DISTR. : LIMITEDDISTR. : LIMITEEWORLD HEALTH ORGANIZATION

ORGANISATION MONDIALE DE LA SANTE

ENGLISH ONLYBS/Ol.1946

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION--~

Geneva, 26 to 30 November 2001

REPORT ON THE CALmRAnON OFTHE PROPOSED TIllRD INTERNA nONAL STANDARD

FOR FACTORS D, VD, IX, X, PLASMA(99/826)

Elaine Gray, Dawn Sands, and Anthony R. Hubbard

National Institute for Biological Standards and ControlBlanche Lane, South Mimms

Potters Bar, Herts. EN6 3QG, UK

SUMMARY

Nineteen laboratories participated in an international collaborative study to calibrate theproposed 3rd International Standard (IS) for Factors II, VII, IX, X, plasma (99/826). Forall four factors, inter-laboratory variability was low for assay of the proposed 3rd ISagainst the 2nd IS (GCV

BS/Ol.1946Page 2

INTRODUCTION

The 2nd International Standard (IS) for Factors II, VII, IX, X, plasma (94/746) wasestablished by the Expert Committee on Biological Standardisation (EGBS) of theWorld Health Organisation (WHO) in October 1996. Low stocks of the 2nd IShave made it necessary to prepare a replac~ement preparation. This reportdescribes the calibration of the proposed 3rd I~~, in an international collaborativestudy, by assay against the 2nd IS and local normal plasma pools.

PARTICIPANTS

Twenty-one laboratories agreed to participate in the study and results werereturned by 19 laboratories from 11 different countries. Each laboratory hasbeen assigned a code number which does Inot reflect the order of listing inAppendix 1.

e~~ .".-~~,~! !'"i~iR I i~

Coded samples included in the study were:

A 2nd IS Factors II, VII, IX, X, plasma, (94/746) with assignedpotencies of 0.93 IU FII, 1.,25 IU FVII, 0.90 IU FIX, 0.95 IUFX per ampoule

B Proposed 3rd IS Factors II" VII, IX, X, plasma, (99/826)prep~.red from a pool of 24 donors, each tested and found

",.negative for anti-HIV 1/2, J-iBsAg and anti-~CV. Each CPO

C "',.

anticoagulated donatipn was buffered with HEPESto a final, .co~centration of 0.04 mol/L and stored overnight at 4°C.After po9ling, the plasma was filled and ampouled{glass dinampoules} under conditions used for international biological',' ',' ',' "standards (Campbell, 19Z4}. "The coefficient variation of thefill ~a,.§ 0.09%. The residual moisture after Iyophilisation andseconda!-y desiccation wa~; 0.17% with mean d!-y weight of

.c" .c" C

92.76mg/ampoule. 4200 ampoules are available fordistribution.

N 1 -N4 fresh normal plasma pools prepared locally according to theinstructions given in the protocol (Appendix 2).

ASSAY M,ethods

Factor II: 13 laboratories used a prothrombin 1:ime based clotting method with avariety of thromboplastin reagents (human pla(~enta, rabbit brain or recombinanthuman) and FI'-deficient plasma; 2 laboratoriE~s used a Taipan Venom clot1:ingassay; 4 laboratories also used a clotting method but did not identify the reageints

BS/Ol.1946Page 3

used; 1 laboratory also performed a chromogenic assay using unspecified in-house reagents.

Factor VII: 15 laboratories used a one-stage clotting method with a variety of

thromboplastin reagents (human placenta, rabbit brain or recombinant human)and FVII-deficient plasma; 4 laboratories used a clotting method with unspecifiedreagents; 2 laboratories performed chromogenic assays (1 using theChromogenix Coaset FVII kit and 1 using unspecified reagents) for information

only

Factor IX: 15 laboratories used a one-stage clotting method based on theactivated partial thromboplastin time (APTT) , using a variety of activators,phospholipids and FIX deficient plasma, 4 laboratories used a clotting methodwith unspecified reagents.

Factor X: 13 laboratories used a one-stage clotting method based on theprothrombin time, with a variety of thromboplastin reagents (human placenta,rabbit brain or recombinant human) and FX deficient plasma, 1 laboratory usedan APTT based clotting assay, 1 laboratory used a Russell's Viper Venomclotting assay, 4 laboratories used a clotting method with unspecified reagents,and 1 laboratory also performed a chromogenic assay using unspecified in-house

reagents

ASSAY DESIGN

Participants were requested to carry out four assays for each factor (II, VII, IX, X)using fresh ampoules of samples A and B and a normal plasma pool (N) in eachassay. Laboratories were requested to assay factors VII and IX on one set ofampoules and factors II and X on a second set of ampoules, and, if possible, touse fresh plasma pools for all assays. The same fresh plasma pool could beused for all 4 factors assayed within one session, but it was preferable to preparea different fresh pool for each session. However, if this was not possible, it wasrequested that the assays for factors VII and IX should be carried out using thefresh pools and the assays for factors II and X carried out using frozen aliquots ofthe pools. Some laboratories that were unable to prepare the fresh plasma poolsused a single frozen plasma pool instead.

