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CLIN. CHEM. 31/2, 296-298 (1985)
The Upper Limit of Normal for Thyrotropin Is 3 or 4 milli-int. units/L
A Paul Durham
Recently
Musto et al. (C/in Chem 30: 329-330, 1984) noted
importance
of accurate measurement of thyrotropin at low concentrations, the upper limit of
normal is well below the stated upper limit of normal of most
commercial tests. Here I amplify their statement.
that, despite the clinical
Additional
Keyphrases:
ods
pituitary hormones
reference
interval
‘kit”
meth-
In a recent discussion of thyrotropin (thyroid-stimulating
hormone) measurement,
Musto et al. (1) draw attention to
the clinical importance of good performance at low concentrations. A thyrotropin
assay that has adequate reproducibility throughout the “normal” range and is essentially free
of nonspecific matrix effects can be expected to confer two
benefits. First, it will minimize the number of borderline
results that will require the thyroliberin
(thyrotropin-releasing factor, TRF) test as followup. Second, it will provide
more meaningful baseline values for comparison with values obtained after TRF stimulation.
The high upper limit of normal that is characteristic
of
most commercially available thyrotropin
assays is symptomatic of their inability to provide precise and accurate
measurements
of thyrotropin
at low concentrations.
Musto
et al. (1) state without documentation
that, according to the
literature, the true upper limit of normal for thyrotropin is
less than 5.0 milli-int. units/L.
Clinicians who regularly
encounter only results for thyrotropin
generated by commercially available kits may well be inclined to doubt this
assertion. But the point made by Musto et al. is an important one, and this Note is intended to document its truth.
Studies aimed at determining
the reference interval for
normal individuals can provide important insights into the
acceptability
of an assay system. This is especially true of
assays for thyrotropin,
where an upper limit of normal
significantly higher than those reported for fully optimized
radioimmunoassays
may well be symptomatic of shortcomings that can seriously compromise clinical efficacy (1).
There is, in fact, a striking contrast between most but not
all thyrotropin
kits now on the market and the several
“research” radioimmunoassays
(2-6) tabulated
in Table 1.
The latter characteristically
show an upper limit of normal
of approximately
3 or 4 milli-int. units/L. The stated normal
range for most kits, on the other hand, has until recently
extended up to 10 milli-int. units/L.
While there has been an encouraging trend towards more
accurate thyrotropin
kits, an upper limit of 7 milli-int.
unitsfL
represents,
even today, the norm for nearly all
commercially
available assays. This shows that the great
majority of thyrotropin kits still err, on borderline samples,
by a factor of two relative to more definitive assay systems.
Indeed, it is rare to encounter any thyrotropin
procedure
with a reasonable assay time (same-day or overnight) that
Diagnostic Products
Corp., 5700 West 96th St., Los Angeles, CA
90045.
Received October 18, 1984; accepted November 27, 1984.
296 CLINICAL CHEMISTRY, Vol. 31, No. 2, 1985
Table 1. Upper Limits of Normal in Research
Assays for Thyrotropin
Thyrotropin,
mull-mt. unlts/L
Assay (and ret.)
Radioimmunoassays
Melbourne, Australia (2)
Newcastle, U.K. (3)
Munich, F.R.G. (4)
Boston, MA (5)
Los Angeles, CA (6)
Other methods
Wick chromatography/RIA (16)
Adsorption to concanavalin A/RIA (17)
Immunochemiluminescence assay (18)
Cytochemical bioassay (19, 20)
measures
accuracy
Research
3.1
2.8
3.8
3.0
3.5
3.0
4.0
4.5
2.4, 3.7
up to standards
set by the research
assays
and precision
at low concentrations
(7, 8).
Assays
for
vs Kit Methods
The systems here designated as “research” assays have
strong claims to being regarded
as reference
methods
for
thyrotropin.
First, they were established
more than a decade ago, and
since then have contributed significantly to our understanding of the physiology and clinical relevance of this pituitary
hormone. This is reflected
not only in countless journal
articles but also in standard medical textbooks and reviews,
where values ranging from 3.0 to 5.0 milli-int. units/L are
usually quoted as the accepted upper limit of normal (1, 5,
9-11).
Second, these research assays resemble the typical thyrotropin kit of today in being nonequilibrium,
competitive,
double-antibody
radioirnmunoassays.
They differ prinulpally in the degree to which they have been optimized for
sensitivity
and specificity. Comparisons
between the research assays and the kit methods are therefore in order.
Characteristically,
the research assays involve use of
meticulously purified tracer, incubation times of six days or
more, and standard curves prepared with human serum.
These features can be expected to minimize nonspecific
“matrix effects,” resulting
in lower and more accurate
values throughout the normal reference interval (12).
Some of these features appear to be essential for meaningful results, particularly
the steps taken to purify the tracer
and to eliminate matrix differences between standards and
unknowns.
