25 Clinica Chimica Acta, 60 (1975) 25-32 0 Elsevier Scientific Publishing Company, Amsterdam -Printed

in The Netherlands

CCA 6905

SEMI-AUTOMATED RADIOIMMUNOASSAYS THYROXINE AND TRIIODOTHYRONINE

G.S. CHALLAND, Radioimmunoassay

W.A. RATCLIFFE Unit, Stohhill

FOR TOTAL

SERUM

and J.G. RATCLIFFE

General Hospital,

Glasgow G21 3UW (U.K.)

(Received September 21, 1974)

Summary Single stage semi-automated radioimmunoassays for total serum thyroxine (T4) and triiodothyronine (T3) are described which employ an automatic pipetting station, automatic gamma counter, and a programmable calculator with paper tape reader and printing facility. Both assays require only a small volume of unextracted serum, and are specific and sensitive. Their sample capacity, precision, speed, and cost are comparable with the measurement of serum protein-bound iodine. Both assays therefore have significant advantages over previous methods for the assessment of thyroid function in the diagnostic laboratory. A simple method of automating the calculation of results is described, which is applicable to any radioimmunoassay in which the standard curve is approximately linear on a plot of the free/bound fraction against the antigen concentration. In addition, a general method is reported which reveals the relative contributions of intrinsic, systematic, and random error to radioimmunoassay precision.

Introduction The assessment of thyroid status in vitro requires the measurement of circulating thyroid hormone levels. Competitive protein binding assays and radioimmunoassays have now been described [l---16} which have advantages of specificity and sensitivity over previous methods such as protein-bound iodine. Currently, however, such methods are labour intensive and are thus unsuited to clinical biochemistry laboratories with large workloads. We report here singlestage semi-automated radioimmunoassays for total thyroxine (T4) and triiodothyronine (T3), which match the speed, precision, cheapness, and sample capacity of the automated measurement of protein-bound iodine while retaining the advantages of specificity and sensitivity.

26

Methods

A Micromedic automated pipetting station, Model 24002, adapted for duplicate sampling of sera, was used in conjunction with a Micromedic high speed automatic pipette, Model 25006 (Chemlab Instruments Ltd, Ilford, Essex). The volumes dispensed by Micromedic pumps are readily varied, and the combination of 50 ~.tland 1 ml pumps on the Station, and a 100 ~1 pump on the Automatic pipette enabled sera to be sampled in duplicate, and all reagents to be added simultaneously to the assay tubes for both assays. Sera were sampled by the 50 1.11pump, and Reagent 1 (see Table I) was dispensed from the 1 ml pump, both pumps being adjusted to the appropriate volume. Reagent 2, containing the antiserum, was dispensed from the 100 ~1 pump on the Automatic pipette acting in conjunction with the Pipetting station. All reagents could be added to 430 assay tubes in one hour by this method. T4 radioimmunoassay Thyroxine antiserum was raised in rabbits as described by Ratcliffe and co-workers [7], and was used at a final dilution of 1 : 3000. [’ ” I] T4 of specific activity 20.--50 #&‘~g was from the Radiochemical Centre, Amersham. The sodium salt pent~ydmte of thyroxine (Sigma Chemical Co. Ltd) was used as standard. &Anilinonaphthalene-l-sulphonic acid (ANS), obtained from Sigma, was used to inhibit the binding of thyroid hormones to serum binding proteins in both T3 and T4 assays. The separating agent was polyethylene glycol 6000, (PEG), obtained from British Drug Houses and used at a final concentration of 13%. T3 rad~oimmu~oassay