Within each assay, participants were requested to assay three dilutions of eachof the samples A, Band N, in replicate, according to balanced assay designs.

Raw assay data were returned together with calculated estimates for sample B(proposed 3rd IS) relative to sample A (2nd IS) from each individual assay.

BS/Ol.1946Page 4

STATISTICAL ANALYSIS

All assays were analysed as parallel line bioassays (Finney 1978), comparingassay response with log concentration. VarioLls transformations of the response(eg. linear, log, etc.) were used in order to obtain suitably linear and paralleldose-response relationships when plotted against log concentration. Thestatistical validity of linearity and parallelism of the assays was assessed byanalysis of variance.

Any individual responses or pairs of responsles that contributed significantly toheterogeneity of variance between groups of responses, within an assay, Vlrereomitted (Jess than 0.1% of the total response~s). For one laboratory, the threelowest doses of sample A (2nd IS) were omitted to restrict analysis to the areawhere the same response range was commorl to all preparations included in theassays.

For the majority of the laboratories that performed FII assays, the log of theobserved responses was found to give best linearity with log dose, with theexception o(five laboratories where the reciprocal of the responses and onelaboratory, where the untransformed responses were found to be moresatisfactory. For approximately half of the laboratories that performed FVIIassays, the square root of the observed responses gave best linearity with logdose and for the other half of the laboratories a log transformation was found tobe more satisfactory, with the exception of one laboratory where the reciprocal ofthe observed responses was used.. For all of the laboratories that performed FIXassays, the actual observed responses were taken as linear 'with log dose. For

c c

the majoti.o/ of the laboratories th~tcperfor~ed FX assays, a log transformation ofthe ops~rved responses gave best linearity with log dose, with the exception of

c c,

two labgratories where the actuaJrespbnsesand the reciprocal of the responseswere,fougd to be more satisfactol:y.

';1;;. ,'~', " "

Foreachfa~or, the potencies qf ~ampl~ B,(proposed 3rd IS) were calculated" '

relative to sample A (2nd IS) and relative to sample N (normal plasma pools) for'" , "each ~ss~y. Additionally,the,ppt~ncyofsample N was calculated against sampleA. For e'ach laboratory I combined potency estimates were obtained by taking

'0" , ' " .geometnc means of results from all assays (NI.BSC calculations) and compared, , ,. cwiththegeometric~ean of laboratories OWr, estimates. For each factor, overallpotency estimates were calculated as geometric means of laboratory means.Comparisons among groups of estimates were made using unwei,ghted analysisof variance of the logs of the estimates compared, with unpaired or paired t-tests,where a~:ptopriate.

Variabilit~' within laboratories (between assays) and between laboratories wasmeasured by calculating geometric coefficient:~ of variation (% GCV's) (Kirkwood1979).

BS/Ol.1946Page 5

RESULTS

Assay data

The nineteen par1icipants returned a total of 319 assays which comprised 79 FIIassays, 84 FVII assays, 76 FIX assays and 80 FX assays.

Assay validity

Laboratory 10 only returned data from three FII assays since the first assayfailed. Laboratory 12 did not assay any of the materials in duplicate or replicate,in any FII, VII, IX or X assays. In the first FII and FIX assay by laboratory 02,responses from the replicates were not returned because of analyzer problems.Therefore, the assumptions of linearity and parallelism could not be tested inthese assays.

Dose-response slopes were compared across all assays, for each assay method.The mean ratios of the slopes of samples Band N relative to the slope of sampleA, for each of the assay methods, together with the corresponding number ofassays, are shown in Table 1. The mean ratios of slopes for sample B relative tosample A, in the assays for all four factors, ranged from 0.99 to 1.01. The meanratios of slopes for the fresh and frozen normal plasma pools (N) relative tosample A, in the assays for all four factors, ranged from 0.99 to 1.04. No assayswere excluded from the analysis on the basis of non-parallelism of the dose-response slopes.

Locally collected normal plasma pools

Nine laboratories carried out assays using fresh plasma pools, prepared from atleast 186 different donors, and 8 laboratories used frozen plasma pools preparedfrom at (east 155 different donors. Two other laboratories (12 and 16) used fresh

>-and fro:zen pools in assays for each of the four factors. The total number ofdifferent donors used to prepare all of the plasma pools exceeded 382."Potency estimates of B vs N have been grouped, in the results tables, dependingon the use of either fresh or frozen local normal plasma pools. Statistical analysisindicated that there were no significant differences between the estimatesobtained from the laboratories using fresh or frozen plasma pools for any of thefour factors. Combined estimates for B re~ative to all plasma pools (fresh and

~frozen) are therefore also presented.