It has been shown repeatedly that neither buffered human serum albumin nor an animal serum matrix is
an adequate substitute for a human serum matrix (8, 1215).
Further corroboration for an upper limit of approximately
3 or 4 milli-int. unitsfL comes from studies performed
with
various “nonstandard”
methodologies (16-20), including immunoassays
based on chemiluminescence,
separation
by
wick chromatography, or sample concentration,
as well as
the modern cytochemical
In addition, experiments
bioassay (Table 1).
in which samples were collected
from normal adults and assayed by radioimniunoassay,
both
before and after suppression by treatment with thyroid
hormones, provide evidence that the true median value for
circulating
thyrotropin
is barely 1 milhi-int. unitJL (21).
Clinical
Significance
From studies performed with the research assays, a great
deal has been learned as a result of optimizing for sensitivity and specificity, thanks to the superior delineation of the
normal reference interval that this entails.
Thus, we now know that thyrotropin exhibits a circadian
rhythm, with lower values in the morning and higher
values near midnight (15, 22). We know more exactly the
pattern exhibited by thyrotropin
during pregnancy (16,23).
We know also that there are significant age- and sex-related
variations,
with an increased
prevalence
of moderately
increased
thyrotropin
concentrations
in ostensibly normal
elderly women (24-26).
The very concept of “subclinical
hypothyroidism”-a
condition characterized
by “normal”
concentrations
of thyroid hormones in the presence of an
increased
thyrotropin
concentration-had
to await the development of fully optimized radioimmunoassays
for thyrotropin (27).
Of even greater clinical significance
is the fact that
improvements
in thyrotropin methodology have reduced the
need for TRF tests to clarify borderline results. Optimizing a
thyrotropin assay not only tends to lower values throughout
the reference interval for normal, it also tends to improve
the precision and discrimination
that can be achieved in this
region, rendering more meaningful the comparison of basal
values with those measured after TRF stimulation
(14, 28,
29). Indeed, Bigos et a!. (30) showed excellent correlation
between such values, as measured with one of the research
assays. Wide and Dahlberg
(29) also showed improved
correlation when steps were taken to optimize their thyrotropin assay for low-end sensitivity. Furthermore,
although
perfect discrimination
of hyperthyroid
samples is not yet
possible, in well-optimized thyrotropin assays a basal value
exceeding the median for norma! persons renders a diagnosis of hyperthyroidism
highly unlikely (31).
Practical
Considerations
major practical considerations
bear on the interpreannotation
of thyrotropin
results at the stage of
them to physicians.
First, reference intervals for thyrotropin that are derived
from blood donors or laboratory
volunteers
are clearly
inappropriate
for newborns
and may be inappropriate
for
certain other groups as well. In particular, they should not
be blindly
adopted
for the interpretation
of results on
samples from a maternity
ward (16,23) or a geriatric unit
(25, 26). Thus, depending
on the reference
group, 3 or 4
miuli-int. units/L may not represent the true upper limit of
normal. Accordingly,
each laboratory
should establish
by
experiment
its own expected
values (32).
Moreover, clinical decision limits should respect the distribution of values and disease in the local patient population. Decision
limits designed to optimize
the predictive
value of a test may not correspond to conventional
reference
interval
limits based on 95% coverage for normal individuals. Use of the thyrotropin
assay as a primary
screen for
congenital hypothyroidism
provides a classic illustration
of
this point (33).
Finally,
one must remember
that in some contexts
a
thyrotropin
result that is only slightly above the reference
interval
for normal individuals
may not by itself warrant
taking any corrective action (27).
Three
tation al
reporting
In deciding whether to accept or reject a thyrotropin
kit,
an important
criterion
at the stage of method evaluation
ought to be its ability to yield values for a normal
adult
population
that are similar
to values found by well-established, fully optimized referencemethods forthyrotropin
(1)..
The true upper limit of normal for young men and nonpregnant women in good health is close to 3 or 4 milli-int.
unitsfL,
according
to the literature
summarized
in this
report.
References
1. Musto JD, Pizzolante
thyrotropin measurement
(1984).
JM, Chesarone VP. A comment on the
and evaluation. Clin Chem 30, 329-330
2. Patel YC, Burger HG, Hudson B. Radioimmunoassay
of serum
thyrotropin: Sensitivity and specificity. J Clin Endocrinol Metab 33,
768-774 (1971).
3. Hall R, Amos J, Ormston BJ. Radioimmunoassay
of human
serum thyrotropin. Br Med J 1, 582-585 (1971).
4. Marschner I, Erhardt FW, Scriba PC. Ringversuch zur radioimmunologischen Thyrotropin-bestimmung
(hTSH) im Serum. J Gun
Chem Clin Biochem 14, 345-351 (1976).
