Clinical Chemistry 44:1

116 123 (1998)

Automation and
Analytical Techniques

Statistically accurate estimation of hormone

concentrations and associated uncertainties:
methodology, validation, and applications
Martin Straume,* Michael L. Johnson,1 and Johannes D. Veldhuis
ple, common assay data reduction procedures used in
clinical and research laboratories often disregard the
experimental uncertainties in the calibration curve replicates, the fitted parameters of the calibration curve, the
replicates of unknown samples, and (or) the variability in
zero-dose calibrators. Beyond intrinsic physical imprecision in the instrumentation (e.g., time-resolved fluorescence or photon counting), the foregoing sources of experimental variation impart finite uncertainties to each
unknown sample determination. Such uncertainty should
be known to a reasonable degree of accuracy to distinguish true assay sensitivity from low-end assay noise, i.e.,
the apparent blank of the assay, and also for numerous
subsequent applications of the assay results. For example,
calculation of endogenous or exogenous hormone kinetics, endogenous secretion rates, and statistics based on the
regularity of hormone pattern reproducibility typically
rely on variably weighted nonlinear fits or Monte Carlobased estimation of asymmetric confidence intervals for
parameters with within-sample uncertainty predictions.
The latter are commonly estimated from duplicate, or
occasionally singlet or triplicate, measurements of the
unknown sample analyte concentration.
Here, we present a comprehensive effort to define
sample uncertainty based on combined experimental variations inherent in: (a) replicates of the calibration curve;
(b) replicates of the zero-dose calibrator; (c) uncertainty in
the calculated calibration curve parameters; and (d) replicate measurements carried out on unknown samples
(e.g., duplicate, triplicate, etc.). In assigning response
uncertainty estimates, however, we depart from the conventional approach in which some theoretical variance
function is used to attempt to analytically relate response
variance to assay response [16]. Instead, because the
distribution of response variance to assay response is so
highly variable and therefore generally poorly analytically determined [16], we adopt an approach in which
response errors are estimated on a case-by-case basis via
an empirical procedure. And, because our means of
estimating response errors is empirical, we present exten-

We describe a data reduction procedure to assign statistically accurate estimates of unknown hormone concentrations, with associated uncertainties, based on experimental uncertainties in sample replicates and the fitted
calibration curve. Three mathematical calibration curve
functions are considered. The one providing optimal
statistical characterization of reference calibrators is
chosen for unknown evaluation. Experimental error is
addressed by assigning and propagating uncertainty
estimates for each measured response (including zerodose responses) by an empirically determined discrete
uncertainty profile and by propagating calibration curve
uncertainty. Discrete uncertainty profiles account for
both response precision (replicability) and accuracy (deviation from predicted calibration curves) without relying on assumed theoretical response varianceassay
response relations. The validity of assigning variable
response weighting by this procedure was assessed by
Monte Carlo simulations based on chemiluminescence
growth hormone calibration curves. Much-improved
accuracy and estimated precision are achieved for unknown hormone concentrations, particularly extremely
low concentrations, by using this variable response
weighting procedure.
Since the development of RIAs in the early 1970s and their
widespread application to clinical chemistry, enhanced
sensitivity and precision have been achieved by nonradioactive indicator techniques such as fluorometry and
chemiluminescence. Empiric fitting of the calibration
curves so generated has not always kept pace with the
enhanced physical precision of these methods. For exam-

Department of Internal Medicine, Division of Endocrinology and Metabolism, National Science Foundation Center for Biological Timing, Gilmer Hall,
University of Virginia, Charlottesville, VA 22903.
1
Department of Pharmacology, University of Virginia Health Sciences
Center, Charlottesville, VA 22908.
*Author for correspondence. Fax 804-982-4505; e-mail ms3g@virginia.edu.
Received May 1, 1997; revision accepted September 5, 1997.