?

BS/O1.1946Page 6

The proposed 3rd IS Factors II, VII, IX, X, plasma, (99/826)

Potencies

Potency estimates for sample B (proposed 3rd IS) were calculated relative to theassigned values for sample A (2nd IS) and relative to an arbitrary value of 1.0unit per ml for the local normal plasma pools.

Mean potency estimates from the individual laboratories for sample B (proposed3rd IS) relative to sample A (2nd IS) and relative to sample N (normal plasmapools) are listed in Tables 2 -5, along with 95% confidence limits. The geometricmeans of the individual assay potencies relative to A (2nd IS) according tolaboratories' own calculations are also given. The data are also shown inhistogram form (for the NIBSC calculations) in Figures 1 -4. The histograms donot indicate whether fresh or frozen normal plasma pools have been used. ~The histograo-is illustrate good agreement betvveen laboratories for sample Brelative to sample A, for all four factors, but the spread of the results for sample 8relative to sample N is much wider. There is also a tendency for the estimates ofsample B relative to sample N to be somewhat lower than those relative tosample A. The histograms also illustrate differences in absolute potencyestimates between the different factors.

Most of the estimates for sample B, relative to sample A, (~alculated by thelaboratories lie within the 95% limits calculated by NIBSC. E:(ceptions are theestimated FII, VII and X potencies by the clotting assays of laboratory 09, theestimated FVII potencies by laboratories 04 and 12 and thle estimated FIXpotencies by laboratories 04 and 21. The cause of the discrepancies is not clear.

Outlier detection employing Duncan's multiple range test (Duncan, 1975)indicated that the FVII estimate of potency of sample B relative to sample N bylaboratory 14 (clotting assay) was significantly higher than all other FVIIestimates. This result was therefore classified as an outlier and excluded fromcalculations of mean estimates.

(:'"")

The overall mean potencies for sample B (proposed 3rd IS) relative to sample A(2nd IS) and relative to sample N (normal plasma pools) are shown in Table 7 forall four factors. The overall mean potencies for FII and FX irlclude the resultsfrom the clotting assays and the one laboratory estimate ot)tained using thechromogenic method since the single chromogenic estimate fell within the rangeof estimates from the clotting assays for both factors. The overall meanpotencies for FVII have been calculated only from the results of the clottingassays since the chromogenic assays were not carried out for calibration of theproposed 3rd IS. Estimates from the two laboratories which carried out bothchromogenic and clotting assays for FVII are given in Table 8. For bothlaboratories the estimates by the chromogenic method were higher than the

BS/Ol.1946Page 7

clotting estimates when calculated relative to the 2nd IS; the reverse was truewhen estimates were calculated relative to the normal plasma pools.

Considering the assays used for potency assignment (ie. excluding the FVIIchromogenic assays) the overall mean potency estimates for sample B(proposed 3rd IS) relative )0 s~mple N (n~rmal plasma pools) are significantlylower than the overall mean estimates relative to sample A (2nd IS) by 3%, 10%,8% and 6% for FII (p

BS/O1.1946Page 8

calculated separately for the fresh and frozen normal plasma pools and as acombination of both fresh and frozen normal plasma pools.

Estimates of inter-laboratory variability for sample N relative to sample A, foreach factor, are summarized as % GCV's in Table 10. The variability betweenlaboratories is lowest for FII and FX and highest for FVII and FIX. The variabilitybetween laboratories using either fresh or frozen normal plasma pools wassimilar for all factors, but tended to be higher with fresh than with frozen plasmas.Inter-laboratory variability for estimates of sample N relative to sample A weregreater than the inter-laboratory variability for estimates of sarnple B relative tosample A for all four factors (Tables 7 and 10).

STABILITY STUDY

Three laboratories participated in the accelerated degradation study of theproposed candidate, 99/826. Each laboratory performed 6 assays involvingan-ipouies of 99/826, vvhich have been stored at -20, +4, +20, +37 and +45°C for11.75 months. All assays were analysed as multiple parallel line bioassays,comparing assay response to log concentration. There was good inter-laboratoryagreement for the potency estimates. For each factor, the estimated geometricmean potencies of 99/826 stored at the elevated temperatures relative to -20°Cwere calculated. The long-term stability was predicted using the Arrheniusequation (Kirkwood, 1977). The predicted % loss per month and per year aregiven in the following tables.

(\

Predicted % Loss of Activity per Year at -20°C

Factor Mean %1055 per year Upper 95% limit

0.09 0.14

0.23 ('"

VII

'-'oV'

0.250.13I/\.

I

0.21x 0.45

BS/Ol.1946Page 9

Predicted % Loss of Activity per Month

II

I

0.01

0.160.985.280.020.281.30

5.490.01I0.23 1.32

6.85I 0.02

0.351.899.18

-20

42037

VII

IX

r- -20

420

I 37!