Triiodothyronine antiserum was raised in rabbits as described by Ratcliffe and co-workers [16], and was used at a final dilution of 1 : 2500. ,[I *’ I] T3 (Abbott Laboratories) of specific activity 40-70 pCi/pg or specific activity of up to 600 ,uCi/iug was used. Triiodothyronine (free acid, from Sigma) was used as standard. A second antibody separation method using donkey anti-rabbit serum (RD17 from Burroughs Wellcome) at a final dilution of 1 : 96 and carrier non-immune rabbit serum at a final dilution of 1 : 2000 gave maximum precipitation of the bound fraction. Standards

and quality controls

Standards for each assay were prepared in bulk by adding weighed amounts of T4 and T3 to T4-free or T3-free serum prepared from pooled serum by charcoal treatment [ 121. Commercial quality control sera, or pooled human sera were included at the beginning of each assay. In addition a pooled serum from euthyroid patients was included at regular intervals throughout each assay to monitor assay drift. All standard sera and quality control sera were stored as aliquots at -20°C. Assay procedures

Assay constituents for both T4 and T3 radioimmunoassays Table I. Assay procedures are summarised in Table II.

are shown in

27

TABLE

I

ASSAY

CONSTITUENTS

FOR

SEMI-AUTOMATED

T4

Serum

(standard

Reagent and

1 (Tracer, separating

or sample) ANS

RADIOIMMUNOASSAYS

T3

Radioimmunoassay

50 !.I1 850

~1 of

Tracer

2 (Antiserum)

T3

20 /ll T4

~1 of

(500

pg)

Tracer

ANS

(320

pg)

ANS

PEG

(106

mg)

Donkey

in diluent

buffer

buffer

8.6

pH

bovine Reagent

AND

Radioimmunoassay

680

agent)

T4

100

(0.05

M barbitone

containing albumin)

~1 of T4

antiserum

(1

:

375)

volume

of incubate

800

buffer

buffer

8.6

pH

100

serum (0.05

containing albumin)

11 of T3

antiserum rabbit

:

(1

82)

M barbitone

serum

1.00

~1

pg)

in diluent

Non-immune

buffer

50 pg)

anti-rabbit

in diluent Total

(

(100

bovine

serum

in diluent

0.1%

T3

0.1% (1

:

serum

250)

(1

:

200)

buffer

ml

Analysis of results 1. Verification of the standard curve For both assays, the plot of the free fraction divided by the bound fraction against antigen concentration [17,18] (the F/B plot) gave standard curves which were close to linear, shown in Figs 1 and 2. Little distortion was therefore involved if each standard curve was considered as a series of straight lines connecting adjacent points, and this linear interpolation method was used to derive results automatically using a programmable calculator. However, before results were calculated, the standard curve was submitted to automated verification in an attempt to exclude any unsatisfactory standard points, that is, standard points which lay some way from their expected position. After input of the series of standard values and the counts corresponding to each, the F/B fraction was stored for each standard point. The gradient of the line through each pair of standard points was usually close to the gradient between the neighbouring pair. More than 20% difference in gradient was considered unsatisfactory. This arbitrary cut-off imposed tight limits on each stanTABLE

II

ASSAY

PROCEDURES

FOR

SEMI-AUTOMATED

T4

AND

T3

Radioimmunoassay

Incubation

overnight

Separation

Centrifugation

at 4’C

minutes

or 3 h at 37’C

at 1000

Precipitate seconds

RADIOIMMUNOASSAYS

T3

at 4’C

Supernatant Counting

T4

for

30

Radioimmunoassay

overnight

at 4’C

as for

assay

T4

X g.

aspirated counted

for

on Wallac

30-60

Autogamma

Counter

Precipitate (T3 30-60 600

counted

tracer

seconds fiCi/ug)

analysis

and

By paper

of results

Counter Model

from

Autogamma

Hewlett

Packard

10 programmable

calculator Model

tape

fed into

via Hewlett

9863A

paper

Packard tape

reader

as for

T4

(T3

assay

2-4

minutes

pCi/ug) tracer

on Wallac

Counter) Calculation

for

of 40-70

or of

Autogamma

28

dard point, usually equivalent to less than 1% difference in the bound fraction. If a bad standard point was identified, the calculator derived a new point from the equation of the regression line around that section of the standard curve. In addition, standard points were examined by calculating a weighted difference between duplicates, discussed below. If significant disagreement occurred between duplicates, that standard point was rejected and a new point was derived by linear interpolation between the adjacent standard points. 2. Calculation of results Random errors were detected from the difference between the observed counts of radioimmunoassay duplicates, weighted to allow for counting error. Weighted difference