116

117

Clinical Chemistry 44, No. 1, 1998

sive empirical validation of our concept of error propagation via Monte Carlo simulations, which examine the
properties and behavior of this data reduction procedure.
The basis for the Monte Carlo simulation experiments is a
set of highly consistent assay data from 14 growth hormone (GH) chemiluminescence assays [7], wherein high
sensitivity and well-defined experimental uncertainty estimates were desirable.2

Methods
To accommodate the largely monotonic increases or decreases in immunologically based calibration curves, we
initially evaluated three empiric algebraic functions, with
the forms given below. These functions include modifications of the widely used four-parameter logistic function
[1, 8, 9] and a modified four-state Adair expression.

three calibration curve functions

Three functional forms are considered for analyzing hormone assay calibration curves.
The most flexibly accommodating monotonically sigmoid function of the three is a modified four-state Adair
expression (referred to as MONOTONE):

MOD4P 2 ^R i& 5

Y` 2 Y0
^R i& 5 Y 0 1
N

D O
j51

11

j~@H# i/j
N
j51

Here, ,Ri. is the estimated assay response of reference

calibrator i, Y0 is the response at zero hormone concentration, Y` is the response at infinite hormone concentration, the Oj are fitting parameters, [H]i is the concentration
of reference calibrator i, and N may assume values of 1, 2,
or 3 (defining the order of fit). Order N 5 3 is automatically tried first. If unable to fit to the N 5 3 condition, an
order N 5 2 fit is automatically tried next. If unable to fit
to the N 5 2 condition, an order N 5 1 fit is automatically
tried finally.
A second function is a modified four-parameter logistic
function [1, 8, 9] (referred to as 4PARMS):
4PARMS 2 ^R i& 5

A2D
1 1 ~@H# i/10

logC 10 logB

1D

A is the response at zero hormone concentration, D is the

response at infinite hormone concentration, and logB and
logC are fitting parameters. Fitting to the logarithms of
parameters B and C obviates estimating unrealizable
negative values.
A third function is a modification of 4PARMS (referred
to as MOD4P):

2
Nonstandard abbreviations: GH, growth hormone; SSR, sum of squared
residual; and LH, lutropin.

~1 1 @H# i/10 logC ! 10

logB

1D

estimating assay response error (empirical

discrete response uncertainty profile)
Replicate-based assay response uncertainties (response
SDs) are estimated for each response at every reference
concentration. The process is performed iteratively in
parallel with successive evaluations of calibration curve
parameter values. The first estimation of calibration curve
parameter values is performed with unit-weighted responses at each calibrator dose. A model-independent
discrete response uncertainty profile is then calculated at
each reference concentration i by first calculating the
root-mean-square response deviation, SDi, relative to the
expected response predicted by the current calibration
curve, ,Ri., as

10 O j)j

~@H# i/j 10 O j)j

A2D

All calibration curve parameter estimations are performed by a modified GaussNewton nonlinear leastsquares parameter estimation algorithm [10, 11] to a convergence criterion of ,1026 relative change in variance
of fit.

MONOTONE 2
N

SDi 5

O ~R 2 ^R &!
ni

ij

j51

ni

1/ 2

where ni is the number of replicate responses at concentration i, and Rij is the jth response at concentration i.
Distance-proportional nearest-neighbor smoothing is then
used to generate smoothed estimates for a discrete uncertainty profile, ,SDi., by smoothing interior points as
^SDi& 5

~^R i11 & 2 ^R i&!SDi21 1 ~^R i& 2 ^R i21 &!SDi11

1
SDi 1
2
^R i11 & 2 ^R i21 &

and endpoints as
^SD1 & 5

~^R n& 2 ^R 1 &!SD1 1 ~^R n& 2 ^R 2 &!SD2

~^R n& 2 ^R 1 &! 1 ~^R n& 2 ^R 2 &!

^SDn& 5

~^R n& 2 ^R 1 &!SDn 1 ~^R n21 & 2 ^R 1 &!SDn21

~^R n& 2 ^R 1 &! 1 ~^R n21 & 2 ^R 1 &!

