IMMULITE® Tumor Marker Assays

D P C T e c h n i c a l R e p o r t
®
IMMULITE Tumor Marker Assays
Multicenter Reference Range Data
for Diagnostic Products Corporation Kits
Paul E. C. Sibley, Ph.D.
International Marketing Manager, Tumor Markers
IMMULITE® Tumor Marker Assays: Multicenter Reference Range Data for
Diagnostic Products Corporation Kits
Preface
Table of Contents
In keeping with our commitment to provide ongoing
support for the clinical application of DPC’s cancerrelated assays, through establishing, verifying and
updating reference range information, the fourth edition of
this technical report has been enhanced in several respects.
Introduction............................................................. 3
The content has been updated to reflect additional results,
notably for IMMULITE® AFP, a sequential immunometric
assay which received FDA clearance for use as an aid in the
management of nonseminomatous testicular cancer.
Disclaimers ............................................................. 9
New to this edition is a direct, graphical comparison of
IMMULITE PSA and IMMULITE Third Generation PSA
results for 563 normal adult male serum samples. The
centile curves testify to the excellent agreement between
these two assays. (Though optimized for the very low
levels of interest after radical prostatectomy, IMMULITE
Third Generation PSA has a working range extending
beyond the 4–10 µg/L “gray zone” up to 20 µg/L, making
it appropriate for routine use in other contexts as well.)
PSA...................................................................... 10
By way of introduction, we have added an overview of the
significance of normal range studies, which figure
prominently in the validation of tumor marker assays as
well as (directly or indirectly) in the interpretation of
patient results. With specific examples drawn from AFP,
CEA, OM-MA (CA125) and PSA, the discussion herein
supplements the tables and graphs in the document’s
analyte-specific reference sections.
CEA...................................................................... 17
Subjects.................................................................. 7
Methods.................................................................. 8
Data Analysis.......................................................... 9
References ........................................................... 22
Analytes
Third Generation PSA........................................... 11
Nomograms ........................................................ 12
Free PSA.............................................................. 13
PAP...................................................................... 14
AFP ...................................................................... 15
Beta-2 Microglobulin ............................................. 16
BR-MA (CA15-3).................................................. 19
GI-MA (CA19-9).................................................... 20
OM-MA (CA125) ................................................... 21
In addition, this document has been substantially reorganized
to make it a still more convenient and comprehensive (printed
or electronic) reference on normal ranges and related
information for DPC’s automated tumor marker assays. It is
being made available, along with other data and discussions
of clinical interest, on DPC’s Web site, www.dpconline.com
— under Technical Documents, Technical Reports — in
Adobe Acrobat PDF format.
The document features detailed analyses of normal
reference range data obtained with IMMULITE tumor
marker assays for the following analytes: alphafetoprotein (AFP), beta-2 microglobulin, BR-MA (CA153), carcinoembryonic antigen (CEA), GI-MA (CA19-9),
OM-MA (CA125), prostatic acid phosphatase (PAP), and
prostate-specific antigen (PSA).
We have aimed throughout at characterizing — as
definitively as possible — the distribution of values to be
expected for the IMMULITE assays used to generate the
data and, by implication, for the corresponding
IMMULITE® 2000 assays as well, which are highly
similar to their IMMULITE counterparts in both design
and performance.
— Paul E. C. Sibley, Ph.D.
2
Results reported to the physician should therefore be
accompanied by a characterization of normal limits
applicable to the assay in use. Laboratory report forms
usually supply this as a centile (either the 95th or 97.5th
for most tumor marker assays) indexing the upper limit of
normal for adults, or for each of several relevant
subgroups.
Introduction
The Significance of Normal Range Studies for
Tumor Marker Assays
Normal range studies serve two main purposes. First, they
help to characterize an assay’s performance in a
fundamental way (method verification), and may prove a
valuable source of clinically relevant insights. Second,
they provide one basis for making sense of individual
patient results (interpretation), even in certain contexts
where adopting the upper limit of normal as a decision
level would be inappropriate.
Method Verification/Validation
Precision and accuracy represent the two fundamental
dimensions of assay performance which can be assessed
via analytical studies — “analytical” in the sense of
requiring no special attention to the clinical status of the
samples employed. Reference range studies make a third
basic contribution towards validating the assay, usually by
exploring the assay’s ability to reproduce well-established,
clinically relevant, group-based distinctions or trends.
Examples are given below for IMMULITE CEA,
IMMULITE Third Generation PSA, and IMMULITE
OM-MA.
These two benefits are especially evident for tumor
marker assays — though only if the normal range studies
are carefully designed and executed. (The value of such
studies depends critically on the quality of the assays
involved, on the character and size of the study
population, and on proper data analysis.)
Accuracy is typically assessed via recovery studies, based
on a reference preparation, and/or by comparison studies
against a reference method. For several tumor markers
including CA15-3, CA19-9 and CA125, however, neither
reference methods (“gold standards”) nor reference
preparations exist. These assays report in “arbitrary” units,
making it imperative to generate assay-specific normal
range limits as a guide for the physician faced with
interpreting patient results. Moreover, the significance of
the usual analytical approach to assessing accuracy is
substantially reduced in this context, forcing method
verification to rely more heavily on a combination of
precision and reference range studies.
Accordingly, DPC has undertaken extensive multicenter,
multivariate reference range studies for several
IMMULITE® tumor marker assays. The main results
established thus far are summarized in this technical
report, while this introduction focuses on the nature and
significance of normal range studies in the context of
tumor marker assays, drawing on the IMMULITE-based
study for illustrations.
