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EXPERT OBUTIEE CN BIOr.cx:;ICAL STANDARDIZATION Geneva, 27 September - 3 October 1983

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~TIVE S'1UDY OF '!HE PRQl(l;E[) SJD:IID IN'lERNATICHU. REFEEREKE PREPARATICE OF ~ 'lBYROID STIMUIATIR; IDM:Im Em :JHottJRl\SSAy

Draft Report

INDEXED

WHO International Laboratory for Biological Standards National Institute for Biological Standards and Control

Lcndon NW3 6RB, En;Jland

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yyu-l-eA~ " J

Contents SUM>1ARy

IN'IRODUCTION

PARTICIPANTS IN 'lliE STUDY

MA'IERIALS

- Preparation of ampoules of candidate materials - Preparations included in the study

ME'lHOOO

- Assay systems used - Statistical analysis

RESULTS

- Characterization of candidate materials - Log dose-response relationships

- Variability of linmunoassay estimates

- Comparison of 80/558 and 81/502 with the MRC Research Standard A by bioassay

- calibration of the proposed Second IRP,80/558, in terms of the First IRP by imnunoassay

- Canparison of the proposed IRP with various local standards by linmunoassay

- Canparison by imnunoassay of the ampouled TSH preparations with the First IRP, 68/38, and with the proposed Second IRP, 80/558

- Stability of the ampouled TSH preparations, 80/558 and 81/502 - TSH content of the serum and plasma preparations by linmunoassay DISCUSSION

- Canparison of linmunoassay and bioassay data with 80/558 and 81/502 - Serum and plasma samples

- Suitability of 80/558 to serve as standard and proposed unitage - Proposed use of 81/502

~rnIOOS

ACKNOWLEOOEMENTS

REFERENCES

'lABLES AND FIGURES

The issue of this document does not constitute formal publication. It should not be reviewed, abstracted or quoted without the agreement of the World Health Organization. Authors alone are responsible for views expressed in signed articles.

Ce document ne constitue pas une publication.

II ne do it faire I'objet d'aucun compte rendu ou resume ni d'aucune citation sans I'autorisation de l'Organisation mondiale de la Sante. Les opinions exprimees dans les articles signes n'engagent que leurs auteurs.

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WHO/BS/83.1402

page 2

S~Y

Four ampouled freeze-dried preparations of human pituitary 'ISH, coded 80/558, 81/502, 81/565 and 81/615 have been evaluated by international collaborative study for their suitability to serve in replacement of the International Reference Preparation of Thyroid Stimulating Hormone (Pituitary 'ISH), Human, for Imnunoassay (code 00. 68/38). Seventeen laboratories provided bnmunoassay data and four laboratories provided bioassay data. The geanetric mean potency estimates obtained by inmunoassays expressed as miu of the IRP per ampoule were: for 80/558, 36.2 miu; for 81/502, 7.7 miu; for 81/565, 11.5 miu; for 81/615, 11.4 miu. Potency estimates obtained by in vivo bioassay of 81/502 and 80/558 were

oonsistent with these figures. The assay characteristics O::etween-assay and between- laboratory heterOJeneity, slopes of dose response lines) and stability were acceptable for 80/558; it is proposed that this material is established as the 2nd International Reference Preparation of human Thyroid Stimulating Hormone (Pituitary 'ISH), Human, for Imnunoassay with a unitage of 37 miu per ampoule. Preparations coded 81/502, 81/565 and 81/615 were oonsidered sui table to serve as working standards for imnunoassay. Preparation 81/502 may not be suitable to serve as a standard for in vitro 'ISH bioassay, and neither 81/502 not 80/558 appears to be an appropriate standard for use in cytochemical bioassays.

Irnnunoassay of humarit;hyroid stimulating hormone ('ISH) is widely used in clinical diagnosis and in basic research. There is also extensive interest in bioassay of 'ISH. The 1st International Reference Preparation for Thyroid Stimulating Hormone (Pituitary 'ISH), Human, for Irnnunoassay (in ampoules coded 68/38), was established in 1974 (World Health

Organization, 1975), and this has been widely used as a standard for the irnnunoassay of 'ISH. This IRP had been calibrated in tenns of an earlier preparaticn, MRC Research

Standard A, set up in 1963 and since used as a bioassay standard. Stocks of both have run low and this report describes the international collaborative study of candidate

replacement preparations.

After evaluation of a mnnber of h'ISH preparations fran different sources, (Bristow, Sutcliffe, Ayling & Bangham, 1983) two preparations were ampouled in four batches (see Table 1 and Materials). An international oollaborative study of these preparations was organi zed wi th the following aims:

1. to calibrate each of the four batches of ampouled h'ISH preparations against the current International Reference Preparation of human Thyroid-Stimulating Hormone (hTSH; 68/38) by bnmunoassay and to assign a uni tage to ampoules of each batch;

2. to examine the sui tabili ty of the ampouled 'ISH preparations to serve as standards for the assay of 'ISH in clinical serum samples by comparisons of these ampouled

preparations of h'ISH with (coded) preparations of sera containing a range of ooncentrations of 'ISH;

3. to relate the ampouled 'ISH preparations to the current 'ISH bioassay standard 63/14 (MRC Research Standard A) by bioassay;

4. to assess the stability of the ampouled 'ISH preparations by comparisons of ampoules of each preparation that had been subjected to accelet"ated thermal degradation with ampoules stored oontinuously at -20°C.

Based on results of this study it is proposed that the preparation in ampoules coded 80/558 should be established as the Seoond International Reference Preparation of Thyroid

Stimulating Hormone (Pituitary 'ISH), Hunan, for Irnnunoassay. It is also proposed that the preparation in ampoules coded 81/502 oould be used as a working standard.

PARTICIPANTS IN '1HE S'IUDY

Of twenty-four laboratories invited, twenty-one laboratories in nine oountries took part in the study. They are listed below. Throughout this report each laboratory has been

represented by a number between 1 and 24 and these numbers 00 not ooincide wi th the order of listing.

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Belgium

Dr Georges Hennen Institute de Pathologie

aru

4000 Sart Tilman Liege 1

Holland

Dr R.D. Gaag & Dr H.A. Drexhage Lab. for Imnunopathology

Dept of Pathology

Free University Hospital De Boelelaan 1117

1007 ME Amsterdam Italy

Dr R. Ermanno

Biodata SpP.

Via Tiburtina Valeria, KIn 19.600 Guidonia Mantecelio

Rare

New Zealand

Dr W.A. Sadler

Nuclear Medicine Dept Christdlurdl Hospital Private Bag

Christdlurdl SWeden

Dr L. Axelsson

Pharmacia Diagnostics AB

BaK 17

S-75l 03 Uppsala 1

Dr L. Wide

Dept of Clinical Chemistry University Hospital

S-750 25 Uppsala 14 SWitzerland

Dr L. Binet Hypolab SA Case Postale 10 1267 Coinsins United Kingdan

Dr J. Seth & Mr N.S. Brown*

Imnunoassay Section

Dept of Clinical Chemistry University of Edinburgh 12 Bristo Place

Edinburgh EHl lED

Dr N. Marshall

Thorn Institute of Clinical Science Middlesex Hospi tal Medical Sdlool Mortimer Street

Landon WlN 8AA Professor D.S. Munro University of Sheffield Clinical Sciences Centre Northern General Hospital Sheffield S5 7AU

* Present address:

Dept of Clinical Pathology Central Pathology Laboratory Hartshill

Stoke on Trent ST4 7PA

Professor W.R. Butt

Dept of Clinical Endocrinology

Birmingham & Midland Hospital for Wanen Showell Green Lane

Sparkhill

Birmingham Bll 4HL

Dr P .G. Malan

Amersham International Ltd Forest Farm, Whitchurch Cardiff CF4 7Y!r

Professor R.P. Ekins Dept of Nuclear Medicine

Middlesex Hospi tal Medical Sdlool Nassau Street

Landon WlN 8AA

Dr A.F. Bristow & Mr N. Sutcliffe National Institute for Biological

Standards and Control Hampstead

Londen NW3 6RB

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WHO/BS/83.1402

page 4

United States of America

MATERIALS

Mr G. Wong

Calbiochem-Behring Corp 10933 North Torrey Pines Road

La Jolla

California 93037 Dr G.E. Lavke

Nuclear Medical Laboratories Inc POBox 47864

8700 N. Stemmons Freeway Dallas

Texas 75247

Mr B. Shah

Meloy Laboratory Inc 6715 Electronic Drive Springfield

Virginia 22151

W. Gennany Dr W. Nagale Byk-Mallinckrodt von Hevesy Str. 1-3 6057 Dietzenbach 2

Preparation of ampoules of candidate materials

Dr P.M. Shapiro, Dr C.A. Burns

& Dr J.M. Andrews

Clinical Assays 620 Memorial Drive Cambridge

Massachusetts 02139

Dr S. Krutzik & Dr J.S. Sangha Nuclear Medical Systems Inc 1533 Monrovia Avenue

Newport Beach California 92663

Dr C. Bradley Hager Micramedic Systems Inc 102 Wibner Road

Horsham

Philadelphia 19044

Preparations of highly purified human pituitary TSH were donated to WHO fram the National Honrone and Pituitary Program of the NIAMMD, USA, extracted in the laboratory of Dr A F ParlaN, California, and fram the British Pituitary Collection, extracted in the laboratory of Professor W R Butt, Birmingham, UK.

Same 70mg of bulk material was received at NIBSC fram Dr Parlow as a freeze-dried paNder and stored at -70°C. Material fram Professor Butt was received as a frozen

solution, and was dialysed against 50mM ammonium acetate, lyophilized, and stored at -70°C until it was dissolved for distribution into ampoules.

~hree batches each of some 4000 ampoules, coded 80/558, 81/565 and 81/615 were prepared containing TSH fram Dr ParlaN's laboratory. One batch of 3800 ampoules, coded 81/502, was prepared containing TSH fram Profesor Butt's laboratory. All four batches of ampoules were prepared according to the procedures used for international standards (World Health

Organization, 1978). Weighed portions of the TSH were dissolved in a sterile solution containing 0.2% w/v peptidase-free human serum alb.lmin and 1% w/v lactose. This solution was passed through a rrenbrane filter (rrean pore diameter 0.45un) and distributed in 0.5ml aliquots into ampoules. The ampouled solution was lyophilized, and after secondary

desiccation, the ampoules were sealed under nitrogen by heat fusion of the glass and stored at -20°C in the dark.

Preparations included in the study

The ampouled preparations included in the study are listed in Table 1. In addition, laboratories were asked to incluoo their local standards i f they were available.

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Assay systans used

!he assay systans used in the collaborative study are given in '!able 2. Assays contributed by each participant have been numbered consecutively. Where b.o assay systems were used by the same participant, these have been identified by the laboratory number and an upper case letter A or B. Each assay number is followed by a lower case letter (a,b,c or d) to

indicate \\hich of the two (in exCEptional cases rrore) independent sets of aIlp)ules were assayed.

statistical analysis

Results of bioassays (exCEpt for those fran laboratory 21, for \\hich only qualitative results were obtained), were analysed as parallel line assays (see, ego Finney, 1978) with the responses transformed, if necessary, to give linearity of log dose-response

relationships. Immunoassay responses were transformed to logits and analysed using

weighted linear regression (Gaines Das & Tydeman, 1980), with potency estimates cx:mputed as for parallel line bioassays. Responses equivalent to either the minimum or maximum

response obtainable with the assay system were anitted fran analysis.

For estimates of potency obtained by bioassays, weights were canputed as the reciprocal of the variance of log potency, based on the residual error of the assay analysis.

Estimates of potency obtained by iImiunoassays were canbined within each laboratory to give the gearetric nean potency and the weight of the laboratory rrean v.as based on the

reciprocal of the variance of the log potencies combined.

Estimates of potency were assessed for heterogeneity using a chi squared test.

Hanogeneous estimates were combined to give a weighted geanetric nean. Heterogeneous estimates were examined to determine the SOurCE of heterogeneity; i f they were canbined, such estimates were combined as unweighted geanetric rreans, and the variability cx:mputed as the variance of the canbined log potencies.

Relative degradation rates were assumed to be related to temperature of storage of thermally degraded samples by the Arrhenius equation and a maximum likelihood approach was used to obtained the predicted rates (Kirkwood, 1977).

RESULTS

Characterization of candidate materials

Both extracts of 'ISH \\hich were used in the preparation of aIlp)ules have been extensively characterized biochemically and immunologically. When analysed by sucrose-density gradient isoelectric focusing, and 'ISH radioimmunoassay, the preparation fran Dr Parlow's laboratory exhibited five charge variants with isoelectric points between 5.3 and 6.7, in approxi- mately similar amounts. The preparation fran Professor Butt's laboratory contained silnilar charge variants, although with a predaninance of forms with isoelectric points of 5.7 and 6.05. Sirnilar results were obtained by gel-electrofocusing and gel-electrophoresis at pH 8.90. Silnilar patterns of heterogeneity were seen with the IRP (68/38) and MRC Research Standard A (63/14). Cootarnination with free subunits, estimated by gel-electrophoreis and size-exclusion HPLC was negligible in both preparations. Contamination with LH and FSH was estimated by radioimmunoassay. 80/558 contained 2.515 units of LH & 0.137 units of FSH per ampoule. 81/502 contained 2.095 uni ts of LH and 0.0265 uni ts of FSH per ampoule. Full details of biochemical, biological and immunological characterization of candidate materials will be published separately.

Log dose-reSponse relationships

Bioassay. Log dose-response lines obtained by bioassays did not deviate significantly fran linearity but the slopes (Table 3) given by the TSH preparations showed significant

differences. In the in vivo bioassays of laboratory 18, 80/558 gave significantly flatter log dose-response lines than either Research Standard A or 81/502. In the in vitro assays of laboratory 20A, slopes for the log dose-response lines for 81/502 were less than those for Research Standard A or 80/558. Also qualitative differences between 80/558 and the other two preparations, Research Standard A and 81/502 were noted in the thyroid growth assay of laboratory 21.

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WHO/BS/83.1402

page 6

Inmunoassay. '!he transformed responses did oot show consistent departures fran linearity.

Slopes of log dose-logit response lines for the purified TSH preparations (Table 4) did not show any significant oon-parallelism.

Slopes of the log Cbse-logit response lines for the serum samples are shown in Table 5.

However, these were generally based 00 only a fStl points, often near the lower end of the response range, and are consequently less reliable than those for the 'ISH preparations. No marked instances of non-parallelism were seen although there was a suggestion in a few

laboratories that the slopes of the log dose-logit response lines for the serum samples were flatter than those for the 'ISH preparations.

variabili ty of Inmunoassay Estimates

'!he values for the relative potencies of the coded duplicates (Table 6) have l:een used to provide an independent measure of within-assay variability and hence of the minimum

variability inherent in calibrations based 00 these assay systems.

The expected potency of eadl coded duplicate relative to the identical material is 100%

and the observed values are in good agreanent with this. '!he geanetric m:an of potencies for H relative to 80/558 was 101% (95% limits 99.4-104%) and for G, relative to 81/565, anitting results fran lB which showed unusual bias, was 98.8% (94.4-102%).