-20

420

i 37I!

-20

420

37

""

These preliminary data indicate that the proposed 3rd IS, 99/826 is stable, withless than 0.3 % estimated % loss per year at the storage temperature of -20°Cfor all 4 factors. The estimated loss per month at transit temperatures of 20 and37°C, are somewhat higher and will be taken into account when the ampoulesare sent to hotter climatic countries.

NIBSC will undertake further accelerated degradation studies to ascertain moreaccurate estimates of degradation. The proposed candidate, once establishedas an International Standard will also be monitored in a real-time degradationstudy, where the -20°C ampoules will be assessed against ampoules stored at-150°C at regular time intervals.

DISCUSSION

The main aim of this study was to calibrate a replacement International Standardfor Factors II, VII, IX, X, plasma. It also provided an opportunity to re-examinethe continuity of the "plasma unit" by comparison with local normal plasma pools.Information regarding the effect of different methods on potency estimation waslimited since most laboratories employed prothrombin time based assays for FII,VII and X and APTT type of assays for FIX whereas only one or two laboratoriesused alternative venom based methods.

Fur1hermore, as only one laboratory carried out chromogenic FII and FX assays,it was not possible to comment on differences between clotting and chromogenicassays. Nonetheless, there were no observable differences in potencies

BS/01.1946Page 10

obtained by the different methods and reagents for these two factors. Therefore,the potencies obtained by chromogenic and clotting methods, against the 2nd ISor normal plasma pools, were combined.

The ratio of FVII potency estimates from the clotting and chromogenic methodscan give an indication of the activation status of FVII, ego a clotting/chromogenicratio >1.0 indicates that FVII in the test sample is relatively more activated thanthe reference standard. In the calibration of the 2nd IS against the 1 st IS aclotting/chromogenic ratio of 1.14 was obtained which indicated that FVII in the2nd IS was more activated than the 1st IS. The increased activation of FVII inthe 2nd IS meant that the assigned value of 1.25 IU per ampoule, derived onlyfrom clotting assays was not valid for use in chromogenic assays. As a result thechromogenic assays for FVII, in the present study, were not included in the meanpotencies for calibration of the proposed 3rd IS. The two laboratories (09 and19), which used both assay methods in the present study, obtained identicalclotting/chromogenic potency ratios of 0.92 for the proposed 3rd IS relative to the2nd ;S (Table 8). This is consistent Vv'ith FVii in Ihe proposed 3rd is being jessactivated than the 2nd IS. However, clotting/chromogenic ratios of 1.08 (lab 09)and 1.13 (lab 19) were obtained for estimates of the proposed 3rd IS relative tothe normal plasma pools and these indicate that FVII in the 3rd IS is slightly moreactivated than the normal plasma pools.

Although the mean FVlllevel, by clotting assay, in the proposed 3rd IS, is higherthan the other three factors, this discrepancy is much lower than was foundduring the calibration of the 2nd IS, ie. 2nd IS vs 1 st IS gave a FVlllevel of 1.25IU per ampoule whereas the proposed 3rd IS vs the 2nd IS gave a FVII level of1.05 IU per ampoule. Considering that estimates of Fil, IX and X are very similarfor both the 2nd IS and the proposed 3rd IS this is a further indication that FVII isless activated in the proposed 3rd IS.

For all four factors, no significant differences were found between potencyestimates calculated relative to the fresh or frozen local plasma pools and it wastherefore valid to combine potencies against all plasma pools.

With some exceptions, estimates of intra-laboratory variability (between assays)were below GCV's of 5% when the proposed 3rd IS was assayed against the 2ndIS, indicating good reproducibility and robustness of the assays. When theproposed 3rd IS was assayed against the local plasma pools, the intra-laboratoryvariability was generally higher and several laboratories obtained % GCV'sgreater th,3n 10%. This is probably due to the variability in the plasma poolsrather than the poor reproducibility of the assays. This is also reflected in thehigher between assay variability found with fresh plasma pools than with frozenpools and can be explained by the different fresh pools, containing plasmas fromdifferent donors, used over the 4 days of the study as opposed to the tendency ofusing the same frozen pools for the whole study.

BS/Ol.1946Page 11

Low inter-laboratory variability (GCV < 4%) was observed for estimates of theproposed 3rd IS when assayed against the 2nd IS. Greater variability was foundfor estimates of the proposed 3rd IS when assayed against the normal plasmapools, although the inter-laboratory variability for estimates relative to both freshand frozen pools combined did not exceed 10 %.