Interpretation
It is widely accepted that tumor marker assays are
generally inappropriate for population screening, due to
inadequate clinical sensitivity and/or specificity. Many
such assays have more firmly established applications in
the monitoring and follow-up of various types of
treatment.
Validation: AFP
Alpha-fetoprotein (AFP) assays provide an excellent
example of how normal range studies can be essential
even when analytical studies of accuracy do apply. Thus,
for AFP, there exists a well-established reference
preparation,4 with a well-defined conversion between
mass and International Units; and major collaborative
efforts have aimed at improving and standardizing various
aspects of AFP immunoassay design.5
Consequently, some authorities have advocated that upper
reference limits be established for certain groups other
than normals, e.g. for curative prostatectomies,2 where an
upper limit for PSA on the order of 0.10 µg/L or less is
expected. (This is well below the upper limit for normal
men and underscores the need for exceptional, low-end
precision — “third generation” sensitivity — if an assay
for PSA is to yield precise and meaningful results when
monitoring a subject’s progress after radical prostatectomy
and other therapies.3)
Even so, a 1996 survey of laboratory practice in the
United Kingdom revealed a surprisingly broad range of
variation in the upper limits of normal quoted for AFP
results.6 Evidently, the same limits cannot be assumed to
apply to all assays for this tumor marker in spite of a
common approach to standardization — even if some of
the highest values in the survey are dismissed as the
legacy of an older technology. (We expect an evolution
towards lower AFP reference limits as assays become
more sensitive and specific, and less susceptible to socalled matrix effects.)
Nevertheless, normal range studies for tumor markers are
also relevant to the interpretation of individual patient
results. The upper limit of normal often represents a major
landmark, even when not serving as a cutoff. Thus,
successful treatment for a cancer is often followed by the
return of relevant tumor markers to normal circulating
levels, as with CEA. Likewise, for most prostate cancer
treatments, a urologist would look for PSA levels to drop
to well below normal.
The survey also stands as a reminder of how difficult it
can be to disentangle the impact of genuine demographic
3
Table 1.
Population-based IMMULITE® CEA centiles, µg/L
differences from differences attributable to variation in the
design and quality of reference range studies, or to
variation in data analysis and presentation; for the survey
also showed considerable variation across laboratories in
the limits quoted for the same assay.
Group
n
5%
50%
95%
97.5%
Smoking
166
0.52
1.8
6.3
8.9
Nonsmoking
312
0.37
1.9
3.3
4.3
Smoking
98
0.42
1.3
4.8
5.4
Nonsmoking
346
0.21
0.73
2.5
3.0
Adult Males
No doubt this can be explained largely by decisions to
establish, retain or adapt in-house reference limits, instead
of relying on “expected values” claims from the
manufacturer. Some variation in outcome from one
reference range study to another is inevitable; but too
small a sample size and/or too casual a selection of
subjects can aggravate the problem. Hence, rather than
attempting to establish their own normal values,
laboratories with limited resources may be better off
adopting the limits from a large, well-designed reference
range study, after verifying their applicability through
suitable review and experimentation.7
Adult Females
Because these differences tally with well-established
expectations in the literature, the statistics provide clinical
evidence that IMMULITE CEA has the accuracy and
precision needed to yield appropriate results under
conditions known to affect baseline CEA levels.
Competing Conventions
Where tumor markers are concerned, we are faced with
two competing conventions for characterizing the
distribution of results for normal subjects.8 Many
laboratories prefer to quote the observed 95th centile,
partly because the lower limit of normal for most tumor
markers is either below the detection limit of current
assays, or of no apparent clinical interest, or both; and
partly to buffer against “contamination” (the presence of
abnormal subjects) in the reference group. Others prefer to
quote the 97.5th centile, as is customary for many analytes
commonly measured by immunoassay.
Indeed, it would be a source of concern if a study were
unable to reproduce the expected male-female, smokernonsmoker differences in CEA levels. Assuming that
demographic factors could be ruled out, such a failure
might be due to the assay (inadequate specificity or
precision); to the study design (too small a data set, or too
weak a criterion for distinguishing smoker from
nonsmoker); or to the data analysis (transcription errors,
genuine outliers, or inappropriate statistics).
The population-based information encapsulated in Table 1
is clearly also relevant to the interpretation of individual
results. Thus, for example, given these range limits, a
physician would be likely to evaluate a CEA result of 6.0
µg/L for an otherwise apparently normal male subject in
the light of his smoking status.
In effect, the first approach characterizes the distribution
of values in terms of the lower 95%, the second in terms
of the central 95%; so both aim at the same degree of
coverage. Figures 1 and 2 show, however, that for
distributions skewed towards higher values, there can be a
considerable difference between the concentration levels
associated with these two centiles. (Given the competing
conventions in this field, DPC has opted to tabulate both
centiles in this technical report.)
Visual representations of the study results and statistical
analysis can serve both as a valuable safeguard against
faulty or overly simplistic data reduction and as a source
of insights into the distribution of real-world data that
cannot be adequately captured in a simple tabulation.
Validation: CEA
Carcinoembryonic antigen (CEA) levels for normal adults
cannot be adequately characterized by a single set of
reference limits. Men tend to have higher levels than
women; smokers tend to have higher levels than
nonsmokers. The differences due to sex and smoking
status observed in a multicenter study based on the
IMMULITE CEA assay are summarized in Table 1, in
terms of selected centiles.
4
Figure 1.