Deviations of the observed values fran the expected 100% anitting results fran lB gave a pooled within-assay m:an square, in each case, of about 0.03 and an approximate 95%

interval for comparisoo of identical materials in these assay systems of 85%-117%.

For either rraterial the l:etween-assay variance was about twice the within-assay variance. However, the l:etween-laboratory variance was simliar to the l:etween-assay variance and exCEpt for results fran Laboratory lB in the case of 81/565 and lOA in the case of 80/558, the laboratory mean estimates did not differ significantly fran one another.

canparison of 80/558 and 81/502 with MRC Research Standard A by bioassay

80/558 was compared with Research Standard A by McKenzie in vivo bioassays in two

laboratories. Individual estimates are given in Table 7. '!he mean 'ISH activity was 39.9 (95% interval 34.4-46.2) units (u) of Researdl Standard A. Preparation 81/502 was also examined in these assays and found to have mean activity of 9.70 (95% interval 8.34-11.28) u Research Standard A per ampoule.

Only qualitative data were available fran the in vitro thyroid growth stimulating assays of laboratory 21, but these were in agreement with the naninally assigned unitages.

Canparison in laboratory 20A by CEll culture bioassay of the proposed IRP with Research Standard A (63/14) gave a sanewhat higher estimate of 'ISH activity than that given by in vivo bioassay (Table 7). The mean (95% interval) fran three assays was 46.1 (42.2-50.3) u Research Standard A per ampoule 80/558. '!he arnpouled preparation 81/502 was also examined in this systan rut dose-response lines for it may differ fran those for 81/558 or 63/14.

'!he rrean activity per ampoule estimated fran three assays was 15.4 (5.5-43.2) u of Research Standard A. Estimates by cytochemical bioassays in laboratory 20B (Table 7) differed rrarkedly fran those obtained by other bioassay rrethods •.

calibration of 80/558 in terms of the First IRP

9Y

~oassays

Individual assay estimates for 80/558 and H, identical to 80/558, in terms of the IRP

(68/38) are listed in Table 8 and shown in Figure 1. Laboratory mean estimates and overall means are given in Table 9. Excluding laboratories 4 and lOB and with log potency

estimates weighted inversely proportionally to within~laboratory l:etween-assay variability, the dli square value for haoogeneity did not differ significantly fran its expected value.

However, there was a tendency for estimates fran sane assay systems to I:e consistently larger (eg. 19A) or smaller (eg. 9B3) than the overall means. '!his was reflected by the comparisoo of l:etween-laboratory variance with between-assay within-laboratory varianCE which gave a significantly large ratio of 11, or anitting estimates fran laboratories 4 and 10, of 5. The l:etween-assay variatioo was similar to that seen for canparisons of

identical rraterials.

canparison by imnunoassays of 80/558 with various local standards

Estimates of the 'ISH content ~ 80/558 in terms of the various local standards are given in Table 10. 'lhese estimates varied, depending on the local standard. However, the overall results were similar to those given by the calibratioo in ter.ms of the IRP (68/38).

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Individual assay estimates for each of the ampouled 'ISH preparations in terms of the First IRP are given in Table 8 and sha-m in Figures 2-5. Laboratory geanetric means for each preparation are given in Table 9 together with overall geometric means of the laboratory means. As can re seen fran the figures, the estimated potencies appeared to re normally distributed about the mean values. 'nlere WiS sane heterogeneity of estimates retween laboratories, but this was highly significant only in a few instances (Laboratories 4,10A,11,16). The retween-assay within-laboratory variation for each preparation was similar to that seen for canparisons of identical materials. Between-laboratory variation was in each case somewhat larger (similar to that given by 80/558).

Individual assay estimates of TSH in each arnpouled TSH preparatioo., in terms of the TSH in an ampoule of the proposed second IRP, 80/558, are given in Table 11. Ap:rrt fran

results fran laboratories 11 and 16 there was no significant heterogeneity of estimates retween laboratories, although laboratories lOA and 4 estimated unusually low values for 81/502. The retween-assay within-laboratory variation of estimates of each preparation was similar to that seen with cnnparisons of identical ampoules. Between-laboratory variation was also similar to that obtained in canparisons of identical materials for estimates of 81/502, if results fran laboratories 4, lOA and 11 were anitted and for estimates of 81/565 and 81/615 if results fran laboratories 11 and 16 were anitted.

Stability of the ampouled TSH preparations

Stability of 80/558. Materials in ampoules of the proposed second IRP, 80/558, were examined by irrmunoassay and bioassay after they had

reen

stored for 151 or 261 days at 20, 37, 45 or 56°C. Slopes of the log dose-response lines for the samples stored at 45 or 56°C

(Table 12) tended in sare irrmunoassay systems to re flatter than those for the samples stored at the lower temperatures. 'nlese samples also showed some loss of reactivity when canpared with samples stored at -20°C (Table 13). Irrmunoassay estimates of samples stored at 45 or 56°C were more variable retween laboratories than estimates of samples stored at 20 or 37°C, which showed less loss of activity. This variability may reflect the different specificity of the assay system for different degraded forms of TSH. Based on the aambined estimates fran all irrmunoassays, the predicted loss of irrmunoreacativity is 0.2% per year at -20°C. Biological activity of the samples stored at 20°C apparently increased and even excluding data for these samples the remaining data did not show a linear relationship relating temperature and degradation rate. This suggests that biological activity was not lost in a way corresponding to a first order reaction related to temperature of storage.

Stability of 81/502. The materials in ampoules of preparation 81/502 were examined by imnunoassayand bioassay after 177 days storage at 4,20, 37, 45 or 56°C. Slopes of log dose-response lines for these samples (Table 14) did not show any significant deviations fran parallelism, although there was a tendency in laboratory 12 for the slopes of the more degraded samples to re flatter than those of the less degraded samples. The relative activity of the thermally degraded samples compared with samples stored at -20°C (Table 15) estimated by irrmunoassay did not differ significantly fran 100% although with the irrmuno- assay system of laboratory 4 there was a trend of estimates suggesting that some loss of inmunoreactivity detectable with this assay system was occurring. In contrast, examination by bioassay of the samples stored at 37, 45 or 56°C showed significant loss of biological activity compared with samples stored at -20°C, although estimates of samples stored at 4 or 20°C tended to re greater than 100%. Predicted degradation based on the bioassay data is about 1% per year at -20°C.

TSH content of the serum preparations by imnunoassay

The laboratory mean estimates of concentrations of TSH in serum samples expressed in terms of iu of the First IRP are given in Table 16 and in terms of 80/558 (assuming 37

iu/ampoule) are given in Table 17. The numerical values for these estimates fran different laboratories differ. 'nlere was, nevertheless, agreanent amongst the laboratories upon the ranking order of TSH concentrations. All laboratories agreed that C contained more 'TSH' than E and exCEpt for laboratories 10, 19A and 22 that F contained less TSH than E and more than B, D and A. The TSH concentrations in these latter three samples were generally at or near the detection limit of the assay systems included in this study.

The assay systems could re subdivided into those in which D was found to contain more 'ISH than A and B (laboratories 12, 14 and 16) and the remainder. On the basis of estimates

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WHO/BS/83.1402 page 8

obtained for C and E, the only

twO

samples which contained sufficient TSH irrmunoactivity for reliable estimation, the assay systems could J::e grouped into three groups; those giving homogeneous estimates for TSH concentration of about 70 miu per ampoule for C and about 20 miu per ampoule for E (laboratories 2,6,14,17,19B, 2 and possibly 15), those in Yklich the estimates for E were greater than 40 miu per ampoule (5,10A,10B and 12) and those in Yklich estimates of C were more than 85 miu per ampoule (the previous four systems and 4,16 and 19A). These relationships and ranking orders were the same for estimates in terms of either the First IRP or 80/558; laboratory 4 was the only laboratory to show a substantial change in its relative position in the ranking order of estimates among laboratories with the change of standard.