5. Ridgway EC, Kourides LA, Maloof F. Thyrotropin. In Nuclear
Medicine In Vitro, B Rothfeld, Ed., J.B. Lippincott, Philadelphia,
PA, 1974, pp 205-219.
6. Pekary AE, Hershman JM, Parlow AF. A sensitive and precise
radioimmunoassay for human thyroid-stimulating
hormone. J Clin
Endocrinol Met.ab 41, 676-679 (1975).
7. Bigos ST, Pekary AE, MacLean J, et al. A thyrotropin radioimmunoassay kit evaluated vs two reference assays. Gun Chem 30,
437-439 (1984).
8. Wood WG. A “same day” TSH radioimmunoassay kit with
acceptableprecision and accuracy. NucCompact 11, 60-63 (1980).
9. Hershman JM (Ed.). Endocrine Pathophysiology:
A PatientOrien,ted Approach. Lea and Febiger, Philadelphia, PA, 1977, p 39.
10. Williams RH (Ed.). Tcrtbook of Endocrinology,
6th ed., W.B.
Saunders, Philadelphia,
PA, 1981, p 137.
11. Wintrobe MM (Ed.). Harrison’s Principles of Internal Medicine,
7th ed., McGraw-Hill, New York, NY, 1974, p 470.
12. Erhardt FW, Marschner I, Wood WG. Quality control and some
sources of error in radioimmunoassays.
Pediatr Adolesc Endocrinol
12, 170-180 (1983).
13. Hall R. Separation of bound from free labelled antigen. In
Radioimmunoassay
Methods, KE Kirkham,
WM Hunter, Eds.,
Churchill Livingstone, Edinburgh, U.K., 1971, pp 313-314.
14. Kubasik NP, Ricotta M, Hunter T, Sine HE. Clinical evaluation of two thyrotropin radioimmunoassay kits: Human serum
matrix calibrators and bovine serum matrix calibrators. Clin Chem
27, 504 (1981). Letter.
15. Parker DC, Pekary AE, Hershman JM. Effect of normal and
reversed sleep-wake
cycles upon nyctohemeral rhythmicityof
plasma thyrotropin: Evidencesuggestiveofan inhibitory
influence
in sleep. J Clin Endocrinol Metab 43, 318-329 (1976).
16. Weeke J, Dybk,jr L, Granlie K, et al. A longitudinal study of
serum TSH, and total and free iodothyronines during normal
pregnancy. Acta Endocrinol 101, 531-537 (1982).
17. Nisula BC, Louvet J-P. Radioimmunoassay
of thyrotropin
concentrated from serum. J Clin Endocrinol Metab 46, 729-733
(1978).
18. Weeks
I, Sturgess M, Siddle K, et al. A high sensitivity
immunochemiluminometric
assay for human thyrotrophin.
Clin
Endocrinol 20, 489-495 (1984).
19. Petersen V, Smith BR, Hall R. A study of thyroid stimulating
activity in human serum with the highly sensitive cytochemical
bioassay. J Clin Endocrinol Metab 41, 199-202 (1975).
20. Faglia G, Bitensky L, Pinchera A, et a!. Thyrotropin secretion
in patients with central hypothyroidism:
Evidence for reduced
biological activity of immunoreactive
thyrotropin.
J Clin Endocrinol Metab 48, 989-998 (1979).
21. Adams DD, Kennedy TH, Utiger RD. Comparison of bioassay
and immunoassay measurements
of serum thyrotropin (TSH) and
study of TSH levels by immunoassay of serum concentrates. J Clin
Endocrinol Metab 34, 1074-1079 (1972).
CLINICAL CHEMISTRY, Vol. 31, No. 2, 1985
297
22. Weeke J. The influence of the circadian thyrotropin rhythm on
the thyrotropin response to thyrotropin-releasing
hormone in normal subjects. Scand J Glin Lab Invest 33, 17-20 (1974).
23. Braunstein GD, Hershman JM. Comparison of serum pituitary
thyrotropin and chorionic gonadotropin concentrations
throughout
pregnancy. J Clin Endocrinol Metab 42, 1123-1126 (1976).
24. Nystrom E, Bengtsson C, Lindquist 0, et a!. Thyroid disease
and high concentration
of serum thyrotrophin
in a population
sample of women: A 4-year follow-up. Acts Med Scand 210, 39-46
(1981).
25. Sawin CT, Chopra D, Azizi F, et a!. The aging thyroid:
Increased prevalence of elevated serum thyrotropin levels in the
elderly. JAm Med Assoc 242, 247-250 (1979).
26. Tunbridge WMG, Evered DC, Hall R, et a!. The spectrum of
thyroid disease in a community:
The Whickham
survey.Glin
Endocrinol 7, 481-493 (1977).