In this matter, the article published in JAMA 1993 by
Oesterling et al. was a major landmark.11 The authors
succeeded in characterizing the age-related distribution of
PSA values in normal men — both graphically, in a
“nomogram” conveying the apparently continuous
variation in PSA levels as a function of age, and in a table,
based on a manageable number of age brackets and
suitable for laboratory report forms.
Figure 2.
Figure 2 displays a comparable nomogram based on
IMMULITE Third Generation PSA results from a study
conducted by Dr. A. Semjonow (Münster, Germany) in
1997. The 563 subjects included (a) men with PSA values
less than 4.0 µg/L and no evidence of disease by digital
rectal examination, and (b) men who failed to qualify
under the first criterion but showed no evidence of a tumor
on transrectal ultrasound-guided sextant biopsy. (These
PSA results were subsequently combined with data from
other sources for tabulation in this technical report. See
pages 10, 11 and 13.)
Thus, in Figure 1, the “ladders” show precisely how the
centile estimates relate to the mass of data points in the
study. (The horizontal lines correspond to the centiles in
Table 1.) Moreover, the figure suggests one additional
factor of possible relevance: there appears to be an
upward, age-related trend for CEA in both male and
female smokers. If genuine, this trend might be due to the
duration of smoking or the amount of tobacco consumed;
but this aspect of the data has not been pursued.
In the analysis summarized in Figure 2, contour lines
representing the 50th, 95th and 97.5th centiles were fitted
globally to the PSA results as a function of age, that is,
using methods which avoid the initial partitioning of
results into bins by age.12,13 (See also page 12.)
Validation: PSA
Several similar studies have made it clear that age is a
critical factor in PSA normal range studies for adult males.
Indeed, for many years, there has been no justification for
believing in a single concentration level representing the
(age-independent) upper limit of normal.
For prostate-specific antigen (PSA), as for CEA, there are
relevant categorical variables. Sex, obviously, is one such
factor, and race is emerging as another;9 but age, a
continuous variable, is an even more fundamental
determinant of PSA reference limits in normal men.
It does not follow, however, that an age-related analysis
should be utilized for interpretation of results. It is widely
understood that the upper limit of normal should not
automatically serve as a cutoff in screening programs,
though the distribution of normal values is highly relevant.
A PSA value of 4.0 µg/L continues to have a great deal of
support when considered as a decision limit applicable in
certain contexts to adult men irrespective of age.14
The traditional decision limit of 4.0 µg/L had its origin as
the upper limit of normal determined in a reference range
study supporting the introduction of Hybritech’s
immunoradiometric assay for PSA. The age distribution of
the subjects included in this study has been questioned;10
but the deeper issue concerns the analysis of results.
5
Moreover, an analysis of IMMULITE OM-MA results for
samples collected during the latter half of the follicular
phase or during the luteal phase in the multicenter
ovulatory cycle study — i.e. excluding just the early days
of the follicular phase — yielded an upper reference limit
comparable to the upper limit observed for the older
women in a large cross-sectional study based on the same
assay.
Sampling Conditions: OM-MA (CA125)
Some studies of the ovarian marker CA125 have yielded
upper limits of normal for postmenopausal women which
are lower than the limits established for adult women
generally; moreover, it has been suggested that this
postmenopausal reference limit might serve as a better
decision level even for younger women in certain
contexts, e.g. after ovariectomy.15
This raises the possibility that the quality of reference
limits determined in population studies of CA125 may
depend more on certain aspects of study design than on
the choice of subgroups during the data analysis stage. In
particular, it argues for closer attention to the conditions
for proper sample collection in women of reproductive
age.
Setting aside issues relating to decision limits, one
assumption here is that either age or reproductive status is
a factor which must be taken into account in determining
CA125 reference ranges for women. However, results
generated with the IMMULITE OM-MA assay for CA125
underscore the need for further examination of this matter.
A multicenter study of several IMMULITE assays
followed normally cycling women on a daily basis
throughout one complete menstrual cycle and showed that
many such women have distinctly higher CA125 levels
during menstruation and the days immediately thereafter
than in the remainder of the cycle.16,17 (See page 21.)
Conclusion
Substantial, carefully designed, appropriately analyzed
normal range studies for an assay can greatly enhance the
value of individual results reported by the laboratory, as
well as provide evidence for the validity and clinical
usefulness of the assay.
These results confirm and extend other studies in the
literature,18 providing a somewhat more precise
delineation of the magnitude and frequency of
menstruation-induced CA125 elevations than was
previously available.
The multicenter studies summarized in this technical
report reflect DPC’s commitment to generating and
supporting extensive clinical studies for its automated
tumor marker assays; and to regularly updating its library
of reference materials dealing with the studies, enhancing
the content, presentation and availability of these essential
resources.
Like age in studies of PSA in adult men or gestational age
in studies of HCG in pregnant women, ovulatory cycle
position could be treated as a continuous covariate, and is
naturally treated as such in studies of several reproductive
hormones. For CA125, however, it is more natural to
regard the early days of the cycle as a transient phase to be
avoided when collecting samples for determinations of
this ovarian tumor marker.
In this manner, DPC continues to strive for improvements
in its service to laboratories using the IMMULITE and
IMMULITE 2000 systems, and thus also to the physicians
who rely on these laboratories for precise and accurate
patient results and for the basic orientation needed in their
interpretation.
From the clinician’s point of view, an appreciation for the
sharp, transient increases in CA125 levels which can be
induced by menstruation has an obvious bearing on
individual patient results, no matter whether these are
interpreted longitudinally, in terms of within-subject
trajectories, or against population-based limits.
