Download were balanced by increased serum cystatin C

Clinical Chemistry 44, No. 12, 1998
were balanced by increased serum
cystatin C, which in addition to kininogens and a2-macroglobulin is the
most important inhibitor for controlling the proteolytic activity of extracellular cysteine proteases. In melanoma we found significant increases
(P 5 0.02) in the cystatin C concentration among patients with metastatic disease and smaller increases
in patients with primary melanoma
(Fig. 1), indicating the up-regulation
of cystatin C in later events of tumor
progression. In colorectal cancer, serum concentrations of cystatin C
were significantly increased (P
,0.0001) in patients at all Dukes
stages, correlating weakly with patient age and gender (unpublished
data). The correlation between cystatin C and creatinine serum values (7),
however, was much weaker in cancer patients than that reported for
healthy controls (3), suggesting the
influence of nonrenal factors on the
concentration of cystatin C in malignant sera. The creatinine values, not
significantly changed in cancer patients, suggest that patients’ renal
function had not been altered at the
time of sample collection.
In our opinion the number of patients included in previous studies
was too low to provide relevant information about changes in the cystatin C serum concentration during
malignant progression. The results
of our studies, which involved 401
patients with colorectal cancer, 97
patients with melanoma, and 124
healthy controls, strongly support
the need for further evaluation of
cystatin C as a marker for glomerular filtration rate determination, at
least in cancer patients, to determine its potential for use in clinical
practice.
References
1. Kyhse-Andersen J, Schmidt C, Nordin G,
Andersson B, Nilsson-Ehle P, Lindstrom V,
Grubb A. Serum cystatin C, determined by a
rapid, automated particle-enhanced turbidimetric method, is a better marker than serum
creatinine for glomerular filtration rate. Clin
Chem 1994;40:1921– 6.
2. Finney H, Newman DJ, Gruber W, Merle P,
Price CP. Initial evaluation of cystatin C measurement by particle-enhanced immunonephelometry on the Behring nephelometer
systems (BNA, BN II). Clin Chem 1997;43:
1016 –22.
3. Newman DJ, Thakkar H, Edwards RD, Wilkie
M, White T, Grubb AO, Price CP. Serum
cystatin C measured by automated immunoassay: a more sensitive marker of changes in
GFR than serum creatinine. Kidney Int 1995;
47:312– 8.
4. Sloane BF. Suicidal tumor proteases. Nat Biotech 1996;14:826 –7.
5. Lah T, Kos J. Cysteine proteinases in cancer
progression and their clinical relevance for
prognosis. Biol Chem 1998;379:125–30.
6. Kos J, Nielsen HJ, Kras̆ovec M, Christensen
IJ, Cimerman N, Stephens RW, Brünner N.
Prognostic values of cathepsin B and carcinoembryonic antigen in sera of patients with
colorectal cancer. Clin Cancer Res 1998;4:
1511– 6.
7. Kos J, S̆tabuc B, Schweiger A, Kras̆ovec M,
Cimerman N, Kopitar-Jerala N, Vrhovec I. Cathepsins B, H, L, their inhibitors stefin A and
cystatin C in sera of melanoma patients. Clin
Cancer Res 1997;3:1815–22.
Janko Kos1,2*
Borut S̆tabuc3
Nina Cimerman2
Nils Brünner4
1
Department of Biochemistry
and Molecular Biology
Joz̆ef Stefan Institute
Jamova 39, 1000 Ljubljana, Slovenia
2
Research and Development Division
KRKA, d.d.
8000 Novo mesto, Slovenia
3
Institute of Oncology
1000 Ljubljana, Slovenia
4
Finsen Laboratory
Rigshospitalet
Copenhagen 2100, Denmark
*Author for correspondence.etters
Anti-Thyrotropin Antibody Interference
in Thyrotropin Assays
To the Editor:
We read with interest the paper by
Després and Grant (1) on antibody
interference in thyroid assays. Thyroid
hormone autoantibodies, heterophile
antibodies, and rheumatoid factors are
certainly the main sources of artifacts.
As mentioned by the authors, antithyrotropin (anti-TSH) antibodies are
more uncommon but may nevertheless deserve additional comments.