Numerical values for estimates of serum TSH concentrations in terms of 80/558 assuming it to contain 37miu per ampoule (Table 17), were generally in reasonable agreement with values obtained by the same laboratories in terms of international units of the IRP (Table 16). Seven of the 13 laboratories showed a difference of estimates for these samples of less than 10% and 9 laboratories showed a difference of less than 15%. The three

laboratories whidl showed differences of greater than 20% (4,10A and 16) were also noted either to J::e outside the group of laboratories giving homogeneous estimates for serum samples C and E, or to have contriruted excessively to the chi square for heterogeneity of estimates of 80/558 in terms of the IRP.

DlSCUSSlOO

COmparison of immunoassay and bioassay data for 80/558 and 81/502

TSH extracted from human pituitaries is heterogeneous and preparations obtained by different methods of extraction and purification rre.y also differ (Bristow et al, 1983).

Nevertheless estimates by immunoassay of the TSH content of the ampouled preparations as miu of the First IRP, with limited exceptions, agreed well; moreover cx:mparisons of the other ampouled TSH preparations appeared generally homogeneous with the exception of only a few estimates. It is not known Yklether the few rre.rkedly heterogeneous estimates reflected different specificities of these assay systems (notably laboratories 4,10,11 and 16) for different cx:mponents of TSH in these preparations. Bioassay data in this study have

revealed both discrepancies in the estimates obtained by bioassay and immunoassay, and also differences in the potency ratios of 80/558 to 81/502 determined by linmunoassay and

bioassay. It should J::e noted that estirnates by bioassay were rre.de in terms of MRC Research Standard A Yklereas estimates by irrmunoassay were in terms of the First IRP, and the units defined by these two reference rre.terials rre.y not J::e directly cx:mparable. However, the First IRP was originally calibrated in terms of MRC Researdl Standard A by immunoassay and bioassay, and these were cx:mpared again recently (BristOW', unpublished data) and the respective units rre.y J::e considered approxirnately the same. Potency estimates obtained by irrmunoassay and in vivo bioassay were in reasonably good agreement for both 80/558 and 81/502, although in the case of 81/502 the in vivo biopotency was somewhat higher than the linmunoreactivity. Potency estimates obtained with in vitro cultured cell assays of 80/558 were also similar to irrmunoreactivity estirnates, whereas in the case of 81/502 they were almost three-fold higher. Potency estimates obtained by cytochemical bioassays were anomalous for both preparations.

The ratios of estirnates, ampoule/ampoule, of 80/558 to 81/502 were approximately 4 by irrmunoassay and in vivo bioassay. In the cultured cell assays this ratio was approximately 3 and in the cytochemical bioassays it YaS more than 500. The reasons for this remarkable discrepancy are unknown rut it rre.y reflect the ability of the cytochemical bioassay to detect subtle differences in the TSH preparations not detected by other assay systems used in this study.

Serun and plasrra samples

Estimates by imnunoassay of the TSH concentration in the serum and plasrre. samples were not haoogeneous. This is not surprising in view of the likely heterogeneity of TSH in serum and the possibility of different specificities in the variety of assay systems included in

this study. With the exception of laboratory lOA however, the lowest estirnates for serum C were at least 10-fold higher than those for F, and using 80/558 as a standard, the TSH radioimmunoassay could J::e successfully used as a diagnostic test. It is notable that the laboratories showing rre.rked heterogeneity in oamparisons amonq the extracted TSH

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obtained such values for the sen:m samples. '!he causal relationship of these results to the specificity of these assay systans is not known.

Suitability of 80/558 to serve as standard, and proposed unitage

The results of this study have not shown any rrarked significant non-parallelism of imnunoassay OOse-response lines for 80/558 with those for the First IRP or other TSH preparations in use as local standards. The slopes of dose-response lines for 80/558 were flatter than those for MRC Research Standard A in the in vivo bioassay of one of the t\\1O laboratories carrying out such assays. However, the in-vivo bioassay estimates of 'ISH conC€Iltraticn were in agreement with one another and with the estimates by imnunoassay which, with few exCEptions, were hanogeneous. Estimates of the 'ISH concentration for the sen:m samples by imnunoassay in t.errcs of either the First IRP or 80/558 were variable.

However, the variability of estimates in terms of 80/558 did rot differ significantly fran that of estimates in t.errcs of the First IRP. Assays of ampoules of 80/558 after storage at elevated temperatures suggested that the material stored at -20°C has satisfactory

stability. It is therefore suggested that the preparaticn in ampoules coded 80/558 is suitable to serve as a standard for lx>th bioassays and for imnunoassays, and proposed that it be established as the Second International ReferenCE Preparation of Thyroid Stimulating Honrone (Pituitary 'ISH), Human, for Imnunoassay. However, in view of the results obtained by cytochemical bioassay, it should be noted that 80/558 may not be suitable as a standard for this assay system. It is further proposed that the unitage assigned to 80/558 be 37 miu per ampoule; this value is canpatib1e with the observed means of estimates in t.errcs of the iu of the First IRP by imnunoassays and in terms of units of MRC A by in vivo bioassay, and it is likely to maintain reasonable continuity of estimates of 'ISH concentration for serum samples.

Proposed use of 81/502

I t is proposed that ampoules of 81/502 are made available, calibrated in terms of the Second IRP, 80/558, with an assigned unitage of 7.75 miu per ampoule; this estimate agrees with the mean of estimates by imnunoassay in~rms of either the First IRP or 80/558 with an assigned potency of 37 miu/ampoule. I t should be noted that 81/502 may not be suitable for use in in vitro bioassays with this unitage since cultured CEll bioassays in this study gave significantly greater estimates of this preparation, whereas in the cytochE!llical bioassays this preparation appeared to have very little activity.

A~

\OlIO gratefully acknowledges Dr S. Raiti of the USA National Pituitary Agency; Dr A.F.

Parlow and Professor W.R. Butt in whose laboratories the 'ISH preparations were purified;

Mr S. Sufi and Dr B. Gledhill for imnunoassays of IE and FSH; Professor W.S. Munro for providirg TSH sera and for helpful advice; Mr N. Sutcliffe, Ms C. Ayling and Dr P .L.

Storring for initial characterization of candidate materials; and the participants of the co1lalx>ra ti ve study.

(10)

WHO/BS/83.1402 page 10

Bristow AF, Sutcliffe N, Ayling C & Bangham IR (1983). '!he evaluation of candidate materials for the replacement of the International Reference Preparatioo of human

'lhyroid Stimulating Honrone ('ISH) for irrmunoassay. Proceedings of Joint lEP/F'DA. Workshop

00 insulins, growth honnone and thyroid axis drugs. USP Press, Bethesda, U~

Drexhage HA, Bottazzo GF, Doriach 0, Bitensky L & Chayen J (1980). Evidence for thyroid- growth-stimula tiD3' irrmunoglorolins in sane goi trollS thyroid diseases. Lancet August 9, 287-282.

Finney ill (1978). Statistical Method in Biological Assay, edn 3. Griffin: London.

Gaines-Das RE & Tydeman MS (1980). Iterative 'Weighted regressoo analysis of logit responses. A ccmputer program for analysis of bioassays and irrmunoassays. Canputer Programs in Biomedicine 15:13-22.

Kirk\o)d 'lBL (1977). Predicting the stability of biological standards and products.

