p) were desp3.tched to enable the p3.rti-cipants to carry out six assays by each methcrl wed. It was r~nded that the six assaysbe performed in three sessions, each of two assays, against three different fresh p:>Ols ofnormal plasma. Balanced desi~s in which the three mater1.als, P, N and L, were assayed, pre-ferably in replicate, were offered to the~ticii;)iir1ts.'1'heraw data fran eacli la'f:xjiat.Orywere returned to NIBSC for analysis.

STATISTICAL MErtI]:>s

All assays were analysed using parallel line methods (4) generally relating lag-responseto lag dose. Validity of assays was OOsed on the linearity and parallelism of lag dose-response lines. The potency of the proposed standard (p) relative to fresh normal plasma (N)was calculated as a function of the horiwntal distance l:etween the lag dose-response lines.Potencies frcm indi vidual assays were combined to gi ve geometric means and 95% confidencelimi ts .Canbinations of all potencles from a particular method and for the overall meanpotency were calculated frcm the indi vidual assays ( total of 214 assays) .

INrRA-I.AOORATORY VARIABILITY OF ~IESBS/87.1561Page 3

Within each laOOratory , p and L were oonsistent throughout all the assays, unlike N,whidh usually consisted of three separate pools of fresh plasma. By taking estimates of Lrelative to P, it is possible to calculate intra-laboratory assay variability in terms of ageometric coefficient of variation (gcv %} .

RESULTS

a) Protein C activity

Potency estimtes for the prq>OSOO standard (p) relative to normal plasm (N), togetherwith estimtes of intra-laboratory variability (gcv %) are given in Table l and Figures 1 and2, aCa:Jrding to the type of IOOt~ used.

The results frcm one snake venom/clotting assay of laOOratory 7 and all the snake venomclotting assays from laOOratory 14 were excluded due to non-parallelism and/or unacc~tableassay drift.

b) Protein C antigen

Potency estimates for the prq)05ed standard (p) relative to norma.l plasma (N) , togetherwith estimates of intra-lalx>ratory variability (gcv %) are given in Table 2 and Figure 3.Results fran two assays of la1:X:>ratory lS using an ELISA methOO were excluded due to operatorerror.

The Q:)mbinOO results are presentOO in Table 3 acQ:)rding to assay methcxj, together withan overall combinOO IX>terlcy from all assays .

c) Variabili ty of oormal px>ls (N)

Estimates of the Protein C content of the normal pools (N} are expressed as units perml, relative to the overall 1OOan value of the prop:>sed standard (p} ~ each 1OOthod (Table4).

d) Variability of local standards (L)

Estimates of the Protein C content of the local standards (L) are expressed as units permI, relative to the overall mean value of the proposed international standard (p) (Table 5) .This allows the variability of L to be examined independently of variability in the freshnormal p::IOls (N) .

Ci:)!:¥:;IJJS lOOS

The primry obJective of this study was to provide a stable plasma standard for ProteinC to improve inter-lab::>ratory agr~nt by overooming the variability in the currently usedlocal reference preparations. r"'bst lab::>ratories in the study used a locally oollected,frozen p:x>led plasma which, relative to the prCl)()Sed standard (p) , ranged from a Protein Cooncentration of 0.84 to 1.15 units per ml (Table 5) .A similar range was found When thevariability of the mrnal p:x>led plasma (N) was expressed relative to the proposed standard(p) (Table 4) , Where values of N ranged from 0.85 to 1.22 units per ml. This wide range ofvalues for reference materials ~hasises the need for an International Standard for ProteinC in plasm.

The many and varied methcrls of ~asuring Protein C were placed into six groups for thiscollatorative study: iour types of activity ~thcrl and two types of antigen ~thcrl. ThefX:'tency of the prq?C)sed standard (p} relative to the normal pooled plasma (N) was found to bevery similar by all the methods used (Table 3} .Such good agreerent between ~thodsindicates that one overall combined figure of 0.82 units per aI!iX:>ule would be acceptable forall methods used to assay Protein c.

-Accelerated degradation studies carried out in one laOOratory have indicated that theprcposed International Standard (p) is suitably stable (Appendix 3) for its intended use.

BS/87.1561

Page 4

RECn'wMENDAT ION

It is recolnmended to the Canmittee that candidate preparation P (86!622) be establishedas the International Standard for Protein C, plasma, hill~, with an assigned potency of 0.82IU per amp:>ule .

REE""E"K:E8

I. Francis, R.B. and Patch, M.J.Thramb.Res. ~, 605-6i3 (1983)

A functional assay for Protein C in hwnan plasma.

