CLIN. CHEM.

31/8,1264-1271 (1985)

Guidelines for Immunoassay Data Processing1

R. A. Dudley,2 P. Edwards,3

R. P. Eklns,3 D. J. Flnney,4 I. G. M. McKenzie,4 G. M. Raab,4 D. Rodbard,5 and

R. P. C. Rodgers6
These guidelines outline the minimum requirements for a
data-processing package to be used in the immunoassay
laboratory. They include recommendations on hardware,
software, and program design. We outline the statistical
analyses that should be performed to obtain the analyte
concentrations of unknown specimens and to ensure adequate monitoring of within- and between-assay errors of
measurement.

AddItIonal Keyphrasee: statistics

programs
data processing

Third, machine computation

.

quality control

computer

The authors of this paper were convened as a group by the

International
Atomic Energy Agency to make recommendations for assayists and laboratory managers on good practice
in data processing for radioimmunoassay
and related techniques. They were requested to identify those computational
procedures that are appropriate,
especially in a hospital
laboratory
providing a routine service, and to establish
priorities as to their importance.
It is timely to examine this topic. In recent years the
dramatic increase in power and decrease in price of computing hardware have brought the capability of machine computation within the reach of all laboratories,
either as an
integral part of a sample counter or as an independent
device. Many programs have been designed, thus testing a
diversity of approaches but subjecting the user to a bewildering choice among possibilities.
In the opinion of this
group, most commercially available programs for analyzing
immunoassays
lack essential features. Indeed, several prograins that have been developed for programmable
calculators (1,2) show more sophistication
than those supplied as
black box systems by many manufacturers
of beta- and
gamma-counters.
The group agreed on several general principles. First, all
aasayists can benefit from the computational
and statistical
1 This article
should be regarded as the composite view of a
committee of experts, convened by Dr. B. A. Dudley under the
auspices of the International Atomic Energy Agency, Vienna,
Austria. It is neither an official position of the IAEA nor a formal
policy of the AACC. However, it should serve to stimulate thinking
by all in the RIA and immunoassay fields. We hope it will
contribute to an improvement in the overall quality of software
systems for these kinds of analyses.
2 International
Atomic Energy Agency, Wagramerstrasse 5, P.O.
Box 100, A-1400 Vienna, Austria.
3Department of Molecular Endocrinology, Middlesex Hospital,
London, U.K.
4
of Edinburgh, Edinburgh, U.K.
Institute of Child Health and Human Development,
National Institutes of Health, Building 10, Room 8C413, Bethesda,

MD 20205.

6University of California, San Francisco, CA.

Received February 5, 1985; accepted May 31, 1985.
1264

procedures that a good program can offer.Indeed, the less

statistically
experienced
the user, the more he stands to
gain by such assistance. Second, while one goal of computation obviously is to derive the concentration
of analyte in the
samples measured, the main advantages
of machine computation include automation,
speed, improved
accuracy
(through avoidance of gross errors), and detailed statistical
analysis and accounting of sources and magnitude of errors.

CLINICALCHEMISTRY,Vol.31, No.8, 1985

should never be thought

to

relieve the analyst of responsibility

for the reliability of his
measurements;
all it can do is provide results that are
computationally
sound, that are relevant to the assessment
of reliability, and that are displayed in the most comprehensible manner. Fourth, the creation of suitable programs is a
major task that can be accomplished
successfully
only
through the combined efforts of professional analysts, statisticians, and programmers.
Finally, although the user himself need seldom know the details of the mathematical
analysis, it is improper that proprietary
secrecy should
conceal the basic strategy, algorithms,
equations, and assumptions.
Two restrictions
adopted by the group as to the scope of
this paper should be stressed. First, it is not a general
critique of quality control. Although data processing is one
key element in quality control, the latter field embraces
much more than data processing. Second, being directed at
the practicing assayust and laboratory manager, the paper
does not offer detailed algorithms such as would be required
by a programmer.
Instead, we seek to explain the type of
analysis that is desirable in programs, with some approximate mathematical
amplification
in appendices to sharpen
the concepts.