27. Ridgway EC, Cooper DS, Walker H, et al. Peripheral responses
to thyroid hormone before and after L-thyroxine therapy in patients
with subclinical hypothyroidism. J Glin Endocrinol Metab 53,
1238-1242 (1981).
28. Pekary
AE, Hershman
JM, Sawin CT. Linear
modulation
of
serum thyrotropin by thyroid hormone treatment in hypothyroidism. Acts Endocrinol 95, 472-478 (1980).
29. Wide L, Dahlberg PA. Quality requirements of basal S-TSH
assays in predicting an S-TSH response to TRH. Scand J GUn Lab
Invest 155, 101-110 (1980).
30. Bigos ST, Ridgway EC, Kourides IA, Maloof F. Spectrum of
pituitary alterations with mild and severe thyroid impairment. ,J
Clin Endocrinol Metab 46, 317-325 (1978).
31. Kubasik NP, Same DO, Brodows RG, Sine HE. Assay of
thyrotropin in hyperthyroidism.
Clin Chem 29, 1688(1983). Letter.
32. Tunbridge WMG, Hall R. Thyrohf-stimulating
hormone. In
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Loraine, ET Bell, Eds., Churchill Livingstone, London, U.K., 1976,
175-192.
33. Petersen PH, Rosleff F, RasmussenJ,
Hobolth N. Studies on the
required and analytical quality of TSH measurements in screening
for congenital hypothyroidism.
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(1980).
CLIN. CHEM. 31/2, 298-302 (1985)
Preparation of Urine Samples for Liquid-Chromatographic Determination of
Catecholamines: Bonded-Phase Phenylboronic Acid, Cation-Exchange
Resin, and Alumina Adsorbents Compared
Alan H. B. Wu and Terrie G. Gornet
We compared results for the liquid-chromatographic determination of free norepinephrine and epinephrine in urine after
purifying the catecholsby the followingmethods: (a)acidwashed alumina, (b) weak cation-exchange resin (WCX), (C)
a combination of weak cation-exchange resin followed by
alumina (WCX-alumina), and (c commercially available
phenylboronic acid adsorbent. We evaluated analytical
specificity, sensitivity, recovery, and turnaround time. The
WCX-alumina combination produced the most sensitive and
specific chromatograms for urinary catecholamines; the other methods took less processing time. Neither WCX nor
alumina alone was suitable for routine work because of
chromatographic interferences in a significant proportion of
urines. The phenylboronic acid method is adequately sensitive and specific for norepinephrine and epinephrine, and
samples can be assayed faster. Thus it provides a compromise between the high analytical performance of the WCXalumina method and the speed of the WCX and alumina
methods.
liquid chromatography
is widely used for
total and free urinary
catecholamines
with ei-
“High-pressure”
Department of Pathology and Laboratory Medicine, University of
Texas Health Science Center at Houston, Houston, TX 77025.
Presented at the 36th national meeting of the AACC, July 1984,
Washington, DC.
Received September 4, 1984; accepted November 6, 1984.
298
ic towards catecholamines,
they cannot be connected to a
source of low pressure for rapid isolation and elution.
We evaluated a simultaneous
dual-step purification procedure involving the use of chemically bonded materials (6)
(ion-exchange
and phenylboronic
acid adsorbents:
Bond
Elut; Analytichem
International,
Harbor City, CA 90710)
for rapidly isolating free catecholamines
and compared
results by this method with results obtained on using weak
cation-exchange
resin and alumina.
Materials and Methods
AddItIonal Keyphrases: norepinephrine
. epinephrine
.
cer
pheochromocytoma
neuroblastoma
. screening
measuring
ther electrochemical
or fluorometric detectors.
In most electrochemical
methods, the amines are isolated by a two-step
procedure
before injection into the chromatograph.
Materials used for this include alumina, cation-exchange
resins,
and Sephadex (1-4). With each of these techniques
the
catecholamines
are adequately separated
from other urine
components, but each is time consuming.
Boric acid gels, which adsorb compounds
containing
ciadiol groups, have been used recently
to isolate urinary
catecholamines
(2,5). Although these gels are higi’y specif-
CLINICAL
CHEMISTRY,
Vol. 31, No. 2, 1985
Standards
and reagents.
Norepinephrine
(NE),
epineph-
rime (E), dopamine,
and the internal standard,
3,4-dihydroxybenzylaimne,
were from Sigma Chemical Co., St. Louis,
MO 63178, as were all of the compounds
in the interference
study.’ Acid-washed
alumina
(Al203), prepared
by the
method of Anton and Sayre (7), was from Bioana!ytical
Systems, West Lafayette,
IN 47905. The weak cation1 Nonstandard
abbreviations: NE, norepinephrine; E, epinephrine; WCX, weak cation-exchange; PBA, phenylboronic acid; PSA,
primary and secondary amine ion-exchange.