Here, the indices 1 and n refer to the lowest and highest

reference concentrations, respectively.
Repeated rounds of calibration curve parameter estimation are then performed with variably weighted assay
response values based on the current estimated response
uncertainty profile. Iterative, parallel estimation of calibration curve parameter values and response uncertainty
profiles is continued until approximately no change in
calibration curve variance of fit is observed between
rounds. Typically, the relative change in variance of fit
has been observed to be less than ;1026 after 10 11
rounds of estimation. The protocol is currently imple-

118

Straume et al.: Estimation of concentrations and uncertainties

mented by using a fixed number of 30 coupled iterative

rounds of estimation.
At the conclusion of these 30 rounds of estimation, the
uncertainty profile is multiplicatively adjusted so as to
produce a final calibration curve variance of fit of unity,
so that uncertainty estimates for calibration curve parameter values can be then evaluated.

uncertainty in the calibration curve

At the conclusion of the last calibration curve parameter
estimation (to variably weighted assay response values
that produce unit variance of fit), approximate nonlinear
asymmetric joint confidence limits are evaluated for each
calibration curve model parameter at a confidence probability level of 68.26% (the probability corresponding to 1
SD) according to
WSSR~ a 9!
p
511
F~ p, n 2 p, 1 2 prob!
WSSR~ a !
n2p
Here, a is the maximum likelihood vector of calibration
curve parameter values, p is the number of parameters
being estimated, n is the number of calibration curve data
points, prob is 68.26%, F is Fishers F-distribution, a9 is a
vector of parameter values statistically different from a at
probability level prob, and WSSR refers to weighted sum
of squared residuals.
Vectors a9 (4p of them) are sought by searching each
parameter dimension bidirectionally as well as by searching both directions along each axis of the p-dimensional
hyperellipsoid given by
~ a 9 2 a ! T H T H~ a 9 2 a ! # p s 2 F~ p, n 2 p, 1 2 prob!
s2 5

WSSR~ a !
n2p

where the elements of HT H, the Hessian or information

matrix, are given by
, a ! SC~@H# , a !
3
O s F SC~@H#
G
a
a
n

~H T H! jk 5

i51

22

Here, the summation is over all n data points in the

caibration curve, si is the estimated response SD for
reference concentration [H]i, and the partial derivatives
are of the calibration curve function, SC, with respect to
the jth and kth fitting parameters, aj and ak, respectively.
The 4p sets of parameter values, a9, identified in this
way constitute an approximate mapping of a 68.26%
constant probability contour in the p 1 1-dimensional
calibration curve parameter-variance space. Estimated
SDs of derived hormone concentrations in unknown
samples are then generated by calculating concentrations
corresponding to each of the 4p 1 1 sets of identified
parameter values (the vector a and the 4p vectors a9) for
each observed response as well as at the observed response 6 the estimated response SD (as estimated from
the discrete response uncertainty profile). One-half the

difference between maximum and minimum calculated

concentrations is recorded as the estimated hormone
concentration SD.

combining concentration estimates and

uncertainties in multiple-replicate samples
The above description applies to any single assay replicate. Most unknown samples are assayed in duplicate, or
sometimes as higher-order replicates. Mean hormone
concentrations of multiple-replicate samples, ,[H]., are
calculated as variance weighted means [12]

O
^@H#& 5
O
n

i51

~@H# i/ s i2 !

n
i51

~1/ s i2 !

where the summations are over all n replicates, [H]i is the

hormone concentration estimate for replicate i, and si is
the corresponding single replicate hormone concentration
uncertainty. The joint experimental hormone concentration uncertainty associated with multiple-replicate means,
smean, is calculated from the individual replicate hormone
concentration uncertainties, si, as [12]

s mean 5

1
n
i51

~1/ s i2 !