For all four factors, there were significant differences in the overall mean potencyestimates for the proposed 3rd IS when assayed against the 2nd IS and the localnormal plasma pools. Mean potency estimates calculated relative to the normalplasma pools were 3%,10%,8% and 6% lower for FII, VII, IX and X respectivelycompared to potencies estimated relative to the 2nd IS. One reason for thisdiscrepancy could be the degradation of the 2nd IS during its lifetime, but theaccelerated degradation study of the 2nd IS predicted

BS/O1.1946Page 12

Proposed assigned potencies for the 3rd IS, 99/826 (mean of potenciesrelative to the 2nd IS and the normal plasma pools)

COMMENTS FROM THE PARTICIPANTS AND S5C OF THE 15TH

All 19 participants agreed with the proposal to adopt 99/826 as the 3rdInternational Standard for Factors II, VII, IX and X, Plasma and therecommended assignment of potencies, ie the mean of potencies relative to the2nd is and the normal plasma pools. Approval of this recommendation has alsobeen given by the Scientific and Standardisation Committee (SSG) of theInternational Society on Thrombosis and Haemostaisis (ISTH) at the Parismeeting, JlJly 2001.

REFERENCES

Campbell, PJ. International biological standards and reference preparations. II:Procedures used for the production of biological standards and referencepreparations. J Bioi Standardisation 1974, 2, 259-267.

Duncan, DB. T -Tests and intervals for comparisons suggested by the data.Biometrics 1975; 31: 339-359.

Finney, OJ. Statistical methods in biological assay,Griffin 1978.

3rd edn London: Charles

Hubbard AR, Rigsby P and Barrowcliffe TW. Standardisation of Factor VIII andvon Willebrand Factor in plasma: Calibration of the 4th International Standard(97/586). Thrombosis and Haemostasis 2001; 85: 634-638.

Kirkwood, TBL. Geometric means and measures of dispersion.1979; 35: 908-909.

Biometrics

Kirkwood, TBL. Predicting the stability of biological standards and products.Biometrics 1997; 33: 736-742.

APPENDIX: 1 List of participating laboratories

BS/O1.1946Page 13

Mr S LeesUniversity of WalesCollege of MedicineCardiff UK

Dr R BaderA Bianchi BonomiHemophilia & Thrombosis CentreMilan Italy

Drs S Kitchen and B SampsonRoyal Hallamshire HospitalSheffield UK

Dr J GillissenGeorge King BiomedicalKansas USA

Dr P BraunOrganon Teknika Corpn.Durham NC USA

Dr J LloydIMVSAdelaide Australia

Dr C LessardInstrumentation Laboratory LtdLexington MA USA

Dr J IngerslevHaemophilia & Thrombosis CentreUniversity Hospital SkejbyAarhus Denmark

Ing M SeidlerBaxter Hyland ImmunoVienna Austria

Dr H RidgewayHelena LaboratoriesBeaumont TX USA

Dr K DensonDiagnostic Reagents LtdThame, axon UK

Dr L LottinDiagnostica StagoFranconvilleFranceMr D Howarth

MRC Epidemiology and Medical Care UnitSt Bartholomews HospitalLondon UK

Dr M RobersDade Behring Marburg GmbHMarburgGermanyProf F Ofosu

McMaster University Medical CenterHamilton, OntarioCanada

Dr H LehmannDRK-PVG mbHSpringeGermany

Dr F van der MeerStollingslaboratoriumLeiden University Medical CentreLeiden The Netherlands

L Weller, R lampetrio and S ThomasDivision of HaematologyNIBSCPotters BarUK

Mr R ParkinsonNatal Institute of ImmunologyPinetown South Africa

BS/Ol.1946Page 14

APPENDIX 2

INSTRUCTIONS FOR THE PREPARATION OF FRESH NORMAL PLASMAPOOLS

Collect fresh normal plasma as described below, on four separate days, givingpools N1, N2, N3 and N4. The method of collection of the fresh normal plasma isan important part of the study and should be standardised as far as possible,according to the following protocol.

DonorsNormal healthy volunteers, excluding women taking oral contraceptives. Takeblood from as many different individuals as possible, on four separate days. Ifpossible, use a minimum of five different donors for each pool; if this is notpossible, some of the same individuals can be used again, but the aim is to havea total of at least 16 different donors for each laboratory.

AnticoaQulant0.109 Molar tri-sodium citrate, ie 3.2% w/v of the dihydrate (or a mixture of tri-sodium citrate and citric acid with a total citrate concentration of 0.109 M). Ratioof 9 volumes blood to 1 volume of anticoagulant.

CentrifuQationBlood should be centrifuged at 4°C as soon as possible after collection either at50,000 g for 5 minutes or at 2000 g for 20 minutes.

Poolino and StoraoePool equal volumes of plasma from the different donors and mix gently. Keepthe plasma pool in a plastic stoppered tube at 4°C during the assay session. Ifnecessary snap-freeze aliquots of each pool (N1, N2, N3, N4) for assays of FIIand FX.