Biometrics 33:736-742.

McKenzie JM (1958). The bioassay of thyrotrophin in serum. Endocrinology 62:372-382.

Webster BR, Furni val BE & Paice JC (1970). Reference Preparations of human thyrotrophpin:

canparative stability and biological and irrmunological potencies. Prac. 6th International Thyroid Conference, Vienna, W1057-1067.

Wide L (1070). Use of particulate irrmunosorbents in radioirrmunoassay. Methods in Enzym:>logy 73:203-224.

mo

Expert Ccmni.ttee on Biological Standardization (1975). 26th Report. WIO Tech Rep Ser No 565.

~O Expert Ccmni.ttee on Biological Standardization (1978). Annex 4, 29th Report. ~O Tech Rep Ser No 262.

(11)

Table 1. Preparations included in the study

Ampoule Assumed Ampoule Content i I

68/38

147 niiu TSH, by definition IRP for iIlUlluno-

assay 63/14 hTSH

50 niu TSH, by definition Res.Std. A

for bioassay

80/558 Approximately 7. 5J,J.g hTSH, lmg albumin, 5mg lactose

81/502 Approximately 2ug hTSH, lmg albumin, 5mg lactose

81/565 Approximately 2pg hTSH, lmg albumin, 5mg lactose

8li6l5 Approximately 2pg hTSH, lmg albumin, 5mg lactose

TSH sera Coded serum samples derived from TRH-provoked normal A, B, C patients, and from patients with toxic nodular goitre,

toxic Grave's disease and initial hypothyroiditis, and D, E, F samples from a normal plasma pool. Each containing

approx. 5~U hTSH except for C which contains approx.

200l1U hTSH

TSH G Coded ampoule of 81/565

TSH H Coded ampoule of 80/558

TSH I, Ampoules of 80/558 subjected to accelerated thermal J, K, L degradation at +56, +45, +37, +20°C respectively

TSH M, Ampoules of 81/502 subjected to accelerated thermal N, 0, P. degradation at +56, +45, +37, +20°C respectively;

- - -- - - - -

j

(12)

WHO/BS/83.1402

page 12

Table 2

Assay Type

In vivo bioassays

In vitro bioassays

Radioimmunoassays

Two-site

Immunoradiometric assay (IRMA)

(a) McKenzie (1958)

Assay Method

Release of radio- iodine (a) from thyroid

Production of cAMP by cultured thyroid cells.

Stimulation of thyroid growth in vitro. (b)

Cytochemical assay

Solid phase antibody

PEG 2nd Antibody 2nd antibody

Solid-phase 1st antibody (c)

(b) Drexhage et al (1980) (c) Wide et al (1981)

Further details

Incubation times (preincubation, antibody tracer, 2nd antibody)

Laboratory No.

8, 18

20A 21

20B

2, 19

9,13,15,19 3,4,5,6,10,11, 12,14

17, 11

16

(13)

Table 3 Slopes of log dose - response lines by bioassay

for MRC Research Standard A(63/14) and the ampouled TSH preparations.

Bioassal

MRC

Research 80/558 81/502

Standard A 63/14

8 1 0.59 0.52

2 0-.54 0.57

3 0.85 0.59

4 0.46 0.36

18 1 5.28 2.68 4.43

2 4.02 2.71 4.35

20A 1 1. 01 0.86 0.54

2 1.13 1. 76 0.82

3 0.44 0.44 0.45

20B 1 3.35 3.25

2 4.39 5.88

3 3.19 3.33

4 4.04 5.30

(14)

WHO/BS/83.1402 page 14

Table

~

Slopes of log dose-logit response lines by immunoassay f?r the IRP, the ampouled TSH preparations, 80/558, 81/502, 81/565 and 81/615 and varlOUS local standards

Laboratory and Assay

1A 1a 2b 1B 3 2 1a

2a 3a 4a 5a 6b 7b 8b 9b 10b 3 1a 2a+

3a+

4b 5b 6b++

7b++

4 1a 2b 5 1a 1a*

2b 2a+

2b*

6 1a 2b 3b 4b

Preparation IRP 80/558 H,Identical 81/502

68/38 to 80/558

-2.05 -2.09 -1.87

-1.62 -2.13 -2.00

-1.12 -1.33 -1.43

-1.56 -1.59 -1.64 -1.61

-1.54 -1.56 -1. 93 -1. 55

-1.59 -1.58 -1.89 -1.58

-1.39 -1.54 -1.56 -1.46

-1.52 -1.45 -1.65 -1.53

-1.35 -1.29 -1.27 -1.33

-1.47 -1.44 -1.43 -1.45

-1.52 -1.52 -1.54 -1.50

-1.49 -1.41 -1.43 -1.39

-1.24 -1.39 -1.44 -1.30

-2.33 -1.98 -2.35

-2.37 -2.75 -2.34

-2.63 -1.85 -2.23

-2.32 -2.52 -2.60

-2.25 -2.48 -2.60

-2.53 -2.90 -3.21

-2.41 -2.30 -2.45

-1.30 -1.40 -1.34 -1.37

-1.23 -1. 31 -1.30 -1.31

-1.24 -1.26 -1.51 -1.32

-1.42

-1.39 -1.49 -1.38 -1. 73

-1.53 -1.40 -1.43 -1.32

-0.86 -0.92+

-1. 52 -1.48 -1.47 -1.56 -1.56 -1. 56 -1.50 -1.50 -1.47 (-1.47 -1.36 -1.44

( -1.40 ( -1.40

-1.62 -1.54 -1.52 -1.59

81/565

-1. 75 -1.82 -1.48 -1.59 -1.54 -1.62 -1.43 -1.55 -1.44 -1.45 -1. 71 -1.40 -1.47 -2.05 -2.44 -2.29 -2.57 -2.45 -3.14 -2.29 -1.40 -1.31 -1.33

NE -1.49

-1.47 -1.65 -1.44 -1.53

G,Identica1 to 81/565

-1. 91 -2.12 -1.45 -1.52 -1.64 -1.57 -1.49 -1.56 -1.28 -1.48 -1. 57 -1.36 -1.47 -1.94

NE -2.08 -2.51 -2.60 -2.22 -2.35 -1. 31 -1.33 -1.27 -1.22 -1.39

-~.56

-1.55 -1.44 -1.57

* as in 1a except diluent was horse serum instead of phosphate buffer + as in 1a except after freezing

++ as in 4b except after freezing

81/615

-2.18 -1.94 -1.51 -1.59 -1.60 -1.60 -1.42 -1.51 -1.31 -1.50 -1.52 -1.45 -1.36

-2.52 -2.50 -2.98 -2.17 -1.36 -1.30

-1.

19 -1.23 -1.35

-1.52 -1.64 -1 .44 -1.54

Local Standard

-1.75 -1.61 -1.09 -1.59 -1.49 -1.54 -1.43 -1.50 -1.41 -1.45 -1.50 -1.47 -1.38

-1.40

-1.36

-1.23

-1. 15

-1.

51

-0.86

(15)

Table 4 Cont

I

d

Preearation

Laboratory IRP 80/558 H,Identical 81/502 81/565 G,Identical 81/615 Local

and assay 68/38 to 80/558 to 81/565 Standard

-

9A1 1a -1.83 -1.78 -1. 75 -1.72 -1. 70 -1.61 -1.69

2a -1.85 -1.96 -1.82 -1.75 -1.79 -1.75 -1.72

3a+ -1.95 -1.90 -1.72 -1.72 -1.67 -1.68 -1.77

4b

-1.