Hardware and Software

The choice of a computer system for a particular laboratory will depend on several factors. The most important
factor is that the system be able to run a data-analysis
package that at least meets the minimum requirements
set
out here. Secondly, in terms of capacity and speed it must be
able to handle the volume of work done in the laboratory.
In all cases the system should be tailored to fit the needs
of the laboratory and be regarded as a piece of equipment
that is as essential as a gamma counter or a centrifuge. Our
minimum recommendation
would be a computer with 48K
of memory and one or more disk drives. For two reasons we
do not consider programmable
calculators
in any detail
here. First, the best of the existing programs are near the
limit of their capacity (1,2) and, second, the calculators can
now be replaced by very-low-cost microcomputers.
The development
of adequate software is a difficult and
time-consuming
task and should not be undertaken
lightly.
If possible, good existing software should be adopted. Unless

the user is willing to accept a program that will be usable

only during the lifetime of a particular machine, the following criteria should be met:
Programming
language: The program should be written
in a standard version of a well-established
programming
language (e.g., FORTRAN, BASIC, or PASCAL) and in general
should avoid machine-specific
enhancements.
However,
some machine-specific
features will often be necessary and
some enhancements
may be desirable.
Modularity:
The program should consist of well-defined
modules, each performing a few specific tasks. This allows
existing modules to be replaced by new modules if better
algorithms become available, and modules containing new
features can easily be added. All machine-specific
features
and enhancements
should be placed in well-defined and
documented modules so that they can be readily adapted to
new hardware or new operating systems.
Operating
systems:
The problem of moving from one
machine to another may be minimized by a good choice of
operating system. Some operating systems can be used on
machines of different power, and a judicious choice allows a
laboratory to upgrade its computer requirements
with minimum dislocation. Examples of such systems are CP/M, MS/
DOS, the ucun p-system, unix, and PICK.
Documentation:
Program documentation
should always
be clear and detailed. Internal documentation
should be
adequate to facilitate program modification. All algorithms
should be publicly available so that they may be examined
and, where necessary,
criticized. A manufacturer
unwilling
to do this should provide a full description of the methods
used, including appropriate references to the literature and
specimen analyses of several real data sets that illustrate all
the features of the program. Good documentation
on the use
of the programs and on the interpretation
of the output is
essential.
Graphics: The use of high-resolution
graphics is perhaps
the only place where the use of machine-specific
enhancements to standard programming
languages is justifiable,
because good graphical output is frequently
clearer than
any other representation
of information.
However, such
output should always be included in separate modules, and
alternative
replacement
modules,
with low-resolution
graphics produced by a standard printer, should be available. In the absence of an adequate graphical output, the
alternative
of alphanumeric
output should be made available.

Input and Output

Input of Responses
The word response is used in this report for the quantitative measurement
obtained for each sample of standard or
test preparation.
For techniques that involve radionuclides,
the response is counts; for other techniques, it might be
the reading from a spectrophotometer
or some other instrument. Input of responses by direct link from the counter or
other device or by machine-readable
media, such as paper
tape or data-logger cassette, should be the norm for routine
assays. Such an approach eliminates operator error during
data entry. Even so, some errors can occur, and the program
should check that the data have the expected format and
magnitude. Occasionally, manual data entry is unavoidable;
a thorough check that the entry is correct is then essential.
If the computer is connected directly to the measuring
device, the responses should be accumulated
in a ifie and
analyzed as a batch once the assay is complete. This makes
data correction easier and allows more satisfactory
error
analysis and quality-control
procedures.
Moreover, comput-

ing resources will usually be used more efficiently if data

collection can proceed while the computer is being used for
other purposes. A printed copy of the stored data ifies must
always be available for inspection.

Input of Assay Configuration

and Instructions

For the responses to be analyzed, the program will need

information
on the concentration
of the calibration
standards, dilutions of the unknowns, and the order in which the
responses are being entered (assay configuration).
In addition, instructions
are required
on the type of analysis
required. The program should allow some flexibility in the
assay configuration,
which includes the identification
of
quality-control
pool samples (see the section on betweenbatch quality control). The provision of standardi.zed configurations for certain types of assay is useful, so that only the
number of test samples need be entered (3).

Output
The details of output from specific parts of the program
are dealt with in the appropriate section below. Care should
be taken to avoid unnecessary
output, which reduces the
impact of important information.
Numbers should be written in a readable form without the use of exponential
format, and the use of numeric codes or cryptic mnemonics
should be avoided. Instead, clear, concise messages in ordinary language should be used. If the result obtained from a
statistical test on the data from an assay is satisfactory,
a
simple message may be all that is required. However, more
information
should be available
if a test fails or if the
operator requests the information. It may be useful to store
such detailed information in a disk file for later inspection if
necessary.