BS/Ol.1946Page 15

FIGURE LEGENDS

Figure 1. Potency estimates for FII in sample 8, the proposed 3rd ISFactors II, VII, IX, X, plasma relative to sample A, the 2nd IS Factors II, VII, IX, X,plasma, (94n46) and sample N, the normal plasma pools prepared locally. Thenumber in the square denotes the laboratory code. Each square represents thegeometric mean estimate from the laboratory. The shading represents thedifferent standards used. A 'C' accompanying the laboratory code denotes thatthe estimate is from chromogenic assays.

Figure 2. Potency estimates for FVII in sample B, the proposed 3rd ISFactors II, VII, IX, :X, plasma relative to sample A, the 2nd IS Factors II, VII, IX, X,plasma, (94/746) and sample N, the normal plasma pools prepared locally. Thenumber in the square denotes the laboratory code. Each square represents thegeometric mean estimate from the laboratory. The shading represents thedifferent standards used. A 'C' accompanying the laboratory code denotes thatthe estimate is from chromogenic assays.

,..

Figure 3. Potency estimates for FIX in sample 8, the proposed 3rd ISFactors II. VII. IX. X. plasma relative to sample A. the 2nd IS Factors II. VII, IX. X.plasma, (94/746) and sample N, the normal plasma pools prepared locally. Thenumber in the square denotes the laboratory code. Each square represents thegeometric mean estimate from the laboratory. The shading represents thedifferent standards used. A 'C' accompanying the laboratory code denotes thatthe estimate is from chromogenic assays.

Figure 4. Potency estimates for FX in sample B, the proposed 3rd ISFactors II, VII, IX, X, plasma relative to sample A, the 2nd IS Factors II, VII, IX, X,plasma, (94/746) and sample N, the normal plasma pools prepared locally. Thenumber in the square denotes the laboratory code. Each square represents thegeometric mean estimate from the laboratory. The shading represents thedifferent standards used. A 'C' accompanying the laboratory code denotes thatthe estimate is from chromogenic assays.

~

BS/Ol.1946Page 16

Table 1 Mean ratios (number of assays) of slopes of log dose-transformed responselines of samples Band N to the 2nd IS Factors II, VII, IX, X, 94/746.

0.99 (8) 0.99 (4)1.05 (38) 1.01 (38)0.99 (4) I NA1.03 (34) 0.98 (34)

1.01 (4) 1.02 (4) 1.00 (4)N 1.00 (70) 1.04 (72) 0.99 (72)

(combined) Chromogenic 1.01 (71) 1.01 (8) NA 1.00 (4)~ ~ ~ "

NA not applicable

BS/O1.1946Page 17

Table 2a Potency estimates for factor II in sample B relative to sample A, the 2nd ISFactors II, VII, IX, X, plasma (94/746)

i Laboratory~Ode 02

0305

06

10

16

1B

19

i 21I 22

I 01

04

07OBI 09! 12

14

15i 20

i B§~

Assay method

L~~;=~-I of a:~

4! 4! 3I

444

4444444444, 4

4

I

IU/anm:..OUleI ~.~~0.930.86I 0.94I

' 0.93

0.930.910.920.90I 0.910.920.930.960.870.920.900.990.91

I 9.~10.94

95% limits 1

I 0.87 -0.95-

0.87-0.99 I

0.81 -0.92

0.91 -0.98

I 0.82-1.07 0.84 -1.02

0.88 -0.94 I

0.88 -0.96

0.80-1.00I 0.87-0.96

0.84 -1.01 !

0.90-0.970.89 -1.04

I 0.84-0.90

0.88-0.96

0.83 -0.98

0.85-1.15

I ~.~~ -~.~~I Q.85 -0.96

0.89 -1.00

L I-iI

0.92I

0.92 I

NR ,0.94 iI

NR0.930.91 II0.92 0.89

0.910.93 ,0.95I0.96 0.87

1.00

i 0.89~.02 0.90

0.91I 0.94

Clotting

("

Chromogenic

Table 2b Potency estimates for factor II in sample B relative to sample N, the normalplasma pools prepared locally

Assay method Laboratory code I Numberof assays

444431244I :44444444! 2

4I ~

State

I

Unitl:~~OUle0.900.820.860.970.880.861.020.840.860.860.930.830.980.850.930.880.820.960.830.910.85, 0.94

~::~o

li:i~~-0.85 -0.96

0.78 -0.87

0.79 -0.93I

0.92-1.02

0.76 -1.02

OA0.34-3.00I0.77-0.92 0.77 -0.95

0.76 -0.97

0.85 -1.01

0.65 -1.06I 0.95-1.00

0.80 -0.90

0.92-0.940.83 -0.93II 0.79 -0.85

0.88-1.04

0.74 -0.9210.82-1.01

I 9.~~ -0.89I 0.92-0.96

!