2. Bertina, R.M. , Broekmans, A.W. , Krommerihoek-van Es, C. and van Wijngaarden, A. The useof a functional and immunologic assay for plasma protein C in the study of the hetero-geneityof congenital protein C deficlency. Thraffib.Haemostas. ~, 1-5 (1984)

3. Sala, N., O.Ven, W.G. and Col1en, D.Blood ~, 671-675 (1984)

A functional assay of Protein C in human plasma

4. Finney, D.J. Statistical Method in Biological Assay. Griffin & Co., Ltd., London (1978)

BS/87.156

Page 5

TABLE OOE

P<JrENCY ESTIMATES (UNITS PER tvD:..) OF THE pmPcsED sr~ (p)RELATIVE TO FRESH OO~ rooLED PIA91A (N) , AS MEASURED USlOO

PROrEIN C ACr IVITY MErHOIS

LaOOrator:i No.Mean p'tency

(+ 95% confidence limits) GCV%

a) Thrombin activation/clotting assays

7* 0.81 {0.74- 0.91)9 0.92 {0.84 -1.00)

10 0.82 (0.76- 0.87)

8.27.29.0

b) Thrombin acti vation/ chrOIOCIgenic assays

4 0.88 (0.80- 0.97)6 0.81 (0.74- 0.90)

10 0.78 (0.69- 0.88)12 0.76 (0.55- 1.05)13 0.78 (0.75- 0.81)16 0.80 (0.77- 0.83)

4.66.95.85.94.66.0

c) Snake venom actlvation/c1otting assays

1 0.87 (0.84 -0.91)2 0.79 (0.71- 0.89)3 0.76 (0.71- 0.81)5 0.76 (0.72- 0.80)7* 0.78 (0.72- 0.84)7 0.85 (0.81- 0.89)

11 0.91 (0.88- 0.95)

5.16.46.23.88.57.33.4

d) Snake venom acti vation/ chron-r::x;;enic assays

1 0.70 (0.69- 0.71)

2 0.80 (0.79- 0.82)

3 0.76 (0.73- 0.79)

8 0.96 (0.93- 0.98)

11 0.96 (0.92- 0.99)

15 0.80 (0.68 -0.94)

16 0.82 (0.80- 0.84)

17 0.83 (0.72- 0.94)

0.0.6.4.3.2.4.5.

* Colorimetric prr using Thranbcquant reagent

.9

.8

.4

.0

.2

.9

.3:6

BS/87.1561

Page 6

TABLE TWO

porENCY EST IMA.TES ( UNITS PER rvD:, ) OF THE PROPOSED sr ANmRD ( P )RElATIVE TO OO~ KX)LED PlAS'1A (N) AS MEASURED USING

ProrEIN C ANr IGEN ASSAYS

LaOOratory No.Mean p:>tency

:+ 95% confidence limits) GCV%

a) I.aure11 I rocket I e1ectroimmunoassay

2 0.76 (0.72- 0.79)4 0.87 (0.83- 0.90)5 0.80 (0.76- 0.83)6 0.76 (0.68- 0.84)9 0.81 (0.71- 0.94)

11 0.94 (0.94- b.95)12 0.82 (0.60 -1.11)13 0.67 (0.65- 0.70)14 0.86 (0.79- 0.93)

7.15.12.55.05.82.47.57.13.5

b) ELlSA methcx:is

378

1015

0.790.780.880.830.78

(0.74- 0.84)(0.71- 0.86)(0.86 -0.90)(0.75- 0.92)(0.69- 0.87)

2.93.67.85.37.4

T ABLE THREE

OOMBINED PCJrENCY ESTIMATES (UNITS PER ML) OF THE PROPOSEDsrANmRD (p) RELATIVE TO FRFSH OOR-1AL PCX:>LED PI.As:v1A (N)

AS MFASURED BY DIFFERENr MElliODS

No. ofassaysMethcxi

Mean fX)tency

+ 95% coni. limits)

Protein C activity

Thr cxnb i nThrombin

Snake venomSrlake venom

ClottingChr CXOCXJ e. "1 i c

ClottingChr om:>g en i c

(0.81- 0.89)(0.78- 0.83)(0.78- 0.83)(0.79- 0.86)

0.850.810.810.82

18334443

(0.78- 0.84)(0.79- 0.85)

Protein C arltigen

Laurell electroiIlUnunoassay

ELISA

0.810.82

4828

(0.80- 0.83)OVERALL ~lNAT 100 0.82 214

BS/87.1561

Page 7

TABLE FOUR

RAOOE OF £grIMATES FOR PwrEIN C IN m~ POOLED P~ (N)RElATIVE TO THE OVERALL MFAN VALUE FOR THE PROPCSFJ)