Analysis of Responses from a SIngle Assay

Batch
General
Computer programs for the analysis of assay responses
fall into two classes. Programs
of the first type take a
manual (usually graphical) analysis as their starting point.
The routines are designed to mimic the procedure that a
technician might use with a ruler or a flexicurve. Examples
are linear interpolation,
smoothing techniques,
and some
uses of spline functions. Programs of the second type base
the analysis on a statistical
model of the assay, and thus
lead to an assessment
of errors of measurement.
We are
unanimous in recommending
a program that uses a statistical model.
The basic statistical model of an assay assumes that the
response from a particular tube has two components:
#{149}
The expected response for the tube, which depends only
on the amount of analyte in the tube. This is the average
response that would be obtained if a very large number of
measurements
were made for a given dose of the analyte.
The relation between expected response and dose is called
the dose-response curve or calibration curve.
#{149}
A random component due to variability in experimental
procedures and measurements,
which will have an average
value of zero across a large set of measurements.
It will not
be satisfactory to assume that this component has a constant standard deviation at all levels of the response. In
most cases, the size of this random component will increase
with the level of response. A formulation of how the standard deviation (or some other measure of variability)
deponds on the mean response is known as the response-error
CLINICAL CHEMISTRY, Vol.31,No. 8, 1985 1265

relation, or RER.7 Details of how the RER affects immuneassay results have been published (4, 5, 8).
This is an idealized model of an assay, in which the
response in one assay tube is unaffected by the responses in
neighboring tubes; there are no mishaps that have produced
completely erroneous results for certain tubes; no systematic effects are present, such as drift due to time or carrier
position; and the expected response is determined
only by
the concentration
of the analyte being measured.
However,
in practice, satisfactory assays can come close to this ideal.
Any serious departure
from this idealized model of the
assay system may lead to results that are incorrect and
subject to much greater uncertainty
than the calculations
imply. Thus an essential part of the analysis of an assay
batch is to check that the responses
are consistent with
these assumptions about the assay. The extent to which this
will be possible will depend on the assay design, which is
beyond the scope of this paper but has been discussed
elsewhere
(6, 7). The steps described in the section on steps
in the analysis
below suggest how the analysis of an
assay batch can proceed,
incorporating
checks on the assumptions. We have presented them in one possible sequence, but alternative
orderings
are possible.
...

1/ A family of curves in which individual members are

defined by the numerical values of very few (preferably only
four) parameters.
2/ Flexibility of shape, slope, and position to suit the
requirements
of standard
assay techniques.
3/ Monotonic form (i.e., no reversals of slope), and restraint from making detours for outliers.
The four-parameter
logistic curve appears to be the
most generally useful and versatile model that will satisfy
the above requirements,
although nothing said here implies
that it is right and all others wrong. Its parameters
characterize:
(a) expected

count at zero dose, not necessarily identical

with any one observed count (Figure 1);
(b) slope factor, related to rate of change of count with
increasing
dose;
(c) the dose expected to give a count halfway between a
and d, i.e., the EC or IC;
(d) expected count at infinite dose (high-dose plateau or
asymptote),
not necessarily
identical with a count for nonspecific binding.
The general form of equation then is:
Expected

response

at dose

d +

+(dose/c)

Recommended Models
Dose-response
curve. Numerous models have been sugA standard program should embody one acceptable
general-purpose
model; a more sophisticated program might
include alternatives
occasionally needed for special circumstances.
Important
features of a model suited to wide use in
routine assays are:
gested.

Abbreviations

used in this paper:

B0, counts observed for zero dose; B/B0, normalized

response

variable, ranging from 0 to 1, representing

counts bound above
nonspecific, relative to counts bound above nonspecific for zero dose
of analyte. Also commonly expressed as a percentage, %B/B0, on a
scale from 0 to 100%.
B/F, bound-to-free ratio for labeled ligand.
BIT, bound-to-total ratio for labeled ligand.
%CV, coefficient of variation, as a percentage of the mean.
a, b, c, d: parameters of the four-parameter
logistic model, with
a = expected response at zero dose of analyte;
b = slope factor or exponent, with absolute magnitude equal to
the logit-log slope;
c = EC or IC, i.e., concentration of analyte with an expected
response exactly halfway between a and d;
d = expected response for infinite analyte concentration
(often,
though not always, synonymous with nonspecific counts bound).
nASA,
enzyme-linked immunosorbent assay.
EMrr,
enzyme-multiplied
immunological technique.
IRMA. immunoradiometric
assay (generic name for assays involv-

ing labeled-antibody reagents).

J, exponent utilized in power function model for response-error
relationship.
NSB, nonspecific binding.
r, number of replicates.
RER, response-error
relationship, commonly expressed as vanance of the response as a function of expected level of response, e.g.
=

ao y.

RMS, root mean square error. For unweighted regression, the

standard deviation of a point around the fitted curve. For weighted
regression, the ratio of observed error to predicted error, based on
the particular weighting model utilized.
sd,standard
se, standard

deviation.