I 02

03

.050610121618192122

01

04070809

12141516

i 20

i 09

r Fresh

Clotting

Frozen

I Chromogenic t-rozen

LNROA

geometric mean of laboratories' own individual assay potency estimateslaboratories' own individual assay potency estimates not returnedestimate of potency based on one assay only

BS/Ol.1946Page 18

Table 3a Potency estimates for factor VII in sample B relative to sample A, the 2nd ISFactors II, VII, IX, X, plasma (94/746)

toAAssay method Laboratory code NumDer

ofassavs IU/ampOIJle95% limits

1.04-1.130.95 -1.090.92-1.101.02-1.051.02-1.200.99 -1.141.01-1.080.97-1.110.93 -1.110.98-1.190.98-1.101.08-1.121.04-1.151.03-1.11090-1,170.99 -1.070.91-1.121.01-1.150.91-1.061.10-1.161.00-1.25

L

1.081.01NR1.03NR1.031.041.041.021.091.061.151.081.071),861.081.021.070.971.121.07

02030506101618192122010407080912141520

444444444444444

4444

1.081.021.011.031.111.061.051.041.021.081.041.101.091.071031.031.011.070.98

Clotting

C'

Chromogenic 1909

44

1.131.12

Table 3b Potency estimates for factor VII in sample B Irelative to sample N, the normalplasma pools prepared locally

Relative to NAssay method Laboratory code NumDer

of assays4444412444444444444244

State 95% limits

0.81 -1.070.76 -1.210.85 -0.940.96 -1.250.79 -0.96

OA0.31 -4.890.81 -1.040.82 -1.090.63 -0.98

0.84-1.17

0.57-1.381.06-1.140.84 -0.900.87 -0.910.93 -1.070.81 -0.911.32 -1.450.85 -1.071.05 -1.05

0.87 -1.00

0.75 -0.94

0.90 -0.95

Fresh

Clotting

Frozen

Chromogenic I-resn

unit/ampoule

02 0.9303 0.9605 0.9006 1.0910 0.8712 0.9216 1.2418 0.9219 0.9521 0.7822 0.9901 0.8904 1.1007 0.8708 0.8909 0.9912 0.8614 1.38.15 0.9516 1.0520 0.94

19 0.8409 t-rozen 4 0.92

L -geometric mean of laboratories' own individual assay potenc\r estimatesNR -laboratories' own individual assay potency estimates not returnedOA -estimate of potency based on one assay only

BS/O1.1946Page 19

Table 4a Potency estimates for factor IX in sample B relative to sample A, the 2nd ISFactors II, VII, IX. X. plasma (94/746)

~AAssay method Laboratory code

Clotting

Assay method Laboratory code

I 02-

03I 05

06

101216181921220104070809

I1214151620

NumberF44441244444444444424

State

,

unit/ampoule

0.780.780.840.900.820.901.000.720.740.690.870.710.840.790.880.840.770.930.900.980.83

95% limits

0.69 -.870.68 -0.90 I

0.81 -0.87 i0.79 -1.04 iI

0.65 -1.03

OA0.77 -1.30

0.66 -0.78

0.60-0.91 I

0.64 -0.75

0.75-1.02

0.50 -1.010.79 -0.90

0.76 -0.82

0.81 -0.95

0.76-0.92I 0.73 -0.81

0.88 -0.98

0.77 -1.04

0.44 -2.22

Fresh

Clotting

Frozen

LNROA

geometric mean of laboratories' own individual assay potency estimateslaboratories' own individual assay potency estimates not returnedestimate of potency based on one as;say only

i Number

~ssays IU/ampoule95% limits L

02- 4 0.95 0.77-1.18 0.9503 4 0.86 0.81 -0.91 0.8805 'I 4 0.86 0.83 -0.90 NR06 ! 4 0.88 0.84 -0.91 0.87

10 4 0.92 0.71-1.17 NR16 I 4 0.89 0.84 -0.94 0.9018 4 0.87 0.84 -0.91 0.8719 4 0.87 0.83 -0.91 0.8721 4 0.88 0.87 -0.90 0.9222 4 0.89 0.78 -1.02 0.9301 4 0.90 0.76-1.07 0.9304 4 I 0.92 0.89 -0.95 1.0007 4 0.84 0.81 -0.87 0.8308 4 0.93 0.82 -1.05 0.9409 4 0.87 0.81 -0.93 0.9012 4 0.86 0.82 -0.90 0.8614 4 0.90 0.84 -0.97 0.9115 4 0.97 0.91 -1.05 0.9820 4 0.89 0.84 -0.93 0.88

Table 4b Potency estimates for factor IX in sample B relative to sample N, the normal

plasma pools prepared locally

BS/01.1946Page 20

Table Sa Potency estimates for factor X in sample B relative to sample A, the 2nd ISFactors II, VII, IX. X, plasma (94/746)

.Relative 0 A

Assay method Laboratory code Numberofassavs

020305061016181921220104070809+

12 I 14

1152009

.