6
good health. This was part of a larger investigation —
DPC’s Multicenter Ovulatory Cycle Study — designed to
establish detailed reference ranges for several reproductive
hormones in normally ovulating women throughout the
menstrual cycle.16,17 The results, presented graphically,
corroborate the reference limits determined from the
Multicenter Tumor Marker Reference Range Study, and
provide some additional insights. (See pages 19 and 21.)
Subjects
Much of the data presented here derives from the
Multicenter Tumor Marker Reference Range Study which
involved DPC assays in several formats (double antibody
and antibody-coated tube RIAs and IRMAs, as well as
IMMULITE assays).19 Blood samples were collected in
France, Germany, The Netherlands and Portugal; an
independent laboratory in The Netherlands generated
results. Additional samples were collected and assayed in
the UK at a later date. The subjects included men and
nonpregnant women, approximately 20 to 70 years of age,
all in apparent good health based on a questionnaire.
Samples were collected in plain glass tubes without
anticoagulants, gel barriers or clot-promoting additives,
and assayed in singlicate.
AFP
For AFP, the tabulated statistics are based on results
generated with the sequential IMMULITE AFP assay
(LKAP) on serum samples from a total of 178 men and
204 women. The data set consisted of results from the UK
site in the study described above, combined with results
obtained on samples from apparently normal adults in the
US. (Not enough was known about the latter to allow for
plotting concentration against subject age for this analyte.
See page 15.)
PSA
Contributing to the data sets for free and total
immunoreactive PSA were additional results from
Germany and the US obtained by IMMULITE assays on
serum samples from apparently healthy men with normal
prostates by various criteria. The samples collected in
Germany — as part of a study conducted by Dr. A.
Semjonow (Münster) — were analyzed by all three
assays; those collected in the US were processed by the
IMMULITE PSA assay only. (See pages 10, 11 and 13.)
To allow for comparing the IMMULITE PSA and
IMMULITE Third Generation PSA on a single
population, the page devoted to an age-related analysis of
results by these two assays was limited to data from the
Semjonow study: the 563 subjects in this study included
(a) men with PSA values less than 4.0 ng/mL and no
evidence of disease by digital rectal examination, and (b)
men who failed to qualify under the first criterion but
showed no evidence of a tumor on transrectal ultrasoundguided sextant biopsy. (See page 12.)
BR-MA, OM-MA
For BR-MA (CA15-3) and OM-MA (CA125), this
technical report also summarizes pertinent results from a
study carried out at laboratories in four countries
(Belgium, Germany, The Netherlands and the UK) where
daily serum samples were collected throughout one
complete cycle from each of 27 volunteers in apparent
7
IMMULITE® 2000 Tumor Marker Kits
Methods
All results presented in this document were obtained with
IMMULITE® tumor marker assays.
Kit
Catalog Detection
Number Limit
Calibration
Range
Based on the close similarity of their performance
characteristics, the corresponding IMMULITE® 2000
assays can be expected to have comparable ranges.
AFP
L2KAP
0.2 kIU/L
Up to 300 kIU/L
Beta-2
Microglobulin
L2KBM
0.3 µg/L
Up to 500 µg/L
Listed below are the principal “tumor marker” assays
available for the IMMULITE and IMMULITE 2000
platforms. (Other assays are under development.)
BR-MA*
(CA15-3)
L2KBR
0.2 kU/L
Up to 300 kU/L
CEA
L2KCE
0.15 µg/L
Up to 550 µg/L
Not all of the IMMULITE and IMMULITE 2000 assays
mentioned here are available in the US. Kit status is
clarified, where relevant, in the following tables and in the
analyte-specific reference sections of this document. For
detailed information on the assays, refer to the package
inserts.
OM-MA
(CA125)
L2KOP
0.3 kU/L
Up to 500 kU/L
PSA
L2KPS
0.04 µg/L
0.04 – 150 µg/L
Free PSA*
L2KPF
0.02 µg/L
Up to 25 µg/L
Third
Generation
PSA
L2KUP
0.003 µg/L
Up to 20 µg/L
Thyroglobulin
L2KTY
0.2 µg/L
Up to 300 µg/L
®
IMMULITE Tumor Marker Kits
Kit
Catalog Detection
Number Limit
Calibration
Range
AFP
LKAP
0.2 kIU/L
Up to 300 kIU/L
Anti-TG Ab
LKTG
1 kIU/L
Up to 3000 kIU/L
Beta-2
Microglobulin
LKBM
0.3 µg/L
Up to 500 µg/L
BR-MA*
(CA15-3)
LKBR
0.5 kU/L
Up to 300 kU/L
CEA
LKCE
0.2 µg/L
Up to 550 µg/L
GI-MA*
(CA19-9)
LKGI
2.0 kU/L
Up to 1000 kU/L
OM-MA
(CA125)
LKOP
0.2 kU/L
Up to 500 kU/L
PAP
LKPA
0.02 µg/L
Up to 100 µg/L
PSA
LKPS
0.03 µg/L
0.04 – 150 µg/L
Free PSA*
LKPF
0.02 µg/L
Up to 25 µg/L
Third
Generation
PSA
LKUP
0.003 µg/L
Up to 20 µg/L
Thyroglobulin
LKTY
0.2 µg/L
Up to 300 µg/L
TPS*
LKTP
6 U/L
Up to 2400 U/L
* Available outside the US
* Available outside the US
8
what age spectrum any such relationship holds;21 and of
course quite a variety of mathematical models (curve
shapes) are compatible both with the PSA data presented
here and with other published data.
Data Analysis
S-PLUS 2000 (www.mathsoft.com) was used for
calculations and data visualization, and for the graphs
themselves.