Sl'ANDARD (p) I EXPRFSSED AS UNITS PER ML FOR EACH MErHOD

Meth:xi Range

Protein C activity

Activator Detection

ThrombinThranbin

Snake venanSnake venom

ClottingChr CJOCX3eni c

Clotting

ChrOI!k:lgenic

0.89- 1.010.93- 1.080.90- 1.080.85- 1.17

Protein C antigen

L3.urell electroiIrnnunoassay

ELI SA

0.87- 1.220.93- 1.05

TABLE FIVE

~E OF FSrIMATES FOR PRarEIN C IN THE LOCAL srANmRDS (L)RELATIVE TO THE OVERALL MEAN VALUE FOR THE PmPOOED STANmRD (p) ,

EXPRESSED AS UNITS PER ML, FOR EACH MErHOD

Methcrl Range

Protein C activity

ThrombinThranbin

Snake venomSnake venom

ClottingChr aIk:X3 en i c

ClottingChr aIk:X3en i c

0.88- 1.100.84 -1.lS0.95- 1.090.87- 1.08

Protein C antigen

Inurell electroi1tUnunoassayELlSA

0.91- 1.150.92- 0.98

APPENDIX 1BS/87.1561

Page 8

Preparation of Fresh No~l Plasma

The assays were designed to ~ carried out in three sessions, each requiring a fr-eshpool of normal plasma derived from at least four , preferably dif~erent, volunteers ,each ti~.participants were asked to prepare fresh normal plasma acoord1.ng to the follow1.ng spec1.-fications:

a) IX>nors : Normal healthy volunteers, excluding women taking oral contraceptives

b) Anticoagulant:

anticoagulant.

0.109 Molar Tri-sodium citrate. Mix 9 vols. ot blood wi th 1 vol. of

c) Centrifugation: Blood should be centrifuged at 4°C as soon as possible after col-lection. If a high speed centrifuge is available, spin at 50,000 9 for 5 minutes; otherwise,spin at 2,000 g for 20 minutes.

d} Storage: During assays, keep the plasma in a st~ed tube at 4°C.eaCh pool should be snap-frozen for assays of Protein C antigen.

Aliqoots of

BS!87.1561

Page 9APPE}I1DIX 2

Numbers of Ibnors Used for the PCX>ls of Fresh Normal Plasma

LaOOratory No.

1

2

3

4

5

6

7

B

9

10

I:..

12

13

14

15

16

17

~. of donors

9331812151215

512132515*

24

F14

82F

* Frozen ~l of 30 donors used instead of fresh normal plasma

~ I:eta not gi ven

BS/87.1561 Page 10APP~IX 3

Stabillty of the Prop:>Sed Standard (p)

Samples of the pr~ed International Standard have ~en stored at elevated temperatures(+4, +20 and +37°C) since 20th August 1986. Assays were carried out at NIBSC using the snakevenom/ dlrark:Jgenic methcxi on samples which had ~en stored for 11 I[k:)nths .The potencies ofthe stored ampoules are given ~low, relative to ampoules stored at -20°C:

Storage temperature (OC) Potency (% of -20°C sample)

+4

+20

+37

93, 100, 104, 9693, 97, 104, 10083, 82, 81, 89

These results were analysed according to the metb:rl of Kirkwcx:rl and Tydeman (1984) ,which uses a fitted Arrhenius equation to predict the degradation rates as given ~low:-

Storage teIllperature ( °C) Predicted % loss per ~nth

0.000

0.016

0.172

1.605

-20+4+20+37

The standard is stored at -20°C arrl, according to these results, should be suitablystable £Or its intended use.

Reference

Kirkwood, T.B.L. and Tydeman, M.S. J.Bio1.Stand. ~, 207-214 ( 1984)

BS/87.1561

Page 11APPENDIX FOUR

Participants in the CollaOOrati ve Stu:iy

Dr. U. BeckerBehringwerke P{3Research I.a]:x)ratories (M300 )Postfach 1140D-3550 M3.rburgWESrGERMANY

Professor M.J. IaIrieuLaboratoire Central d 'HematologieHOpital Bicetre78 Avenue du General Leclerc94275 Kremlin-BicetreFRANCE

Dr. R.M. BertinaHaeIOC>Stasis & ThromOOsis Research UnitUniversity Fbspita1Building 1, C2-R, Room 140ro Box 96002300 RC LeidenTHENEl'HERIMOO

Professor P.M. l'1annucciA. Bianchi Bonomi HaeIlk)philia arrl

ThranWsis CentreUniversity degli Studi di MilanoVia Pace 920122 MilanITALY

Dr 0" Bo Chambrette

Diagoostic Stago6 ter , rue Denise-PapinF92600 Asnieres-sur-SeineFRANCE:

Dr .R.A. ~rlarBLood Center of S.E. Wisconsin1701 West Wisoonsin AvenueMilwaukee WI 53233USA

Dr. A. DessauerBoehringer Mam1heim Gml:HBiochemical Research Ce.'1treBahnOOfstrasse 9-15D-8132 TutzingWESrGERMANY

Dr. K. MertensCentral La.1:x>ratory for Blcxxi TransfusionPlesmanlaan 1251066 cx: Amsterdci1UTHE~

Professor D. GreenDirector, Athercsclercsis Uni tRehabilitation Institute of Chicago345 East Superior StreetChicago IL 60611USA

Dr. Steffen RosEm

Biochemistry I.a1X>ratoryKabi Vi trum ABDiagrosticaTalj~rdsgatan 3s-431 33 MOlndaJ.&YEDEN

Dr. J.H. GriffinDepartment of ImnunologyScripps Clinic & Research FOundat10I110666 Nccth Tccrey Pines RoadLa Jolla CA 92037USA

Dr. N. SalaServei d 'HanatologiaHospital de la Santa creu i Sant PauAvgda. S. Antooi M.a Claret 1608025 BarcelonaSPAIN

Dr .A. R. Hubta.rdNational Institute for Biological Standards

and ControlBlanche L3neSouth MirmnsPotters Bar, Herts. EN6 3(.):;UK

Prof. Dr .med. I. ScharrerDep:irtment of Internal Medicine

Haus l3A

University HospitalThecrlor Stern Ka.i 7D-6000 Frankfurt./Main 70WFST GERMANY

Dr. H. LangIrmnunOOiagnostika GmtlfIndustriestrasse 72A-1220 ViennaAUSTRIA

Dr .H. VinazzerB1ocrl Coagulation LaOOratoryUntere iX>naulande 12A-4020 LinzAUSTRIA

Dr. s. WasiThe Canadian Rei Cross Scx::iety1800 Alta Vista DriveOttawa, Ontario KIG 4J5CANAOO.

BS/87.1561Page 12 FIGURE OOE

Estimates of Protein C activity in the proposed International Standard (p) ,relative to fresh p:x>led oormal plasma (N) , as measured using

a) thranbin activation/clotting methods arrlb) thranbin activation/chrOIOCJgenic methods

The numbers in the boxes identify the relevant laboratories .

* Colorimetric Prr using ThrOIIlb::)<:lu:s.nt reagent

U)UJHa:01-~a:oaJ~-J

U-O

a6

4

2

a:2: .I 7 * I 10 I 9 I0 -III I I I I I .LU I ~ I I I I I I

0.40 0.52 0.64 0.76 O.BB 1.00 1.12 1.24

PROTEIN C ACTIVITY U/ML

b00UJH£1:O1-<£1:OIJJ<-l

u..o

0z

6

4

~

2,T~=='=,~~=~

0 -III II j 6 4 j I I I I I

0.40 0.52 0.64 0.76 0.88 1.00 1.12 1.24

PROTEIN C ACTIVITY U/ML

BS/87.1561

Page 13FIGURE 'IWO

Estimates of Protein C activity in the proposed International Standard (p)relative to fresh p;:x>led oormal plasma (N) , as lIeasured using

a) snake venom activation/clotting ~thods andb) snake venom acti vat ion/ chrark:>genic ~th:>ds

The numbers in the OOxes identify the relevant laOOratories

* Colori~tric Prr using Thran1:oq\Bnt reagent

cnUJI-ICI:O1-~CI:OaJ~-l

l.L0

0:z

a

7*

5

2 1 11,1 I I I I I I I I I I

0.52 0.64 0.76 0.88 1.00 1.12 1.24

PROTEIN C ACTIVITY U/ML

bU)UJHa:01--:3:a:oaJ"«-1

1.1-0

0z

6

4

2

00.40 0.52 0.64 0.76 0.88 1.00

PROTEIN C ACTIVITY U/ML

1.12 1.24

BS/87.1561Page 14 Fl GURE THREE

Estimates of Protein C antigen in the prqposed International Standard (p) ,relative to fresh fXX)led l"X:>rmal plasma (N) , as neasured using

a) Laurell electroinununoassay nethOO arrlb) ELISA neth:rls

The n~rs in the b:>xes identify the relevant laOOratories

'~

b(/) 6UJ1-40:0.1--d:0: 40CD-d:-.J

~ 2

oz

3 10 80 ~I I I I I -I I I I I

0.40 0.52 0.64 0.76 0.88 1.00 1.12 1.24

PROTEIN C ANTIGEN U/ML