We recommend that the RER be expressed as a product of

two terms. The first term will be a constant-i.e.,
independent of the level of response-for
a particular assay batch (or
for a subset of the batch if standards
and unknowns are to be
treated
separately). The second term will describe how the
standard deviation of the responses varies with the level of
LOG,0
-I

COUNTS
BOUND
B

CLINICALCHEMISTRY, Vol.31, No.8, 1985

COUNTS
2

3
FR!(

8/T

Bo 2.000

B/U.
I #{176}

10,000

IO000

--.,.1eN..

PEDjo

-I 6

#{176}0

2,000

e000

4.000

6,000

4.000

.2

N 2.000

error.

y, slope.
residual mean square between doses.
5, mean square within doses.
w, weight for observation i.
z, estimate of log(dose).
1266

This curve (Figure 1) satisfies all the desiderata and is

adequate for the vast majority
of existing immunoassay
systems. It is continuous and smooth, and the slope factor is
very stable in repeated
assay batches. It can approximate
closely the simple mass-action equation (8). One additional
parameter
for asymmetry
is easily incorporated
to give
even greater versatility
(9, 10).
Response curves are further discussed in Appendix 1, and
greater detail can be found elsewhere (11, 12).
Response-error
relation (RER): Detailed modeling of experimental
errors and their sources is not required. A
program should include a simple formulation of how the
variance or the standard deviation of the random component
of the response
varies with the mean response level. This
will usually require no more than two parameters
(e.g., a
power function, a linear or quadratic relationship
for the
standard
deviation or the variance as a function of expected
response).

le

:
.#{176}!!

0IO

!!!

d.O.(flO.Oec,GccJ

.iei
I

Fig. 1. Schematic

16,000
18.000

20,000

1,

I
10

DOSE

-19-

00

1000

22,000.1

S (104 scsI.)

drawing of a dose-response (calibration) curve

Note smooth, symmetrical sigmoidal shape, characterized by four parameters (a.

b, C, c. Confidence limitstaper in a smooth, consistent manner. Reproduced,
with permission, from ref. 29

the response,

and this relationship

will remain fairly conacross a series of batches (assays). This implies that
the scale of the random errors in the responses may differ
between batches, but that the shape of their relationship to
the mean response would be similar in each batch (4, 5).
An appropriate
shape for the dependence of the standard
deviation on the mean response should be determined from
the data for a series of assays. This information is fed back
into the program either:
#{149}
by the user entering a few numbers (preferably just one)
calculated from a series of assays, or
#{149}
preferably, by the computer performing this automatically by utilizing stored information from previous batches.
Poisson errors due to counting may be estimated directly
from the number of counts and the RER can, if desired, be
specified without this component. The counting error can
then be added to the estimated RER to obtain the total error
(13). Details of how to characterize the RER are given in
Appendix 2.
stant

Steps in the Analysis of a Single Batch

Adjustment
and verification of responses. Certain procedures may yield responses that require adjustment
before
calculation starts. For example, variable counting efficiency
as a result of variable
quenching
in liquid-scintillation
counting gives a response that must be corrected before
other calculations.
Other adjustments,
however, such as
variable recovery in preliminary
separation
procedures,
must be made after the concentrations have been estimated.
The computer
program should be designed
to handle
any
such procedure that the laboratory methods demand.
It may be useful to display any apparent anomalies in the
data-such
as serious discrepancies
among replicates
from
the same sample or inconsistent standard responses-before
the main analysis is undertaken.
Error correction can be
done at this stage, but a record of any changes should be
sent to the quality-control
file (see below) and must be
recorded on the output.
Screening of replicate sets. When some or all of the
specimens have been measured in replicate, the RER may
be estimated from the scatter of the sets of replicates, which
may thus enable individual outliers to be detected. The
term outlier is used with two different meanings
in
immunoassays.
Firstly, the term outlier has been used to
denote a member of a set of replicates that is dramatically
further from the set mean than would be expected from the
estimated HER, and presumably
indicates a blunder. We
use it in this sense here. Secondly, the term is used for the
case when all the responses for a concentration
of standard
deviate from their expected value. This case is dealt with in
the section on testing goodness-of-fit.
The first step is to use the data from the replicate sets to
estimate the HER. Where the shape of the HER can be
assumed to be constant over a series of assays, only the
multiplying
constant
needs to be found. This is easily
estimated
as a weighted combination
of the individual
variances. Practical difficulties
can arise if any gross outliers are present in the data. Robust techniques,
such as
taking medians within sub-groups (5) or a modification of
the method proposed by Healy and Kimber (14), should
prevent these extreme sets from influencing the estimates.
After the RER is estimated from all the data, the scatter
of individual
sets of replicates about their mean can be
compared with what would be expected from the RER. All
sets where the ratio of observed to expected variance is
greater than some value that would be very unlikely to
occur by chance (say, p <0.005) can be classified as outliers.
Such sets would ordinarily
be discarded.
A permanent