Clotting

("\

Chromogenic

4444444444444444444"4

Table 5b Potency estimates for factor X in sample B relative to sample N, the normalplasma pools prepared locally.

Assay method Laboratory code

02030506101216181921220104070809121415162009

Numberof assa~'s

State 95% limits

0.86 -0.950.81 -0.940.85 -0.890.89 -1.130.77~0.91

OA0.44 -2.240.79 -0.990.72-1.060.72 -0.900.80 -0.970.66-1.090.90 -0.980.82 -0.910.91 -0.950.87 -0.970.77 -0.810.91 -1.180.79-0.940.75-1.170.83 -1.000.94 -0.99

unit/ampoule

0.900.870.871.000.830.820.990.890.870.810.880.850.940.870.930.920.791.040.860.930.910.96

4444124444

(~Fresh

Clotting

Frozen

Chromogenic Frozen

444444424""4"

geometric mean of laboratories' own individual assay potency estimateslaboratories' own individual assay poterlcy estimates not returnedestimate of potency based on one assay only I

LNROA

BS/Ol.1946Page 22

Table 7 Summary statistics of sample B relative to sample A, the 2nd IS Factors II,VII, IX, X, plasma (94n46), and sample N, the normal plasma pools prepared locally.

Number of Mean potency Inter-laboratoryestimates (units/ml) variability

GCV%

20 : 3.1

~

FactorStandard

~

0.921.050.890.960.890.890.890.950.950.94 i

0.820.84

-0.820.880.91

0.90

.191920

3.43.82.96.66.66.112.19.09.911.810.010.11.11.5

1.0

AIXX

i 11!

11I

22!

11

9

20

11I 10!

21

i 11

11

i 22

II

*VII

FreshFrozen

CombinedFresh

FrozenCombinedN

("\I-resnFrozenIX

CombinedFresh

FrozenxCombined

Combined*

state of normal plasma pools ignoredexcluding outlier laboratory 14 and chromogenic FVII results

BS/Ol.1946Page 23

Table 8 Comparison of FVII estimates (IU per ml) in the proposed 3rd IS, relative to ~,c~~:~,,;;;~~,the 2nd IS and the normal plasma pools, by clotting and chromogenic methods in two ~~:::~='--'laboratories. --~-"_:=-=:~"~

Lab Proposed 3rd IS vs 2nd IS Proposed 3rd IS vs normal pools

clottina I chromoqenic I ratiociotti no I chromoaenic I ratio -

ratio -clotting/chromogenic potencies

BS/Ol.1946Page 24

Table 9 Potency estimates (IU/ml) for factors II, VII, IX and X in sample N relative tosample A, the 2nd IS Factors II, VII, IX, X, plasma (94/746).

~'... ~~.;;'~~~

~~"' p::~~~,

Lab Number Potency IU/mlState Assay method code of donors FII FVII FIX FX

1.01 1.22 1.071.13 1.10 1.111.01 1.03 1.060.98 0.97 0.981.06 1.12 1.161.13 0.98 1.190.88 0.88 0.951.08 1.22 1.061.07 1.17 1.111.04 1.27 1.210.98 1.02 1.07NA NA NA1.10 1.270.95 1.091.14 1.06093 1 051.05 1.041.10 1.121.03 0.971.09 1.081.06 0.911.07 1.071.00 NA

~?;4820?

22194127172020202720?

263527416

21205527

1.161.061.120.951.271.150.891.14

1.101.301.09

1.341.171.001.251201.031.19

0.73"1.130.981.05

1.21

t

ClottingFresh

Chromoqenic

0203050610$1216181921221901040708091214151620

1 1.14

1.07

1.151011.041.181.001.081.041.050.98

('

ClottingFrozen

Chromoqenic 09

$

NA*

?

estimate of potency based on one assay onlynot applicableestimate classified as an outlier by Duncan's multiple range testnot reported

BS/O1.1946Page 25

Table 10 Summary statistics of sample N relative to sample A, the 2nd IS Factors 11,- -~VII, IX, X, plasma (94/746). ;

Factor State Number ofestimates

Mean potency(IU/ml)

Inter-laboratoryvariability

111122

1.031.051.04

7.66.45.9

IIFreshFrozen

Combined

119

20

1.101.111.11

11.99.39.9

*VIIFresh

FrozenCombined

r 111021

1.081.061.09

12.39.09.8

IXFreshFrozen

Combined

FreshFrozen

Combined

111122

1.091.061.07

7.76.26.4

x

Combined*

state of normal plasma pools ignoredexcluding outlier laboratory 14 and chromogenic FVII

BS/Ol.1946Page 29

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