calibration curve assay response conditions for

monte carlo simulations
Fourteen highly consistent chemiluminescence GH calibration curve data sets [7] were the basis for the conditions outlined below and used in Monte Carlo simulation
experiments. A broad range of simulation conditions was
examined to validate the data reduction protocol and to
elucidate the performance characteristics to be expected
when analyzing calibration curves constructed with one,
two, three, four, or five replicates per reference concentration. Additionally, for each number of replicates, three
data reduction methods were examined with (a) variably
weighted assay responses (via the above discrete response

Conditions for Monte Carlo Simulations

[GH] reference
calibrator (mg/L)

Chemiluminescence
response (relative
light units)

Target
response SD

0
0.0025
0.01
0.04
0.12
0.4
1.33
4.6
13.3
45

620
730
1080
2390
5800
16 500
44 000
98 000
146 000
180 000

50
50
90
270
700
2000
5000
10 000
15 000
19 000

119

Clinical Chemistry 44, No. 1, 1998

uncertainty profile), (b) uniformly weighted responses,

and (c) uniformly weighted responses excluding zerohormone-concentration calibrators.

monte carlo simulation experiments

One thousand synthetic calibration curve data sets were
produced with one, two, three, four, and five replicates
per reference concentration to simulate the desired assay
performance reported in the preceding table. Gaussian
distributed assay response values with the above specified means and SDs were randomly generated by summing 12 uniformly distributed random variables in the
range 0 to 1 and subtracting 6 (producing a standard
normal deviate with zero mean). This standard normal
deviate was multiplied by the specified target response
SD and added to the corresponding chemiluminescence
response value to produce a value for inclusion in the
synthetic calibration curve data set being constructed.
Each of the 5000 calibration curve data sets was subjected to nine data reduction analyses. The functions
MONOTONE, 4PARMS, and MOD4P were applied to
each data set in which assay response values were
weighted either variably, uniformly, or uniformly excluding zero(es). For each of the three response weighting
schemes, the function producing the smallest absolute
sum of squared residuals (SSRs) was selected as the
preferred model. (Absolute SSRs refers to the SSRs of the
fitted curve to the calibration curve response values when
applying unit weight to each response value.)
For each of the 5000 calibration curve data sets, an
additional single-replicate data set was randomly generated as described above. This data set was treated as an
unknown set of assay responses to which data reduction by the selected calibration curve analysis was applied. Estimated hormone concentrations (and associated
uncertainty estimates, in variable weighting scheme analyses) were recorded and summarized to characterize the
performance of these data reduction protocols.

Results
calibration curve analysis of a synthetic gh
calibration curve
Simulations were based on 14 well-characterized GH
chemiluminescence assays [7]. Fig. 1 illustrates our comprehensive calibration curve analysis with the discrete
response uncertainty profile applied to five uniformly
distributed replicates.

Fig. 1. Calibration curve with corresponding discrete response uncertainty profile (inset).
Synthetic calibration curve input data for this example were of uniformly spaced
quintuple replicates representative of the conditions replicated during Monte
Carlo simulations as taken from selected data reported in Chapman et al. [7].
The best fit (lowest absolute SSRs) was obtained with 4PARMS.

a tendency for variable (vs uniform) weighting to favor

fitting with MOD4P, although the MONOTONE function
was still the most adopted function in about two-thirds of
the fits.

prediction of assay (gh) concentrations

Figure 2 illustrates the prediction of GH concentrations by
the three different weighting schemes. Predictions are
shown for one to five simulated replicates. Median and
68.26% confidence intervals are shown compared with
different target ranges. Variable weighting reduced experimental uncertainty at the lowest hormone concentrations
and in the zero-dose calibrators.
Table 1. Model selection results.
Best-fit analysis, %
Weighting scheme