record must be kept of all rejected specimens, and their

occurrence must be printed on the output. Various options
for automatic and manual rejection are possible, and the
program may allow each laboratory to specify its own rules.
After these outliers are excluded, it will usually be
desirable to calculate and display the RER values separately
for standards and unknowns and to test whether they are
consistent. In exceptional circumstances the unknowns and
standards may be subject to different sources of error, for
example, when the unknowns undergo some preliminary
processing. A sophisticated
program would handle the two
estimates of the HER and use whichever is appropriate at
each stage of the calculations.
Fitting the dose-response curve. Fitting of the calibration
curve ought to be performed in terms of the actual quantities independently measured and observed, namely, doses
and responses. Output may be expressed in terms ofB/T,
B/B0, B/F, or other familiar derived quantities.
The response curve should be fitted to the raw counts
obtained from the calibration
standards
by the method of
iteratively reweighted nonlinear least squares. The weights
should be obtained from the HER. An appropriate algorithm
would be the Gauss-Newton
method or the MarquardtLevenberg modification of this technique (15, 16). Robust
regression methods are also acceptable.
Initial estimates of parameters are needed to start the
calculations; they are easily built into the program, either
by reference to values in past runs of similar assays or by
rough initial computations
(e.g., based on the logit-log
method or even simpler approximations,
using the dose
having a response close to 50% B/B0 as the initial estimate
of c).
Testing goodness-of-fit.
The program must perform an
analysis of variance on the responses for the standards,
weighted in inverse proportion to the variance given by the
HER. It must conform to standard statistical methods for
weighted least squares, producing weighted mean squares
and degrees of freedom for:
11 the scatter of mean responses (at individual standard
doses) about the fitted calibration
curve, averaged over all
standards (i.e., residual mean square between doses, sf).
2/ the scatter of responses on replicate standard tubes
about their respective means, averaged over all standards
(i.e., mean square within doses, 4).
The variance ratio 4/4 examines whether the model
chosen for the dose-response
curve gives an acceptable fit to
the responses from the standards. The residuals for means of
individual standard doses help to pinpoint where the lack of
fit occurs. The program should calculate the Studentized
residual
(17) for the mean count at each dose of the
standard; a residual >3.0 warns of poor fit at that dose. A
plot of the standard responses and the fitted curve resembling Figure 2 will be a useful diagnostic tool for the
assayist. It will help to reveal whether there is a systematic
lack of fit-perhaps
caused by an unsuitable choice of model
for the dose-response
curve-or
whether responses at one or
two dose levels are grossly out of line. The latter situation
corresponds
to the second usage of the term outlier,
meaning inconsistent
results from one standard dose relative to other doses, rather than a response inconsistent with
other replicates at a single dose.
Automatic rejection of all the responses from one standard
dose is not recommended,
there seldom being enough doses
to make such a procedure reliable. A minimum of eight dose
levels is recommended.
Manual intervention
should be
allowed to reject an occasional standard dose (but never
more than one) on the basis of a mixture of statistical
grounds and laboratory experience. Excessive manual rejection, however, will lead to biased, over-optimistic estimates
CLINICAL CHEMISTRY, Vol.31,No. 8, 1985

1267

-1

Logl(X)

Fig. 2. Computer generated plot ofthestandardcurvedata,thefitted

logistic
curve,andthe 95% confidence limits for a single observation,
(excluding
uncertainty inthe position ofthecurve)
From IBM-PC RIA program ofM. L Jaffe

of precision.
Finally, the program

should produce a table showing the

of analyte in the standards when
these are treated as if they were unknown
specimens.
Comparison of these results with the actual concentrations
of the standards will suggest how much bias a misfitting
dose-response
curve might introduce into the estimates for
the unknowns. A statistically significant lack of fit, especially in assays of high precision, may be sufficiently small that
the results will remain useful for their intended purpose.
Presentation of precision profile. The above estimation of
the HER provides the program with an estimate of the
random error at any level of response. The combination of
this estimate with the slope of the fitted dose-response
curve permits one to obtain an estimate of the random error
in the estimated concentration of an unknown (Appendix 3).
A representation
of this derived variability
in the estimated
concentration of unknowns against analyte concentration is
known as the precision profile. (The term imprecision
proffle is also used, to emphasize that a larger percentage
coefficient of variation
indicates poorer precision.)
This
should be presented either as a graph (Figure 3) or as a table
of expected precision (e.g., the percentage
coefficient
of
variation or standard error for a test preparation) vs concenapparent

concentration

tration.