77 -1.78 -1.64 -1.68 -2.00 -1.59 -1. 79

5b -1.73 -1. 75 -1.79 -1.80 -1. 73 -1.58 -1.65

9A2 6a -2.01 -1.98 -1.81 -1.89 -1. 70 -1. 71 -1. 73

7a -1.94 -1.93 -1.48 -4.63 -1. 75 -1.64 -1.74

8a+ -2.10 -2.05 -1.87 -1.91 -1.66 -1.63 -1.94

9b -2.27 -2.26 -2.64 -2.47 -2.26 -2.02 -1.96

10b -2.09 -2.10 -1.59 -1.80 -1.85 -1.66 -1.82

9A3 11a -2.02 -2.13 -1.62 -1.72 -1.68 -1. 75 -1.81

12a -2.27 -2.21

-1.

75 -2.03 -1.98 -1.92 -1.97

13a+ -2.23 -2.27 -1.77 -2.14 -1.93 -2.04 ':'2.19

14b -2.26 -2.00 -1.99 -1.96 -1.98 -1. 76 -1.95

15b -2.41 -2.55 -2.34 -2.19 -2.10 -2.02 -2.25

9B1 16e -2.21 -2.05 -2.49 -2.26 -2.89

NE

-2.58

17e -2.10 -2.08 -2.25 -1.79 -2.18 -1.89 -1.86

18e+ -2.49 -2.19

NE

-2.72 -2.74 -2.53 -2.39

19d -1.97 -1.83 -1.43 -1.67 -1.60 -1.41 -1.81

20d -2.37 -2.49 -3.25 -2.13 -2.32 -2.34 -2.22

9B2 21e -2.29 -2.35 -3.13 -2.39 -2.37 -2.57 -2.00

22e -2.43 -2.55 -3.41 -3.94 -2.68 -2.52 -2.23

23e+ -2.94 -2.43

NE

-2.77 -3.78 -3.12 -2.78

24d -2.36 -2.42

~3.

10 -2.82 -2.77 -2.46 -3.49

25d -2.43 -2.38 -2.50 -2.77 -2.30 -2.53 -2.25

9B3 26e -2.14 -2.22 -1.95 -1.80 -2.00 -1.61 -2.08

27e -2.47 -2.34 -1.33 -1.93 -1. 76 -1.95 -2.25

28e+ -2.63 -2.43

-1.

74 -2.16 -2.07 -2.03 -2.58

29d -2.21 -2.12 -1.58 -1.67 -1.62 -1. 17 -2.01

-f 30d -2.35 -2.30 -1.64 -2.10 -1.94 -1.84 -2.11

10 1a -1.40 -1.39 -1.29 -1.52 -1.18 -1.29 -1.39 -1.22

2a -1. 33 -1.40 -1.18 -1.56 -1.23 -1.42 -1.41 -1.31

3b -1.52 -1.31 -1.37 -1.39 -1.48 -1.44 -1.29 -1.34

4b -1.40 -1.24 -1.37 -1.37 -1.39 -1.33 -1.24 -1.26

11 1a -2.00 -1.68 -2.18 -1.87 -1.91 -1.96 -1.92

2b -2.06 -1.86 -1.96 -2.04 -1.64 -2.11 -2.14

12 1a -1.49 -1.48 -1.43 -1.55 -1.60 -1.42 -1.62 -1.49

2b -1.54 -1.60 -1.41 -1.51 -1.52 -1.48 -1.43 -1.34

(16)

WHO/BS/83.1402

page 16

Table

4

Cont'd

I:..

a bora tory Prepa ra t ion

and Assay IRP 80/558 H,Identical 81/502 81/565 G,Identical 81/615 Local

68/38 to 80/558 to 81/565 Standard

13 1a -1.44 -1.41 2a -1.49 -1.46 3b

-1.

53 -1.40 4b -1.41 -1.38

14 la -1.31 -1.38 -1.43 2a++ -1.26 -1.32 -1.32 3b -1.35 -1.37 -1.43 4b++ -1.41 -1.56 -1.55 15 1a -2.23 -2.28

2a -1. 91 -2.09 3b -2.04 -1.81 4b -1. 79 -2.04

16 1a 1.35 1.28

2b 1.42 1.40

17 la -1.26 -1.43 2b -1.22 -1.28

19A 1a -2.22 -2.17 -2.14

2b -2.01 -2.04 -1.94 19B 3a -1.69 -1. 72 -1.66

4b ( -1. 52

(-1.50++ -1.60

22 1a -1.74 -1. 70

-1.

79 2b -1.84 -1. 71 -1. 74

++ As in preceding assay after freezing +++ Different diluent

-1.45 -1.39 -1.39 -1.44

-1.53 -1.45 -1.49 -1.47

-1. 52 -1.44 -1.44 -1.42

-1.50 -1.38 -1.42 -1.41

-1.33 -1.34 -1.41 -1.29 -1.41

-1. 21 -1.29 -1.33 -1.30 -1.28

-1.45 -1.40 -1.42 -1.45 -1.43

-1.51 -1.54 -1.49 -1.45 -1.65

-2.33 -2.12 NE -2.06 -2.08

-1.96 -1.04 -2.08 -2.02 -1.88

-2.03 -2.01 -2.48 -2.12 -1.92

-1. 91 -1.87 -2.18 -1.90 -1.85

1.18 1.63 1.49 1.47 1.47

1.34 1.60 1.68 1.47 1.51

-1.26 -1.30 -1.27 -1.32 -1.26

-1.22 -1.28 -1.26 -1.25 (-1 .21 (-1. 23 (-2.12 -2.25 -2.10 -2.16 -2.10 (-1.77 ( -1.69 (-2.10 -2.13 -2.07 -1.94 -2.12 (-2.13 (-1.93 (-1.94 -1.47 -1.67 -1.88 -1.62 ( -1.89 (-1.77

-1.49 -1.61 -1.65 ( -1. 67

( -1 .61

-1.

71 -1.65 -1.69 -1.69

-1.77 -1.81

1.

74 -1.65

(17)

+

solutions from preceding assay stored frozen

(18)

WHO/BS/83.1402 page 18

Tab 1 e 5 co n t'd Laboratory

and Assay C Initially Hypothyroid

17 1a -1.30

2b -1. 24

19A 1a -1. 74

2b -1.67

19B 3a -1.57

4b -1.38

22 1a -1.48

2b -1.61

Preparation

F E

Normal TRH

Pool Stimulated Normal

* -1.18

-1.57 -1.26

- * -1. 73 -0.91

-1.

91

* -1.69

-1.64

* -1.00

* -1.44

Code and Source

B

D A

Toxic Toxic Toxic

Graves Multi -noduler Single Nodule

Disease

~oiter

Goiter

* * *

* * *

-1.21* -0.87* * *

-1.89* * -2.01

-1.22* * *

-1.76*

* * * *

* * -1.51

(19)

Table 6 Comparison of coded duplicates G and H with the identical materials 81/565 and 80/558 respectively

)dule - Laboratory G H

and assay as % of 81/565 as % of 80/558

1A 1a 91.9 128.8

1

2b 180.6

1B 3 213.4 213.4

2 1a 95.8 99.8

2a 94.8 98.1

3a 95.1 101.5

4a 98.3 100.1

5a 97.1 98.6 98.2

6b 110.6 95.5

7b 95.7 98.1

8b 103.2 94.5

9b 104.4 103.7

10b 92.3 91.3

3 1a 90.5

2a 99.0

3a 93.7

4b 98.6 97.1

5b 91.6

6b 109.1

7b 98.0

4 1a 100.2 117.8

2b 82.4 90.9 88.1

5 1a 83.7 92.3

2b 145.2 99.6 98.4

2c 81.3 114. 1

6 1a 100.4 207.0+

2b 97.0 97.1

3b 90.4 96.5 99.4

4b 98.5 97.6

9A1 1a 100.2

2a 96.4

3a+ 95.5 98.1

4b 97.2

5b 101 ,2

9A2 6a

96.8

7a 96.3

8a+ 98.7 98.2

9b 103.2

10b 96.1

9A3 11a 94.9

12a 98.0

13a+ 99,2 98.6

14b 97.0

15b 104.0

98.0

101. 9 101. 2

98.0

(20)