(First count - Second count) A =J(First count + Second count)

At least 95% of all duplicates for both assays usually fell within a A value of modulus 3.5. A A value of greater than modulus 3.5 was taken to indicate random error in duplicates. If this occurred in the standard curve, the standard point was rejected; in the assay run, the sample was repeated. If the A value was within acceptable limits for a duplicate pair, the result was calculated automatically from the mean count by linear interpolation using the F/B plot of the smoothed standard curve derived by the calculator. Results were printed using the Hewlett Packard calculator’s alpha printer facility. 3. Verification of results The following methods were used to verify results. i. Calculation of the midpoint of the standard curve, that is, the hormone concentration corresponding to half the difference between antibody and diluent blank counts. ii. Examination of the results of quality control samples run early in the assay. iii. Examination of the results of drift control samples placed every fourteen samples throughout the assay. iv. Plotting a histogram of the A values obtained during the assay run. The incidence of random error was indicated by the number of outliers (A values greater than modulus 3.5). The relative level of intrinsic error was revealed by the spread of A values. The presence of systematic error in the handling of duplicate assay tubes was shown by a shift in the mean A value away from zero. Results Standard curves and comparison with manual methods Standard curves for T4 and T3 assays obtained by the semi-automated method (Figs 1 and 2) closely resembled those by manual methods [16]. T4 results, y, obtained by the semi-automated method gave good correlation (r = 0.96) with those by the manual method, x(y = 1.04 x -.- 0.01). Similarly, T3 results, y, by the semi-automated method correlated well with those obtained manually, x(r = 0.95; y = 0.85 x + 0.10). The lower absolute T3 levels

29

T4 Fig.

CONCENTRATION

1. Standard

curve

(n moler’l~tre)

for the

T4

74 CONCENTRATION

(n dcr/l,tre>

radioimmunoassay

obtained by the semi-automated method resulted from the use of serum standards. In the manual method, standards were prepared from a stock solution of T3 in diluent buffer, with T3-free serum added immediately before assay. Several batches of serum standards assayed by the manual method showed a discrepancy from their expected value corresponding to the shift in absolute T3 values observed above. Precision and quality control Fig. 3 shows a quality control chart for the T4 assay for 16 consecutive assays over an eight week period. Over this period the mean between-batch coefficient of variation for all quality control samples was approximately 8% in both T4 and T3 assays. Fig. 3 also shows the standard curve midpoint for the T4 assay. In general this remained approximately constant, but shows a change at Assay 5, probably due to the introduction of a new batch of T4-free serum. Drift occurred in both T3 and T4 assays. The mean drift in the T3 assay is shown in Fig. 4. Drift was not eliminated by keeping both reagents on ice throughout the assay, nor by keeping samples capped until immediately before sampling. When drift was less than lO%, which usually occurred when assay

00.2

1.0

13 CONCENTRATION Fig.

2. Standard

curve

39

10

30

(n moks/litre> for the

T3

radioimmunoassay.

0

5

10

13 CONCENTRATION

15

20

Cnmoledlitrs)

25

30

130

.

High pool 125. MIddIe

pool

120 x _ z 3 P

Curve

modpant

/

115. 110

0 6

105

G

100.

ii 95 4

7

10

Assay

Fig. 3. Quality

control

, 16

13

I, 0

25

Number

50 Number

75 of

100

125

150

sampler

chart for the T4 radioimmunoassay.

Fig. 4. Drift in the T3 radioimmunoassay. the mean result (? S.E.M.) for successive of the first drift control result.