Lucas Plots
Centiles
The BR-MA (CA15-3) and OM-MA (CA125) results from
the Multicenter Ovulatory Cycle Study are shown in
Lucas plots, with the trajectories for two representative
subjects highlighted. This method of depicting menstrual
cycle data forces alignment of results not only at
midcycle, but also at the start of follicular phase and the
end of luteal phase, by rescaling the days before and after
each subject’s observed lutropin (LH) peak to a common
phase length.16,17
The tables provide estimates for relevant centiles,
including the concentration at the 97.5th centile as well as
at the 95th centile. These correspond to the upper limits
for central 95% and lower 95% intervals, respectively,
both of which are frequently encountered in the literature
on tumor marker assays.8
The centiles were calculated using a robust, nonparametric
technique, because most of the distributions were highly
skewed rather than Gaussian or even symmetric, and in
order to accommodate the presence of possible outliers.
(Specifically, they were calculated using an S-PLUS
implementation of the Harrell-Davis function,20 which is
considered the nonparametric method of choice for
univariate reference range analysis in clinical chemistry.12)
Disclaimers
The tabulated centiles represent guidelines only. Each
laboratory should establish or verify the appropriateness of
adopting reference range limits suggested by this
document.7
In general, results for samples from subjects 20 to 70 years
of age were subgrouped by sex and/or smoking status,
when appropriate, prior to analysis. PSA results were
further partitioned by age into “bins” corresponding (in
accord with convention) to successive decades.
Appropriate decision (or "action") limits cannot be
automatically identified with upper (or lower) reference
range limits, no matter how defined.8
The manner of data presentation adopted here should not
be construed as suggesting for any analyte either that a
significant relationship exists between concentration and
age or that age-related clinical decision limits are
appropriate. (For a brief discussion of this matter, see the
Introduction.)
Scatterplots
In cases where subject ages were available for most
samples, graphs are included. These graphs are intended
merely to convey demographic information (age
distributions of the subjects figuring in the study) and a
sense of the uncertainties associated with the highest
centiles tabulated. For uniformity, the graphs show
concentration on a linear scale plotted against subject age,
typically across the entire 20- to 70-year age spectrum
used in the analysis. Limits for the vertical concentration
axes have been regimented, so far as possible, to facilitate
comparisons. Trend lines, where present, are based on
local regression analysis (as implemented by the S-PLUS
loess functions).
Nomograms
In constructing the pair of graphs intended as a direct
comparison of IMMULITE PSA and IMMULITE Third
Generation PSA, contour lines representing the 50th, 90th,
95th, 97.5th and 99th centiles were fitted to the PSA
results as a function of age using parametric methods —
manually implemented in S-PLUS and similar to those
recommended in the modern literature12,13 — which avoid
the initial partitioning of results into bins by age. The
results are comparable to the “nomogram” published in
the seminal paper by Oesterling et al.3 which likewise
sought to represent PSA concentration levels as a
continuous function of age. It must be understood,
however, that even for PSA, it is not fully established over
9
PSA
IMMULITE PSA
Results from the Multicenter Tumor Marker Reference
Range Study, combined with results obtained on serum
samples from men with normal prostates in Germany, and
from apparently healthy men in the US. (See also pages 5
and 12.)
IMMULITE PSA: Males
PSA, µg/L
8
Catalog Numbers
LKPS1 (100 tests)
LKPS5 (500 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
Sample
10 µL serum
Incubation
30 min
Calibration Range
Up to 150 µg/L (ng/mL)
Detection Limit
0.03 µg/L
Hook
None up to 20,000 µg/L
6
IMMULITE 2000 PSA
4
Catalog Numbers
L2KPS2 (200 tests)
L2KPS6 (600 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
Sample
10 µL serum
Incubation
30 min
Calibration Range
Up to 150 µg/L (ng/mL)
Detection Limit
0.04 µg/L
Hook
None up to 22,500 µg/L
2
0
20
30
40
50
60
70
Age, years
IMMULITE PSA, µg/L
Males
n
5%
50%
95% 97.5%
Combined (20–70) 1486 0.20
0.75
2.9
3.7
20–40 years
297
0.16
0.57
1.5
1.8
40–50 years
471
0.16
0.70
1.7
2.2
50–60 years
418
0.24
0.86
3.0
3.9
60–70 years
300
0.27
1.2
4.8
6.9
10
Third Generation PSA
IMMULITE Third Generation PSA
Results from the Multicenter Tumor Marker Reference
Range Study, combined with results obtained on serum
samples from men with normal prostates in Germany. (See
also pages 3, 5 and 12.)
Catalog Numbers
LKUP1 (100 tests)
LKUP5 (500 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
(sequential)
Sample
50 µL serum
Incubation
60 min
Calibration Range
Up to 20 µg/L (ng/mL)
Detection Limit
0.003 µg/L
Hook
None up to 90,000 µg/L
IMMULITE Third Generation PSA: Males
PSA, µg/L
8
6
4
IMMULITE 2000 Third Generation PSA
2
0
20
30
40
50
60
70
Catalog Numbers
L2KUP2 (200 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
(sequential)
Sample
50 µL serum
Incubation
60 min
Calibration Range
Up to 20 µg/L (ng/mL)
Detection Limit
0.003 µg/L
Hook
None up to 112,000 µg/L
Age, years
IMMULITE Third Generation PSA, µg/L
Males
n
5%
50%
95% 97.5%
Combined (20–70) 1075 0.23
0.67
2.6
3.7
20–40 years
253
0.19
0.52
1.3
1.5
40–50 years
328
0.22
0.65
1.6
1.9
50–60 years
306
0.25
0.80
2.6
3.6
60–70 years
188
0.29
1.2
5.6
6.9
11
IMMULITE Third Generation PSA (LKUP)
99%
97.5%
95%
8
Total Immunoreactive PSA, µg/L
7
6
90%
5
4
3
2
50%
1
0
35
40
45
50
55
60
65
70
75
80
Age, years (563 Normal Males)
IMMULITE PSA (LKPS)
99%
97.5%
8
95%
Total Immunoreactive PSA, µg/L
7
6
90%
5
4
3
2
50%
1
0
35
40
45
50
55
60
65
Age, years (563 Normal Males)
12
70
75
80
Free PSA
Results from the Multicenter Tumor Marker Reference
Range Study, combined with results obtained on serum
samples from men with normal prostates in Germany.