Uniform w/o zeroes

Uniform

model selection results

Based on 5000 simulated calibration curves, Table 1
summarizes the best-fitting functional forms. Of the calibration curves, 6290% were best fit via the MONOTONE
function (lowest absolute SSRs), and 9 28% by MOD4P. A
minority (1.312%) showed optimal fitting via the
4PARMS model. All of the 5000 replicated calibration
curves, independent of numbers of replicates per dose,
were fitted by at least one of the three models. There was

Variable

No. of
replicates

MONOTONE

4PARMS

MOD4P

1
2
3
4
5
1
2
3
4
5
1
2
3
4
5

77.6
81.9
86.3
87.5
89.5
79.4
82.5
85.8
87.8
89.4
69.3
62.1
61.9
63.3
62.3

5.5
2.2
2.3
1.4
1.7
4.8
2.1
2.2
1.3
1.9
11.7
9.8
10.8
11.3
11.8

16.9
15.9
11.4
11.1
8.8
15.8
15.4
12.0
10.9
8.7
19.0
28.1
27.3
25.4
25.9

120

Straume et al.: Estimation of concentrations and uncertainties

Fig. 2. Vertical lines show 68.26% confidence ranges

obtained from distributions of hormone concentration
estimates produced by 1000-cycle Monte Carlo simulations.
Points superimposed on the lines represent median and high and
low 68.26% confidence limit concentrations. Each of the three
analytical protocols was examined under conditions in which
synthetic calibration curve data sets were constructed with one,
two, three, four, or five replicates per reference concentration.
Horizontal lines in each graph correspond to expected hormone
concentrations. Negative concentration estimates are not disallowed. They emerge as a consequence of reflecting response
values, R-, that are below Y0 (in MONOTONE) or A (in 4PARMS and
MOD4P) back into the theoretical calibration curve in proportion to
Y0 2 R- or A 2 R- and assigning the derived value as negative.

estimated hormone concentration standard

deviations

calibration curve analysis of high-replicatenumber caibration curves

Figure 3 shows estimated hormone concentration SDs for

variable weighting calibration curve analysis based on
1000 Monte Carlo simulations at each replicate level. For
GH reference concentrations ,1.33 mg/L, increasing replication number beyond singlets increased the reliability
of experimental uncertainty estimation as evidenced by
the narrower range of [GH] SDs at higher replicate
numbers. This effect of sample replication was lost at high
GH concentrations. The horizontal lines in Fig. 3 correspond to SD estimates obtained directly from evaluating
the distributions of hormone concentration estimates produced by the simulations (as in Fig. 2). The generally
higher SDs produced by the variable weighting protocol
reflect the additional uncertainty introduced to concentration estimates as a result of considering the error in the
calibration curve itself.

Figure 4 shows results of high-replicate-number calibration curve analysis of a GH chemiluminescence assay (GH
Chemi) and a lutropin (LH) IRMA. For each set of
calibration curve data, analysis was performed by using
each of the three calibration curve functions in which
assay responses were weighted variably, uniformly, and
uniformly excluding zeroes. For both the GH Chemi and
the LH IRMA, the MONOTONE calibration curve function provided the lowest absolute SSRs for each of the
three assay response weighting schemes. Plotted in Fig. 4
are the three resulting MONOTONE calibration curves
for each weighting scheme. The three curves are nearly
superimposable in each case, but do deviate somewhat at
the lowest (zero) hormone concentration, particularly for
the GH Chemi. The error bars on the points and the plots
of estimated assay response error (insets) are those for the

Clinical Chemistry 44, No. 1, 1998

121

Fig. 3. Vertical lines show 68.26% confidence ranges

obtained from distributions of individual estimated hormone concentration SDs produced by the variable weighting analysis in 1000-cycle Monte Carlo simulations.
Points superimposed on the lines represent median and high and
low 68.26% confidence limit SD estimates. Analysis was performed under conditions in which synthetic calibration curve data
sets were constructed with one, two, three, four, or five replicates
per reference concentration. Horizontal lines in each graph correspond to one-half the difference between high and low 68.26%
confidence limit concentrations as obtained directly from the
distributions of hormone concentration estimates produced by
the simulations (i.e., one-half the lengths of the vertical lines in
the Variable panels in Fig. 2).

variably weighted assay response analysis. A detailed

comparison of the back-calculated results obtained by
each of the three assay response weighting schemes is
presented in Table 2.