WHO/BS/83.1402 page 20

Table

6

cont'd

Laboratory

G H

and assay as % of 81/565 as % of 80/558

9B1 16c '97.5

17c 96.3

18c+ 96.0 96.7

19d 95.8

20d 98.0

9B2 21c 98.7

22c 120.1

23c+ 99.7 103.5

24d 100.2

25d 100.5

9B3 26c 121. 1

27c 100.4

28c+ 100.1 102.8

29d 96.4

30d 97.8

10A 2a 1a 85.8 86.5

86.~

136.6 141.8 139.2 10B 4b 3b 101.6 99.7 100.6 107.0 105.7 106.3

11 1a 128.9

92.2 I

2b 66.0

12 2b 1a 121. 7 81.1 99.3 128.7 90.8 108.1

13 1a 98.1

I

2a 94.6

100.0

3b 107.9

4b 99.9

14 1a 100.5 94.8

2a 98.2

99.8

103.4

98.0

3b 98.5 97.9

4b 102.2 96.3

15 1a 136.4+

1

2a 143.0+

3b 96.5 101. 1

4b 105.9

16 1a 87.6

95.9 I

2b 105.0

17 2b 1a 109.3 96.4 102.6 I

19A 1a 2b 110.5 114.5 112.5 108.1 107.2 107.6

19B 3a 4b 76.6 76.6 84.0 84.0

22 2b 1a 102.3 77 .9 89.3 101.4 85.2 92.9

+ Results from responses near limiting values; omitted from analysis

(21)

Table 7

~ioassays

of the TSH

preparation~

80/558 and 81/502 agpinst MRC Research Standard A, expressed in .m.units of the latter.

Laboratory and Assay

8 1 2 3 4 18 1 2 20A 1 2 3 20B 1 2 3 4

Prepa ra t ion 80/558 81/502

45.9 39.9

8.11 10.35

37.4 10.39

35.7 11.37

46.4 19.25

46.3 9.54

45.7 19.96

0.06 0.37 280.7

2797.5

(22)

WHO/BS/83.1402

page 22

Table 8 Comparison by immunoassays of the TSH preparations with the IRP, 68/38 expressed as miu per ampoule.

Preparation Laboratory

80/558 H, identical

81/502 81/565 G, identical

and assay to 80/558 to 81/565

1A la 37.4 8.75 12.4 11.4

2b 34.6 6.61 11.5 20.8

1B 3 34.8 5.48 3.7 17.5

2

1a 37.4 37.3 7.48 12.0 11.5

2a 38.8 38.1 7.96 12.2 11.6

3a 39.3 39.8 8.04 12.3 11.7

4a 39.8 39.9 8.18 12.3 12. 1

Sa 38.6 37.9 8.23 12. 1 11.8

6b 39.7 37.9 8.33 11.8 12.1

7b 35.1 34.4 7.28 11.2 10.7

8b 36.1 34.1 7.35 10.5 10.8

9b 37.6 39.0 8.00 11.8 12.3

10b 38.8 35.4 8.36 12.2 11.2

3 1a 41.6 9.07 13.1 11.9

2a+ 34.6 8.40 11. 1 11. 0

3a+ 36.4 7.54 11.7 10.9

4b 41.3 8.61 12.0 11.8

5b 39.4 8.94 13.3 12.2

6b+ 41.7 8.43 12.5 13.6

7b + 42.2 9.17 13.3 13.0

4 1a 21.4 25.3 3.42 6.6 6.7

2b 27.1 23.8 4.32 7.9 6.5

5 1a 36.4 33.6 7.89 11.8 9.8

2b 39.5 38.9 9.42 9.3 13.6

2a+ 28.9 33.0 19.46 11.0 9.0

6 1a 18.5 38.2 7.79 11.5 11.5

2b 40.9 39.7 8.86 12.4 12.0

37.4

3b 35.0 36.0 7.57 11.9 10.8

20.2

4b 37.9 37.0 7.66 11.7 11. 5

81/615 11.8 11.5 8.0

10.9 11.6 11.8 11.9 11.6 11.7 11.2 11.2 11.4 . 11.4

13.2

13.5

16.0

17.3

6.7

6.8

'10.5

7.5

7.8

11.3

11. 5

10.9

11.0

(23)

Table 8 cont'd

Laboratory 80/558 H, i dent i ca 1

and Assay to 80/558

9A1 1a 36.3

2a 36.2

3a+ 35.8

4b 36.5

5b 36.2

9A2 6a 36.1

7a 35.2

8a+ 35.3

9b 35.7

10b 35.4 9A3 11a 34.6 12a 35.1 13a+ 36.4 14b 37.7 15b 35.7 9B1 16c 32.4 17c 32.2 18c+ 35.2 19d 32.2 20d 34.4 9B2 21c 30.7 22c 31.6 23c+ 33.9 24d 32.0 25d 33.9 9B3 26c 31.5 27c 29.8 28c+ 33.0 29d 27.7 30d 31.6

10A 1a 23.1 31.6

2a 22.4 31.8

lOB 3b 43.7 46.8

4b 45.3 47.9

11 1a 40.9

2b 30.0

12 1a 31.4 40.4

2b 34.9 31.7

Preparation 81/502 81/565

7.33 11.4 7.85 11.8 7.62 11.7 7.74 11.6 7.43 11.4 7.77 11.5 7.44 11. 1 7.57 11.3 7.60 11. 1 7.43 11.2 6.70 10.8 7.10 10.9 1. 17 11. 1 7.29 10.8 7.51 11.0

?6.35 11.0 7.02 10.7 7.59 11.6 8.02 11.3

?6.88 11.6

?6.36 10.0

?6.93 10.0

?6.28 10.8

?7.61 11.8

?7.10 11.3

b.89 9.8

6.67 9.4

7.07 10.4

5.72 8.9

7.15 10.6

4.06 8.2

3.91 7.8

8.69 13.9 9.24 14.2 15.45 24.1 15.29 22.0 5.82 10.5 7.27 12.2

G, identical to 81/565

11.5 11.4 11.2

?11.3 11.6 11.2 10.7 11. 1 11.5 10.8 10.2 10.7 11.0 10.5 11.4 10.7 10.3 11.2 10.8 11.3 9.9 10.7 10.8 11.9 11.4 10.8 9.4 10.4 8.6 10.4 7.0 6.7 13.9 14.4 31.1 14.5 12.7 9.9

81/615

11. 5

11.8

11.6

11.3

11. 1

11.3

10.8

11.7

10.9

10.8

10.4

10.6

11. 2

10.5

10.9

7.0

10.3

11.3

11.2

12.0

9.6

12.0

10.3

11.3

11. 1

11.9

9.7

10.2

11.3

10.4

7.6

7.4

13.7

14.6

23.0

21.3

10.6

11.5

(24)