Drift results were collected drift control results through

over ten successive the assay is plotted

assay runs, and as a percentage

runs involved less than a hundred samples, its effect on results was ignored. However, when drift was more than lo%, results were corrected accordingly. Fig. 5 shows histograms of A values for a semi-automated T4 assay and for a manual assay. For the semi-automated assay (Fig. 5b), the distribution of values was close to that expected from counting error alone. The few outliers indicated that random errors were uncommon and the mean value close to zero suggested that systematic error was absent. For the manual assay (Fig. 5a) there was a higher incidence of random error, the mean A was not zero indicating that systematic error was present, and the spread of A values was wider than for the semi-automated assay, suggesting that intrinsic error was larger.

a) MANUAL ‘50

b)SEMI-AUTOMATED

ASSAY

h4eana:-0~9 SD.A:2.2

)5l

h&n

Fig. 5. Histograms of the weighted semi-automated T4 assay.

d

ASW

: -0.1

difference

between

duplicates.

A. (a) for a manual &%a~,

and (b) for a

31

Discussion Radioimmunoassays for total T4 and T3 have rapidly established their place in the evaluation of thyroid function in vitro [19]. Automation of these assays is essential in dealing with large numbers of samples rapidly, precisely, and cheaply since current manual methods are demanding in analyst time and skill. Full automation of radioimmunoassays has in general proved difficult [20], and semi-automated systems have been described for few assays of clinical importance (e.g. insulin, human growth hormone, human placental lactogen) [21,22]. The choice of a suitable separating agent is a critical feature of a semiautomated assay. The separating agent, PEG, in the T4 assay has significant advantages over other methods [16] . Thus, it is convenient, gives apparently complete separation of bound and free fractions and is cheap. For the semiautomated T3 assay, the second antibody method has advantages over other separating agents such as Charcoal, Dowex resin and PEG [ 161. The incorporation of all assay constituents and separating agents in only two assay reagents allows these assays to be set up in one operation, significantly reducing analyst time. In addition to the automation of pipetting operations it is important to simplify labour intensive operations such as the calculation of results. The linear interpolation method used here is simple and has the advantage that no assumptions are made about the shape of the standard curve, thus avoiding the distortions which occur when curve-fitting procedures such as the logit-log transform [23] are applied to these assays. The method is optimised by choosing standard curves which are close to linear (the F/B plots) and by identifying outlying standard points. Random errors in standards and samples are identified by the calculation of the weighted difference, A, between the observed counts of duplicate tubes, and rejection of duplicates in which the A value exceeds certain limits (which depend on the levels of intrinsic and systematic error in the particular assay). A also allows recognition of the contributions of different types of error to overall assay precision, and enables easy identification of methods giving best assay precision. This simple technique has not previously been described, and is generally applicable. Automated calculation of results reduces clerical errors and analyst time. For the T4 radioimmunoassay an assay run of fifty samples (with a total counting time of two hours) can be completed and results made available within the working day. Alternatively, with a workload of 500 samples weekly, one junior technician for each assay can easily produce results the following day. The single stage semi-automated assays for T4 and T3 described here are as sensitive and specific as manual assays and offer the same diagnostic accuracy. Only small serum volumes are required, so that the assays are well suited to paediatric practice. The costs of the assays are comparable with the measurement of PBI. These assays therefore have significant advantages, summarised in Table III, over previous methods for the assessment of thyroid status in the diagnostic laboratory.

32

TABLE

I11

COMPARISON

OF

DIFFERENT

METHODS

FOR

MEASURING

CIRCULATING

THYROID

HOR-

MONES Arrows

are used

to indicate

relative

amounts.

PBI

T4

by

CPB

T4

by

semi-

T3

automated

RI.4

Specificity

+

+

+

Sensitivity

+

+

+

r

4

I

T

1

+

+

Sample

size

t

Sample

capacity

I

Cost

1

i

Diagnostic

semiRI.4

1

+

accuracy

by

automated

A fuller description of the programs written calculator is available on application to G.S.C.

for the Hewlett

Packard

Acknowledgements We acknowledge

the excellent

technical

assistance

of Mrs D. Cooper.

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