Discussion
As part of a systematic characterization of experimental
uncertainty inherent in assay measurements, we have examined the performance of three monotonically varying algebraic forms for the assay doseresponse function, and delineated the joint experimental uncertainties inherent in the
fitted curve, assay replicates, calibrator replicates, and zerodose tubes. Among the three common fitting functions
explored (modifications of the logistic and Adair expressions), the modified four-state Adair equation (MONOTONE) was favored by the majority of calibration curve
realizations evaluated here, whether composed of one, two,
three, four, or five replicates per dose. A modified fourparameter logistic function [4PARMS], however, very commonly used for calibration curve analysis [1, 8, 9], was also

adaptable. We further demonstrated that the variable response weighting protocol exhibited performance superior
to that of either the uniform response weighting protocol or
the protocol involving uniform response weighting without
consideration of zero-hormone-concentration calibrators,
since variable weighting provided greater accuracy and
precision, especially in determining low-end hormone concentrations. Indeed, single-replicate conditions with variable
weighting provided better performance than even the quintuple-replicate cases involving uniform weighting with or
without utilization of zero calibrators at concentrations below ;0.04 mg/L in the Monte Carlo-simulated GH chemiluminescence assay. Approximately equivalent performance
among the three protocols was seen at higher hormone
concentrations.
We further observed that all of the protocols were inaccurate and imprecise at 45 mg/L in the Monte Carlosimulated GH chemiluminescence assay, illustrating the
importance of characterizing critically the relevant operating
range of any given assay configuration. With few replicates,

122

Straume et al.: Estimation of concentrations and uncertainties

Fig. 4. High-replicate-number calibration curve analysis of a GH chemiluminescence assay (GH Chemi) and a LH IRMA showing plotted
results of MONOTONE best-fit calibration curves for variable, uniform,
and uniform without zeroes assay response weighting (nearly superimposable curves except near the zero-dose concentration).
Error bars and estimated assay response error inset plots are for variably
weighted assay response analysis.

as might be anticipated intuitively, there was a greater

tendency to underestimate hormone concentrations on average because of a larger number of out-of-range points. In
addition, we observed that whereas the variable weighting
protocol can provide estimates of response and hormone
concentration uncertainty even with single replicates, significant improvements in both the accuracy and precision of
these estimates are achieved by the use of duplicates, with
less remarkable further improvements evident on going to
higher numbers of replicates. Thus, cost constraints vs
precision requirements by the clinical chemist, clinician, and
investigators will determine the desired replication density.
The present work also shows that an empirically based
discrete response uncertainty profile is effective for estimating response errors at all except the highest reference concentration, with greater reliability achieved at higher replicate conditions. Perhaps unexpectedly, despite a general
preference for the use of duplicates or higher numbers of
replicates, even single determinations are moderately reliable below approximately the inflection point of the sigmoid
calibration curve. This remains a persistent consideration in
the (repeated) sampling of infants or children with limited
blood volumes when assay miniaturization is imperfect.
Our analysis further indicates that estimating and prop-