0.1

10
hCG (ngimi)

100

1000

Fig. 3.Precision profile: %CVforan unknown specimenmeasured in

duplicate
(ordinate)
plotted
vs serum analyteconcentration
(logscale)
on the abscissa (3(
The effects of uncertainty in the positionof the standardcurveare not included
in
this example. Also shown: empirical within-batch %CV(#{149})
andbetween-batch
%CV (o)for three quality-controlpools,
analyzed
in triplicate
in each of the past
20 batchesor assays
1268

CLINICAL CHEMISTRY, Vol.31,No. 8,1985

Precision profiles can be calculated for a single measurement for the unknown,
or for the mean of duplicate or
triplicate measurements.
The error in estimating the response curve may or may not be included in the errors used
to calculate the precision profile: this option should be
specified. The program should generate a table or graph of
the precision proffle for the number of replicate measurements and the sample volume ordinarily
used for the
unknowns. The program should also provide an estimate of
the lowest level of reliable assay measurement.
A statistical
estimate of the minimal detectable concentration (18) is
recommended. Alternatives
recently described by Oppenheimer et al. (19) may be useful.
Estimation of concentration for test samples (unknowns).
The program must provide an estimate of the concentration
of each unknown sample and a measure of its precision. The
precision may be expressed as an estimated
percentage
error in the result (% CV) from the precision profile, or as
95% confidence limits. A warning when the estimated error
exceeds a certain threshold (e.g., 10%) is useful, and may be
all that is required for certain applications.
When a sample is analyzed in replicate at a single
dilution, the concentration
corresponding
to the mean response is read from the calibration curve and corrected for
sample dilution. The estimated
precision at this level of
response is used to assign confidence limits or a %CV to the
result.
lithe sample has been included at two or more dilutions, a
combined estimate of concentration should be obtained from
all the responses by using a weighted average, which gives
greater
influence
to the doses that lie within the region of
the curve where estimation
is more precise. Again, estimates of precision should be obtained for this combined
estimate. When two or more dilutions are included, the
program can test whether the concentrations obtained at
different parts of the response curve are consistent. This is a
generalized test of parallelism. An outline of these calculations is given in Appendix 4.
For each test sample, the minimum
output
from the
program should be an estimated concentration,
along with
warning messages about outliers, lack of parallelism, or
poor precision. For outliers or lack of parallelism,
the
estimates from individual responses or individual doses will
help the assayist to interpret the results.
Evaluating assay drift or instability.
Appreciable drift in
responses from the same sample placed in different positions
in the assay batch may seriously invalidate the estimates of
precision described above. Information
will be available
from replicate counts from standards or unknowns placed in
different parts of the sequence of test samples. Tests of
possible distortion of results caused by systematic drift can
be based upon these replicate responses (or upon the corresponding estimates of concentrations). The particular test
adopted will depend on the assay design, but will usually
consist of a regression analysis or an analysis of variance
with suitable weighting. A combined test should make use
of all the available data. Any apparent
drift should be
evaluated in terms of its effect on the estimated concentrations, so that its importance may be assessed.

Between-Batch Quality Control

One important goal of data analysis is to reveal whether
performance is consistent in a series of assay batches.
Automated data analysis is essential if a sufficiently broad
set of indicators is to be followed without excessive labor.
An archive should be maintained
for the most important
indices of consistency, including the results from qualitycontrol pools, the parameters of the dose-response
curve, the