WHO/BS/83.1402 page 24

Table 8 cont

'ct

Laboratory

80/558 and assay

13 1a 37.8

2a 39.2

3b 38.7

4b 38.8

14 1a 39.5

2a+ 39.0

3b 37.6

4b+ 35.6

15 1a 40.8

2a 39.5

3b 41.0

4b 36.2

16 1a 27.4

2b 31.0

17 1a 35.6

2b 49.7

19A 1a 38.0

2b 38.2

19B 3a 39.1

4b 36.4

22 1a 39.7

2b 40.9

H, identical to 80/558

37.4 40.3 36.9 34.3

41.1 41.0 32.8 33.8 41.5

+ Assay of frozen solutions

Preparation

81/502 81/565 G, identical to 81/565

8.00 11.9 11.6

7.79 12. 1 11.5

8.14 11.6 12.5

7.66 11.3 11.3

8.51 11.7 11.8

8.42 12.6 12.3

7.01 11.8 11.7

7.03 11.2 11.5

9.05 14.5 19.8

9.14 13.4 19.2

7.97 13.9 13.4

7.73 11.9 12.6

6.39 16.9 14.8

9.58 18.1 19.0

8.93 13.2 14.4

10.23 15.5 14.9

7.81 11.4 12.6

7.67 10.9 12.5

8.50 12.0 9.2

8.63 11.6

8.76 14.2 11. 1

9.24 13.7 14.0

? Estimate based on counts outside the range 10%-90% bound

81/615

10.9

11.0

12.4

11.2

13.4

12.3

11. 1

10.6

13.5

13.0

11. 5

11. 1

14.2

18.8

13. 1

14.0

11.6

11.0

11.7

10.9

12.6

12.8

(25)

Table 9 Laboratory geometric means of the purified TSH preparations expressed as miu of the IRP per ampoule.

Preparation Laboratory

~0/558

identical to

H

81/502 81/565

80/558

1A 36.0 7.61 11.9

1B 34.8 5.48 3.7+

2 38.1 37.3 7.91 11.8

3 39.5 8.58 12.4

4 24.1+ '24.5+ 3.84+ 7.2 +

5 34.6 35.1 8.62* 10.6

6 37.7* 37.7 7.95 11.9

9A1 36.2

7~59

11.6

9A2 35.5 7.56 11.2

9A3 35.9 7.15 10.9

9B1 33.3 7.15 11.2

9B2 32.4 6.84 10.8

9B3 30.7 6.68 9.8

10A 22.7 31.7 3.98+ 8.0+

10B

44.5+ 47.3+ 8.96 14.0

11 35.0 15.37+ 23.0+

12 33.1 35.8 6.50 11.3

13 38.6 7.90 11.7

14 37.9 37.2 7.71 11.8

15 39.3 8.45 13.4

16 29.1 7.82 17.5+

17 42.1 9.56 14.3

19A 38.1 41.0 7.74 11. 1

19B 37.7 32.8

8.56

11.8

22 40.3 37.5 9.00+ 13.9+

Weighted geometric 36.2 7.78

mean

95% confidence 35.2 7.55

interval 37.1 8.01

Unweighted geometric 35.4 7.52

mean

95% confidence 33.4 6.74

interval 37.4 8.40

identical to G 81/615 81/565

15.4 11.6

17.4 8.0

11.6 11.5

12.0 14.9

6.6 + 6.7+

10.6 8.5

11.4 11.2

11.4 11.5

11.1 11. 1

10.8 10.7

10.9 11.2

10.9 10.8

9.9 10.7

6.8+ 7.5+

14.1 14.1

21.2 22.1

11.2 11.0

11.7 11.4

11.8 11.8

15.9 12.2

16.8 16.3

14.6+

13.5

12.5 11.3

9.2 11.3

12.5 12.7

11.5 11.4

11.3 11.1

11.7 11.7

11.7 11.4

10.8 10.3

12.7 12.6

+ Estimate contributed excessively to

JC2

for heterogeneity and is not included in the weighted geometric mean

* Omitting presumed dilution error

~.----.---- - -- ---~--

(26)

WHO/BS/83.1402

page 26

Table

10

Laboratory geometric means of the ampoule content of the proposed IRP,

80/558

expressed in terms of the various local standards for immunoassay.

Laboratory

1A

18

2 3

4 5 6

9Al 9A2 9A3 9Bl

982

9B3 10

11

12

13 14

15 17

19A 19B

22

Estimated content of one ampoule of

80/558

42 u 41 u

42 u 1l2*u

20

u

1. 05

ng (?factor of

10)

33 u 33 u 3'6 u

28

u 25 u 42 u 15 u

40 u 35 u

33

u 20 u

l2

u 43*u

* Reported. Data fof own standard not sent.

Standard

Highly purified from Calbiochem-Behring

Amerlex TSH reference standard Clinical Assays hTSH (no further

deta

il

s) In-house standard (no details) TSH Hennen

68/38

NnL 08-019 (no further details)

Purified TSH ex-Parlow, calibrated vs.

68/38

Own standard calibrated in terms of 68/38

68/38 supplemented plasma Information not given

68/38

Preparation no. 031182 (no further information given)

In-house standard - no details TSH-supplemented serum, calibrated in terms of

68/38

NML Ki t Amersham kit

TSH supplemented serum, calibrated

in terms of 68/38

(27)

Table 11 Calibration of ·the ampouled TSH preparations in terms of mill; ampoules of the proposed IRP, 80/558, per ampoule

Preparation

Laboratory 81/502 81/565 identical to

G

81/615

and assay 81/565

1A 1a 234 211.4 331 332.0 304 427.4** 314

2b 191 333 601 334

1B 3b 157 157.2 107 107.4** 503 503.5** 230

2

1a 200 320 307 292

2a 205 315 299 299

3a 205 314 299 302

4a

205 308 303 298

5a 213 207.7 314 308.7 305 306.1 300

6b 210 281 329 294

7b 207 319 305 318

8b 204 291 300 311

9b 213 313 326 303

10b 216 314 290 294

3 1a 218 315 285

2a+ 243 323 319

3a+ 207 321 301

4b 208 217.1 291 314.2 287 304.7 319

5b 227 338 309 341

6b+ 202 299 326 384

7b+ 217 315 308 410

4

1a 160 160.0 310 300.3 311 272.6 315

2b 160 291 239 250

5 1a 217 324 269 289

2b 239 227.7 237 308.4 344 306.1 190

2a+ 673++ 382 310 272

6 1a* 204 300 301 295

2b

217 207.9 303 310.0 294 299.0 280

3b 209 330 298 302

4b

202 308 303 290

323.8 230.6

301.0

361.8

280.6 246.3

291.6

(28)

WHO/BS/83.1402

page 28

Table 11 cont'd

Laboratory and assay

9Al 1a 2a 3a+

4b 5b 9A2 6a 7a 8a+

9b lOb 9A3 11 a 12a 13a+

14b 15b 9B1 16c 17c 18c+

19d 20d 9B2 21c 22c 23c+

24d 25d 9B3 26c 27c 28c+

29d 30d

81/502

202 217

213 209.7 212

205 215 211

215 212.8 213

210 202 193

197 198.9 193

210 196 218

216 215.0 249

200 207 220

185 211.3 238

210 219 224

214 217.9 207

226

Preparation 81/565

G

identical to 81/565

315 316

327 316

327 320.4 312 3i4.4

317 308

316 320

320 310

314 303

319 316.2 315 310.7

311 321

317 305

311 295

312 306

304 304.1 302 300.1

287 279

307 320

338 330

333 321

331 337.7 318 326.7

350 335

337 330

326 322

315 379

318 332.2 317 344.3

370 371

335 337

313 379

315 317

313 319.3 314 328.5

321 309

335 328

81/615

318 326

324

316 • ...:

308 306 312 306

333 312.0 306

304 300 303

309 299.2 279

306 216 320

320 305.9

348

348

312 338

:;02 ',< -;(" ~L 1 .. ) .. l

352 329 342 325

309 34

~

.;::

,~(j9

329

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