agating the effects of calibration curve uncertainty contribute noticeable effects on concentration uncertainty estimates
beyond that due solely to response variability. Evidence of
this is provided by our observation that GH concentration
SDs were conservatively estimated by the variable weighting procedure when compared with directly estimated
Monte Carlo [GH] SDs. Monte Carlo estimates were generally lower than those provided by the variable weighting
protocol because calibration curve parameter uncertainty is
not propagated as a contributor in the direct Monte Carlo
estimates. That the Monte Carlo procedure is valid is supported, however, by the observation that Monte Carlo estimates of assay response SDs were extremely consistent (a
situation in which agreement should indeed be expected).
To our knowledge, uncertainty in the fitted parameters of
the calibration curve is not reflected in sample uncertainty
estimates in most available data reduction methods. Hence,
earlier procedures for calculating within-sample SDs underestimate sample variance. This bias is especially significant
in defining assay sensitivity, leading potentially to inferred
greater sensitivity than actually achievable. In addition,
underestimation of (low-end) assay uncertainty may have
nontrivial impact on computer-assisted curve fitting of
(weighted) neurohormone time series, presumptively promoting false-positive (type I) statistical errors. Lastly, estimating the precision of inferred statistics from a time series,
e.g., the SD of an approximate entropy estimate for any
given time series, will lead to an overstatement of precision.
In summary, the variable weighting data reduction protocol
described here provides greater accuracy and precision than
most commonly used hormone concentration data reduction procedures, particularly at extremely low hormone
concentrations. Three monotonically sigmoidal functional
forms for evaluating calibration curves are examined, after
which selection of a preferred model is based on empirical
grounds (lowest absolute SSRs). Assay responses are variably weighted by an empirically derived discrete assay
response uncertainty profile that (a) is specifically tailored to
the particular calibration curve response profile being considered yet free of any constraints applied by assuming a
particular functional form for a variance profile [1 6], (b)
accounts for both response precision (replicability) and accuracy (relative deviation from predicted calibration curve),
and (c) is generated in a manner maximally consistent with
the most probable derived calibration curve. We show that,
in principle, uncertainty estimates for both assay responses
and hormone concentrations can be obtained from even
single-replicate assay protocols. However, the reliability of
measures rises significantly upon increasing to duplicates.
Uncertainty in determination of the calibration curve is also
evaluated and subsequently propagated as a contribution to
derived concentration uncertainty estimates. The explicit use
of zero-hormone reference information during evaluation of
calibration curves also contributes to better determination of
low hormone concentrations. Efforts are currently under
way to fully implement this data reduction protocol into a

123

Clinical Chemistry 44, No. 1, 1998

Table 2. Back-calculated results from high-replicate-number calibration curve analysis.

Variable
Var-wgt
[GH], mg/L

0
1
3
6
10
12
15
20
25
30
35
40

No. of replicates

8
8
8
8
8
8
8
8
8
8
8
8

GHavg

0.000
0.997
3.08
5.57
10.41
11.89
15.14
20.25
22.22
30.6
33.4
40.1

Arith.

0
1.5
2.5
5
7.5
12.5
20
40
80
120
160

LHavg

9
10
11
10
11
11
10
11
11
11
10

20.14
1.32
3.22
5.09
7.14
12.59
19.67
40.6
79.0
120.2
157.6

Uniform w/o zeroes

Arith.

Arith.

SEM

GHavg

SEM

GHavg

SEM

GHavg

SEM

0.029
0.040
0.10
0.18
0.29
0.29
0.28
0.56
0.83
1.5
1.7
2.5

20.0003
1.005
3.087
5.580
10.42
11.88
15.17
20.274
24.04
31.29
34.0
41.3

0.0045
0.027
0.028
0.055
0.14
0.19
0.15
0.092
0.96
0.98
1.1
1.0

20.0933
1.063
3.207
5.628
10.34
11.80
15.11
20.340
24.18
31.41
34.0
40.70

0.0056
0.030
0.028
0.053
0.13
0.19
0.15
0.094
0.98
0.97
1.0
0.93

20.3678
0.935
3.206
5.661
10.35
11.78
15.07
20.274
24.13
31.40
34.0
40.72

0.0061
0.033
0.029
0.054
0.13
0.18
0.15
0.094
0.98
0.98
1.0
0.93

Var-wgt.
[LH], IU/L

Uniform

Arith.
SEM

LHavg

0.32
0.35
0.39
0.42
0.38
0.44
0.66
1.2
2.1
3.4
6.0

20.15
1.47
3.25
5.04
7.13
12.67
19.83
40.92
79.3
122.4
159.8

32-bit Windows operating environment in a manner that

will facilitate maximal ease of user interaction as well as
maximal data throughput capabilities.