RER, the weighted root mean square error, and a summary

of rejected data.
Although subsequent analysis of results from qualitycontrol pools may be carried out by a separate program, this
should not be optional. Recommendations
on the placing of
the quality-control
pools in the assay batch and their
disposition at different analyte concentrations
have been
made elsewhere (20), although guidelines are somewhat
arbitrary.
Use of at least three quality-control pools, with
different analyte concentrations, is recommended.
The results from these pools should be analyzed for trends
or sudden shifts between batches. The analysis should
include the use of control chart methods, mean squared
successive differences, or other appropriate tests for randomness. Details of these methods are discussed in standard
texts (21, 22) and their application to immunoassays
has
been discussed (23-25). Similar tests can also be applied to
other features of the assay such as the parameters of the
standard curve, but these will be of secondary importance.
The program should provide tests that combine information from all the quality-control pools. These test whether
all the pools are changing in a similar manner. In their
simplest form they can be performed by applying the tests
above to the mean of all the quality-control pools; weighting
according to the estimated precision of each pool is desirable.
Assessment of between-batch precision. The quality-control specimens are the only source of information on between-batch precision, which is the essential measure that a
clinician needs for comparing results obtained on different
days. The between-batch
precision includes components
from within-batch
errors and between-batch errors.
The program should compute the estimated
betweenbatch precision for each quality-control
pool. Two components of between-batch
imprecision can be predicted from
the responses from a single batch. The first is the withinbatch variability for each specimen. A second is due to the
uncertainty
in the position of the fitted dose-response
curve
as a result of the variability in responses for the standards.
Both of these components can be estimated from the data
from a single batch, although the second component, which
is relatively small, is part of the between-batch variation as
assessed from the quality-control
pools. The results should
be presented in a manner that compares within- and between-batch precision, estimated from the quality-control
pools, with the precision proffle of the current batch or with
a pooled estimate from several recent batches (Figure 3).
None of these efforts excuses the analyst from participation in a well-designed inter-laboratory quality-control program.
We thank the International Atomic Energy Authority for sponsoring the meeting that led to this paper, and the Department of
Molecular Endocrinology, Middlesex Hospital, for acting as hosts.
We also thank M. L. Jaffe for supplying Figure 2.

AppendIxes
1. Response Curves
The logistic model is applicable to immunoassays
in
which bound or free counts, or both, are measured. (BIT or
B/B0 may also be used as response variable,
although
counts are preferable.) In addition, this method is applicable to assays that involve enzyme-labeled
antigens (e.g.,
EMIT) or antibodies (EusAs);
to labeled-antibody assays (twosite iiu&s, or sandwich-type assays); to assays involving
fluorescent, chemiluminiscent,
electron spin resonance, or

bacteriophage
labels; and to radioimmunodiffusion
methods.
It is also applicable to many receptor assay systems and to
several in vivo and in vitro bioassays. Though some assay
users may be accustomed to-and
thus prefer-other
types
of curves and methods of fitting, they are urged to consider
seriously the procedures described here as the basis of a
general approach.
The logistic response curve is not applicable when the
curve consists of a summation of discrete sigmoidal components, or to non-monotonic curves (with reversals of slope),
or in cases of severe asymmetry (when plotted as response
against log dose). Should such response curves arise, other
methods, either derived from the mass-action law or based
on empirical models (but, unfortunately,
with more parameters) will be necessary. No one method of curve fitting is
likely to be optimal in all circumstances,
nor is there
sufficient experience with some of these assay techniques for
general proposals to be made. For unusual
curves, the
assayist would need to seek special assistance
from colleagues experienced in mathematical modelling and statistical curve fitting. In addition, he should examine the
system experimentally,
to evaluate whether this anomalous
behavior could be removed without damage to assay performance.
A program should provide the option of reducing the
number of fitted parameters by regarding
the expected
response for infinite concentration
as constant, perhaps
constrained to be equal to the experimentally
determined
mean response for nonspecific binding (NSB). In some
assays, however, the expected response for infinite concentration may differ markedly from the observed response for
NSB. This indicates that either the model is unsatisfactory
or the method for measurement of NSB does not give an
appropriate
estimate of the corresponding
plateau or asymptote. In both cases the NSB responses should be excluded. Similar problems are less likely to occur for very low
concentrations
(approaching
zero). However, the assumption that the infinite concentration parameter is known
exactly-or
that the NSB counts can be ignored-should
be
made cautiously and never merely as a convenience. In all
circumstances
the program should prevent any estimation
for samples beyond the highest standard
concentration,
other than an explicitly approximate
one only for the
purpose of indicating the need for an appropriate dilution of
sample before re-assay.
Additional parameters may be incorporated into the logistic ftmction to allow for marked asymmetry
(6, 9, 10).
Estimation of the asymmetry can be difficult, and it may be
advisable to fix the value of the asymmetry parameter, on
the basis of data from several consecutive batches.
The ability to detect lack of satisfactory goodness-of-fit
for a simple curve (e.g., a four-parameter logistic) improves
as the precision of the assay improves. Thus, failure of the
four-parameter logistic to fit may be encountered in exceptionally precise assays. Likewise, increasing the number of
replicates or increasing the number of dose levels leads to
significant improvement
in ones ability to detect lack of fit,
and hence may introduce the need to utilize more complex
models. Conversely,
when experimental
errors are large,
replicates are few, and the dose levels are few and far
between, simple models are likely to be adequate; i.e., the
error of the estimate introduced by an incorrect model for
the standard curve is small relative to the uncertainty
in the
response of the unknown sample itself.
When both the low-dose response and high-dose plateaus
are fixed at arbitrary values (e.g., mean values of B0 and
NSB), then the logistic method becomes identical with the
logit-log method, and the magnitude of the slope factor (b)
CLINICAL CHEMISTRY, Vol.31,No. 8, 1985 1269

is identical with the slope of the logit-log plot.