IMMULITE Free PSA*
Catalog Numbers
LKPF1 (100 tests)
LKPF5 (500 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
(sequential)
Sample
25 µL serum
Incubation
60 min
Calibration Range
Up to 25 µg/L (ng/mL)
Detection Limit
0.02 µg/L
Hook
None up to 10,500 µg/L
IMMULITE Free PSA: Males
Free PSA, µg/L
2.0
1.5
1.0
*Available outside the US
0.5
IMMULITE 2000 Free PSA*
0
20
30
40
50
60
70
Age, years
IMMULITE Free PSA, µg/L
Catalog Numbers
L2KPF2 (200 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
(sequential)
Sample
25 µL serum
50%
95% 97.5%
Incubation
60 min
Combined (20-70) 1072 0.038
0.17
0.50
0.66
Calibration Range
Up to 25 µg/L (ng/mL)
20–40 years
252
0.13
0.33
0.37
Detection Limit
0.02 µg/L
40–50 years
328 0.041
0.16
0.39
0.45
Hook
None up to 10,000 µg/L
Males
n
5%
ND
50–60 years
305 0.058
0.19
0.49
0.58
60–70 years
187 0.084
0.25
0.87
1.1
*Available outside the US
13
PAP
IMMULITE PAP
Results from the Multicenter Tumor Marker Reference
Range Study.
IMMULITE PAP: Males
5
PAP, µg/L
4
3
2
1
0
20
30
40
50
60
70
Age, years
IMMULITE PAP, µg/L
Males
n
5%
50%
95% 97.5%
525
0.89
1.4
2.3
2.7
14
Catalog Numbers
LKPA1 (100 tests)
LKPA5 (500 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
Sample
50 µL serum
Incubation
30 min
Calibration Range
Up to 100 µg/L (ng/mL)
Detection Limit
0.02 µg/L
Hook
None up to 15,000 µg/L
AFP
IMMULITE AFP
Results from the Multicenter Tumor Marker Reference
Range Study (UK site), combined with results obtained on
serum samples from apparently normal adults in the US.
(Not enough detailed information was available on these
samples to allow for plotting analyte concentration against
subject age.)
IMMULITE AFP, kIU/L
n
Males and Females 382
5%
50%
95% 97.5%
0.56
1.21
2.64
Catalog Numbers
LKAP1 (100 tests)
LKAP5 (500 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
(sequential)
Sample
10 µL serum
Incubation
60 min
Calibration Range
Up to 300 kIU/L (IU/mL)
(WHO 1st IS 72/225)
Detection Limit
0.2 kIU/L
Hook
None up to 450,000 kIU/L
Conversion to
Alternate Units
kIU/L x 1.21 → µg/L (ng/mL)
2.97
IMMULITE 2000 AFP
15
Catalog Numbers
L2KAP2 (200 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
(sequential)
Sample
10 µL serum
Incubation
60 min
Calibration Range
Up to 300 kIU/L (IU/mL)
(WHO 1st IS 72/225)
Detection Limit
0.2 kIU/L
Hook
None up to 534,000 kIU/L
Conversion to
Alternate Units
kIU/L x 1.21 → µg/L (ng/mL)
Beta-2 Microglobulin
Results from the Multicenter Tumor Marker Reference
Range Study.
IMMULITE Beta-2 Microglobulin
IMMULITE Beta-2 Microglobulin: Males and Females
Beta-2 Microglobulin, µg/L
3000
Catalog Numbers
LKBM1 (100 tests)
LKBM5 (500 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
Sample
5 µL serum (diluted) or urine
Incubation
30 min
Calibration Range
Up to 500 µg/L (ng/mL)
Detection Limit
0.3 µg/L
Hook
None up to 10,000 µg/L
2000
1000
IMMULITE 2000 Beta-2 Microglobulin
0
20
30
40
50
60
70
Age, years
Catalog Numbers
L2KBM2 (200 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
Sample
5 µL serum (diluted) or urine
Incubation
30 min
Calibration Range
Up to 500 µg/L (ng/mL)
Detection Limit
0.3 µg/L
Hook
None up to 10,000 µg/L
zb148-b, lkbm-fm, 19-feb-99
IMMULITE Beta-2 Microglobulin, µg/L
n
Males and Females 878
5%
50%
95% 97.5%
670
1,496 2,143 2,329
16
CEA
Results from the Multicenter Tumor Marker Reference
Range Study. (See also pages 4–5.)