We acknowledge support from: NSF DIR8920162 (National Science Foundation Center for Biological Timing; M.S.,
M.L.J., J.D.V.); NIH RR00847 (General Clinical Research
Center at the University of Virginia; M.L.J., J.D.V.); NIH
DK38942 (Diabetes and Endocrine Research Center at the
University of Virginia; M.L.J., J.D.V.); NIH RR08119 (Center for Fluorescence Spectroscopy at the University of
Maryland at Baltimore; M.L.J.); NIH GM35154 (M.L.J.);
NIH RCDA1K04 HD00634 (J.D.V.); NIH P30 HD28934
(Reproduction Research Center at the University of Virginia; J.D.V.); Baxter Healthcare Corp., Round Lake, IL
(J.D.V.); The NIH-supported Clinfo Data Reduction Systems; The Pratt Foundation; and The University of Virginia Academic Enhancement Fund.

References
1. Rodbard D, Lenox RH, Wray HL, Ramseth D. Statistical characterization of the random errors in the radioimmunoassay dose
response variable. Clin Chem 1976;22:350 8.
2. Rodbard D. Statistical estimation of the minimal detectable concentration (sensitivity) for radioligand assays. Anal Biochem 1978;90:112.

Arith.
SEM

0.17
0.28
0.25
0.39
0.19
0.29
0.48
0.83
1.1
3.1
2.6

LHavg

20.12
1.43
3.16
4.90
6.95
12.47
19.71
41.04
78.9
121.4
159.7

Arith.
SEM

0.16
0.27
0.25
0.39
0.19
0.29
0.48
0.83
1.1
3.1
2.7

LHavg

20.21
1.37
3.11
4.88
6.95
12.50
19.75
41.03
78.8
121.5
159.6

SEM

0.16
0.27
0.25
0.39
0.19
0.29
0.48
0.83
1.1
3.1
2.7

3. Sadler WA, Smith MH. Estimation of the response-error relationship in immunoassays. Clin Chem 1985;31:18025.
4. Davidian M, Carroll RJ, Smith W. Variance functions and the minimum
detectable concentration in assays. Biometrika 1988;75:54956.
5. Davidian M. Estimation of variance functions in assays with possibly
unequal replication and nonnormal data. Biometrika 1990;77:4354.
6. Hwang L-J. Impact of variance function estimation in regression
and calibration. Methods Enzymol 1994;240:150 70.
7. Chapman IM, Hartman ML, Straume M, Johnson ML, Veldhuis JD,
Thorner MO. Enhanced sensitivity growth hormone (GH) chemiluminescence assay reveals lower postglucose nadir GH concentrations
in men than women. J Clin Endocrinol Metab 1994;78:13129.
8. Rodbard D. Statistical quality control and routine data processing
for radioimmunoassays and immunoradiometric assays. Clin
Chem 1974;20:125570.
9. Rodbard D, Munson PJ, DeLean A. Improved curve-fitting, parallelism testing, characterization of sensitivity and specificity, validation, and optimization for radioligand assays. In: Radioimmunoassay and related procedures in medicine. Vienna: International
Atomic Energy Agency, 1977:1:469 509.
10. Johnson ML, Frasier SG. Nonlinear least squares analysis. Methods Enzymol 1985;117:301 42.
11. Straume M, Frasier-Cadoret SG, Johnson ML. Least-squares analysis
of fluorescence data. In: Lakowicz JR, ed. Topics in fluorescence
spectroscopy, Vol. 2: Principles. New York: Plenum, 1991:177241.
12. Bevington PR. Data reduction and error analysis for the physical
sciences. New York: McGraw-Hill, 1969:73.