The logistic method is a generalization
of, and an
improvement
on, the logit-log method. It will be satisfactory for many assays in which the logit-log is unsatisfactory.
Those who currently use the logit-log method should consider changing to the use of this more general and flexible
method.

2. Details of Response-Error

Relationship

Various forms of the response-error

relationship are
possible, but an exponential model is recommended that has
the form: variance at response
(expected response).
When J = 0, the variance is constant; when J = 1, the
variance is proportional to the response; when J = 2, the
%CV of the response is constant.
This model is preferred to others because the value of J is
usually quite stable from assay toy.
For most immuneassays J is very nearly 1. The computational
routines
require only a knowledge of J and will estimate the proportionality constant for each assay. One of the published
methods for estimating J from a series of assays (4, 5, 26)
should be available. This method should be implemented in
a robust manner, not easily perturbed by outliers.
When one is using this form of response-error
relationship, the background counts or NSB responses must not be
subtracted from the observed counts before processing.
Alternative
models (1, 4, 5, 26) will also be acceptable,
with similar method of calculation.

3. Calculation of Unknown Concentrations and Their

Confidence Limits
The outline of the calculations given here ignores the
contribution
to the error (component of variance) in the
estimate of the unknown
concentration from the curvefitting procedure, and involves approximations
especially
near the asymptotes. These details are intended to explain
the basis of the method. An ideal program should use
methods that do not involve these approximations
(6, 8).
1. A single dose for the unknown sample: Calculate the
mean response for the (r) replicates of the unknown. Interpolate the corresponding log dose from the fitted curve at this
point and add to this the logarithm of the fraction by which
it has been diluted to get the log of the estimated concentration (z). Calculate
the slope (y) of the response plotted
against log dose at this point. The estimated
standard
deviation (ad) of a single response at this point on the curve
is obtained from the predicted RER. The standard error of
the estimated log dose at this point is then
(sd)I\

This standard error can be used to calculate a confidence

interval for z and the corresponding (arithmetic) dose, and a
%CVfor the dose estimate.
2. More than one dose of an unknown: Proceed as for a
single dose to get values zj, (sd)1, (y)1, r1 for each dose. The
weight for each dose is then given by
r

W1

and the combined estimate

with estimated
1270

,WjZj

w1

standard

(yl)2

(sd),2

of log dose by

w1z1 + w2z2 + w3z3 +

w1 + w +

W3 +

error

CLINICAL CHEMISTRY, Vol.31, No. 8, 1985

XL X Xu

Iog,(dose)
dose

Fig.4. Illustration of the principles involved inthe estimation of the

standard error or%CV for an unknown: as an approximation, CV =
se(Iog6 (i)) = se/Iyi
=
(sd/Vt)/IyI,
where y = slopel =
A1o90(4; alternatively, se(s) = se /Islope(, where se = s4 /VF and
slope= dydx

W2 W3 +

An approximate
test of whether the estimates
at the
different dose levels are compatible (generalized
parallelism) is obtained by calculating the quantity
(z1

z)2

(zj

z)2 +

z)2 + (z3

As a first approximation, this quantity should follow a chisquare distribution with degrees of freedom = n
1, where
n = number of doses; any large departure from its expected
value of n
1 would be a basis for suspicion.
-

4. Calculation of Precision Profiles

For any dose (x), we can calculate the expected response
and hence, from the response-error
relationship,
the estimated standard deviation of a single response at this point
on the curve. The next step is to divide by
to obtain the
standard error of the mean response for r replicates. The
component of error for uncertainty
in the fitted curve may
be added in here (in terms of variances).This is desirable,
especially when the curve is based on only a few dose levels.
Usually, this component of variance should be quite small.
This standard error is then divided by the slope of the
response curve, and the estimated coefficient of variation
(%CV) calculated (Figure 4) (23,24,27-30).
it is convenient
to use the approximation
that the CV of dose x, expressed as
a decimal fraction, is equal to the standard error of loge (x).

vi

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Vol.31,No. 8, 1985 1271