IMMULITE CEA: Female Nonsmokers
IMMULITE CEA: Female Smokers
6
CEA, µg/L
CEA, µg/L
6
4
2
4
2
0
0
20
30
40
50
60
70
20
30
40
50
60
70
Age, years
Age, years
IMMULITE CEA, µg/L
Females
n
5%
50%
95% 97.5%
Nonsmoking
346
0.21
0.73
2.5
3.0
Smoking
98
0.42
1.3
4.8
5.4
IMMULITE CEA: Male Smokers
IMMULITE CEA: Male Nonsmokers
12
12
10
10
CEA, µg/L
14
CEA, µg/L
14
8
6
8
6
4
4
2
2
0
0
20
30
40
50
60
20
70
30
40
50
60
70
Age, years
Age, years
zb148-b, lkce_ms, 19-feb-99
IMMULITE CEA, µg/L
Males
n
5%
50%
95% 97.5%
Nonsmoking
312
0.37
1.9
3.3
4.3
Smoking
166
0.52
1.8
6.3
8.9
17
IMMULITE CEA
IMMULITE 2000 CEA
Catalog Numbers
LKCE1 (100 tests)
LKCE5 (500 tests)
Catalog Numbers
L2KCE2 (200 tests)
L2KCE6 (600 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
Methodology
Chemiluminescent enzyme
immunometric assay
Sample
15 µL serum
Sample
15 µL serum
Incubation
60 min
Incubation
60 min
Calibration Range
Up to 550 ng/mL
Calibration Range
Up to 550 µg/L (ng/mL)
Detection Limit
0.2 ng/mL
Detection Limit
0.15 µg/L
Hook
None up to 300,000 ng/mL
Hook
None up to 250,000 µg/L
18
BR-MA (CA15-3)
Results from the Multicenter Tumor Marker Reference
Range Study.
IMMULITE BR-MA*
Catalog Numbers
LKBRZ (50 tests)
LKBR1 (100 tests)
LKBR5 (500 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
(sequential)
Sample
5 µL serum
Incubation
60 min
Calibration Range
Up to 300 kU/L (U/mL)
Detection Limit
1.0 kU/L
Hook
None up to 23,500 kU/L
IMMULITE BR-MA: Females
BR-MA (CA15-3), kU/L
50
40
30
20
10
*Available outside the US
0
20
30
40
50
60
70
IMMULITE 2000 BR-MA*
Age, years
IMMULITE BR-MA, kU/L
Females
n
5%
50%
477
9.2
22
38
42
BR-MA (CA15-3), kU/L
50
40
30
20
10
0
0%
Methodology
Chemiluminescent enzyme
immunometric assay
(sequential)
Sample
5 µL serum
Incubation
60 min
Calibration Range
Up to 300 kU/L (U/mL)
Detection Limit
0.2 kU/L
Hook
None up to 30,000 kU/L
*Available outside the US
IMMULITE BR-MA: Ovulatory Cycles
-50%
L2KBR2 (200 tests)
95% 97.5%
Results from the Multicenter Ovulatory Cycle Study (27
subjects), with the trajectories for two representative
subjects highlighted. In contrast to the situation for OMMA (CA125), cycle position has no apparent impact on
BR-MA (CA15-3) levels.16,17
-100%
Catalog Numbers
50%
100%
Percent of Follicular or Luteal Phase
zb148-b, lkbr_cyc, 19-feb-99
19
GI-MA (CA19-9)
Results from the Multicenter Tumor Marker Reference
Range Study.
IMMULITE GI-MA*
Catalog Numbers
LKGIZ (50 tests)
LKGI1 (100 tests)
LKGI5 (500 tests)
50
Methodology
Chemiluminescent enzyme
immunometric assay
40
Sample
50 µL serum
Incubation
60 min
Calibration Range
Up to 1,000 kU/L (U/mL)
Detection Limit
2.0 kU/L
Hook
None up to 34,000 kU/L
GI-MA (CA19-9), kU/L
IMMULITE GI-MA: Females
30
20
10
*Available outside the US
0
20
30
40
50
60
70
60
70
Age, years
IMMULITE GI-MA: Males
GI-MA (CA19-9), kU/L
50
40
30
20
10
0
20
30
40
50
Age, years
IMMULITE GI-MA, kU/L
n
5%
50%
95% 97.5%
Males
470
2.5
3.1
19
24
Females
435
2.6
4.1
19
25
Combined
905
2.5
3.5
19
24
20
Results from the Multicenter Ovulatory Cycle Study (27
subjects), with the trajectories for two representative subjects
highlighted. The graph indicates that cycle position can have
a significant impact on the interpretation of OM-MA
(CA125) results — whereas no comparable effect is evident
for BR-MA (CA15-3). In this study of 27 normal cycles,
several showed moderate or even striking OM-MA (CA125)
elevations during the early follicular phase. In a clinical
situation, such a result could be mistaken for an abnormal
level, or a clinically significant departure from baseline, if the
sample collection date is not collated against the woman’s
menstrual cycle calendar.16,17 (See also page 6.)
OM-MA (CA125)
Results from the Multicenter Tumor Marker Reference
Range Study.
IMMULITE OM-MA: Females
40
30
IMMULITE OM-MA: Ovulatory Cycles
20
50
10
0
20
30
40
50
60
70
Age, years
zb148-b, lkom-f, 19-feb-99
IMMULITE OM-MA, kU/L
Females
n
5%
50%
474
2.6
6.1
95% 97.5%
18
24
OM-MA (CA125), kU/L
OM-MA (CA125), kU/L
50
40
30
20
10
0
-100%
-50%
0%
50%
100%
Percent of Follicular or Luteal Phase
During the course of the Multicenter Tumor Marker
Reference Range Study, the IMMULITE OM-MA kit was
reformulated: the fifth (UK) site used the newer version.
The data sets were combined because the reformulation
had no apparent impact on the distribution of results, as
shown in the following graph.
zb148-b, lkop_cyc, 19-feb-99
IMMULITE OM-MA
IMMULITE OM-MA: Two Formulations
OM-MA (CA125), kU/L
50
40
30
Catalog Numbers
LKOPZ (50 tests)
LKOP1 (100 tests)
LKOP5 (500 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
Sample
50 µL serum
Incubation
60 min
Calibration Range
Up to 500 kU/L (U/mL)
Detection Limit
0.2 kU/L
Hook
None up to 84,500 kU/L
IMMULITE 2000 OM-MA
20
10
Catalog Numbers
L2KOP2 (200 tests)
Methodology
Chemiluminescent enzyme
immunometric assay
Sample
50 µL serum
Incubation
60 min
Calibration Range
Up to 500 kU/L (U/mL)
Detection Limit
0.30 kU/L
Hook
None up to 80,000 kU/L
0
20
30
40
50
60
70
Age, years
zb148-b, lkop_uk, 19-feb-99
21
14. Dalkin BL, Ahmann FR, Kopp JB, et al. Derivation
and application of upper limits for prostate specific
antigen in men aged 50-74 years with no clinical
evidence of prostatic carcinoma. Br J Urol
1995;76:346-50.
References
1.
2.
Sibley PEC. Tumor marker assays; the significance of
normal range studies. News & Views (DPC) 1999
Fall;13(4):6-8. Available at DPC's Web site,
www.dpcweb.com, under Technical Documents, News
& Views, Fall 1999.
Stamey TA. Lower limits of detection, biological
detection limits, functional sensitivity, or residual
cancer detection limit? sensitivity reports on prostatespecific antigen assays mislead clinicians. Clin Chem
1996;42:849-52.
3.
Diamandis EP, Yu H, Melegos DN. Ultrasensitive
prostate-specific antigen assays and their clinical
application. Clin Chem 1996;42:853-7.
4.
Muenz LR, Sizaret P, Bernard C, et al. Results of the
second international study on the W.H.O. alphafoetoprotein standard. J Biol Stand 1978;6:187-99.
5.
Nustad K, Paus E, Kierulf B, Bormer OP. Specificity
and affinity of 30 monoclonal antibodies against
alpha-fetoprotein. Tumour Biol 1998;19:293-300.
6.
Sturgeon CM, Seth J. Why do immunoassays for
tumour markers give differing results?— a view from
the UK National External Quality Assessment
Schemes. Eur J Clin Chem Clin Biochem
1996;34:755-9.
7.
15. Fritsche HA, Bast RC. CA 125 in ovarian cancer;
advances and controversy. Clin Chem 1998;44:137980.
16. Sibley PEC, Vankrieken L, et al. Impact of the
menstrual cycle on BR-MA (CA15-3) and OM-MA
(CA125) values, as determined by automated
chemiluminescent assays on the IMMULITE
Analyzer [abstract 385]. Clin Chem
1999;45(S6):A109. Full presentation available at
DPC's Web site, www.dpcweb.com, under Technical
Documents, Scientific Posters.
17. Sibley PEC. OM-MA (CA125) and ovarian cancer.
News & Views (DPC) 1999 Summer;13(3):12-4.
Available at DPC's Web site, www.dpcweb.com,
under Technical Documents, News & Views, Summer
1999. Also available, in printed form and at the Web
site, as technical report ZB195.
18. Meden H, Fattahi-Meibodi A. CA 125 in benign
gynecological conditions. Int J Biol Markers
1998;13:231-7.
19. Wilson AP, van Dalen A, Sibley PEC, Kasper LA,
Durham AP, El Shami AS. Multicentre tumour
marker reference range study. Anticancer Res
1999;19(4A):2749-52.
National Committee for Clinical Laboratory
Standards. How to define and determine reference
intervals in the clinical laboratory; approved
guideline. Wayne, PA: NCCLS, 1995. NCCLS
Document C28-A.
8.
Stenman UH. Prostate-specific antigen, clinical use
and staging; an overview. Br J Urol 1997;79 Suppl
1:53-60.
9.
DeAntoni EP, Crawford ED, Oesterling JE, et al.
Age- and race-specific reference ranges for prostatespecific antigen from a large community-based study.
Urology 1996;48:234-9.
20. Wilcox RR. Introduction to robust estimation and
hypothesis testing. New York: Academic Press, 1997.
21. Kirollos MM. Statistical review and analysis of the
relationship between serum prostate specific antigen
and age. J Urol 1997;158:143-5.
10. Dalkin BL, Ahmann FR, Kopp JB. Prostate specific
antigen levels in men older than 50 years without
clinical evidence of prostatic carcinoma. J Urol
1993;150:1837-9.
11. Oesterling JE, Jacobsen SJ, Chute CG, et al. Serum
prostate-specific antigen in a community-based
population of healthy men; establishment of agespecific reference ranges. JAMA 1993;270:860-4.
12. Harris EK, Boyd JC. Statistical bases of reference
values in laboratory medicine. New York: Marcel
Dekker, 1995.
13. Wright EM, Royston P. Calculating reference
intervals for laboratory measurements. Stat Methods
Med Res 1999;8:93-112.
22
ZB148 – D © 1999 DPC All Rights Reserved
Diagnostic Products Corporation
5700 West 96th Street
Los Angeles, CA 90045-5597
Tel: 800.372.1782
Tel: 310.645.8200
Fax: 310.645.9999
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