The existence of anti-TSH antibodies
in patient sera has been reported after
injections of bovine TSH (2, 3). The
2557
antibodies also appear in autoimmune
thyroid diseases such as Graves disease, Hashimoto thyroiditis, silent thyroiditis, and subacute thyroiditis (4–7),
and nonthyroid autoimmune disease
(6). In sera from patients with Graves
disease, the possibility that thyrotropin receptor antibodies (TRAbs)
may be anti-idiotype antibodies
against anti-TSH antibodies or that anti-TSH antibodies may be anti-idiotype
antibodies against TRAbs is controversial (8–12). Most of the reported antiTSH antibodies reacted against bovine
TSH; however, some also reacted
against human TSH (4, 12–14).
The results of published studies on
anti-TSH antibody interference in TSH
assays concerned mainly RIAs. In
those cases, depending on the assay
design and the antibody specificity,
interference may yield lower or increased values. Increased results were
found with the double antibody techniques (4, 5, 13, 15–17). Single antibody techniques with polyethylene
glycol (PEG) precipitation yielded
low values (14, 15). Fewer results
have been reported with the widely
used, “sandwich” immunometric assays (IMAs). IMA results have been
found to be lower (5) or similar to
double antibody results (6). Moreover, different IMA kits may yield
discrepant values (14).
We previously reported (18) TSH
concentrations that we measure (19)
with eight different third-generation
IMAs in four serum samples that contained
anti-TSH
antibodies
as
determined by increased precipitation
of protein-bound 125I bovine or human
TSH. Two samples from patients with
autoimmune
thyroid
disorders
(Graves disease and postpartum thyroiditis) contained only anti-bovine
TSH antibodies. The results of the different TSH kits were not grossly discrepant, ranging from 0.36 to 0.60
mIU/L and from 2.9 to 4.7 mIU/L for
the two samples, respectively. The
other two sera contained both antibovine and anti-human TSH antibodies. In the first case, our suspicion was
aroused because the high serum TSH
contrasted with an apparently healthy
clinical picture. The second case was
from a euthyroid woman who had
given birth to two children with tran-
2558
sient neonatal hyperthyrotropinemia,
a case similar to those presented in
previous reports (17, 20). This patient’s
serum contained neither stimulating
nor blocking TRAbs. The thyroid-stimulating TRAbs were checked by measuring the stimulating activity (cAMP
production) of the serum on thyroid
cells. Blocking TRAbs were ruled out
because, after preincubation of the serum with thyroid cells followed by
two wash steps, added TSH displayed
normal stimulating activity. This serum blocked the stimulating activity of
TSH on thyroid cells only when TSH
and the serum were incubated simultaneously. We considered that this serum contained anti-TSH antibodies capable of inhibiting the stimulating
activity of TSH. Measurements with
the different kits yielded, for these two
euthyroid patient sera containing antihuman TSH antibodies, TSH results
that were highly discrepant, ranging
from 2.2 to 36.6 mIU/L and from 2.1 to
13.9 mIU/L, respectively [see Sapin et
al. (18) for details]. Of 12 values, 9 were
in the hypothyroid range. As measured by the BeriLux kit, estimates of
TSH concentrations in these samples
were within the health-related reference interval after immunoglobulins
were eliminated through pretreatment
with 250 mL/L PEG and centrifugation for 30 min at 4 °C (18). After this
treatment, the mean TSH recovery was
66% in control sera without anti-TSH
antibodies. This decreased recovery
value was taken into account when the
results after PEG treatment were calculated (1.2 mIU/L for both patients).
Our observations underline the possibility of misinformation in TSH assays because of anti-TSH antibodies,
even with recent kits. For the first two
patients, variation between kits could
be explained in part by the bias we
observed between the results of the
different kits in euthyroid control patients (19, 21). The median ranged
from 1.0 to 1.9 mIU/L. This is definitely not the case for the last two
patients. During this study, which has
spanned .5 years, 300 000 TSH determinations have been performed by the
two laboratories in which these four
cases were found. The incidence of
anti-TSH antibodies may exceed 4 in
300 000 because some cases may have
Letters
been missed. However, the incidence
of TSH results showing a gross discordance either with the clinical status of
the patient or between methods because of anti-TSH antibodies could be
only 2 in 300 000.
Despite the forthcoming preclusion of bovine TSH injections (22)
and despite the fact that recombinant
human TSH (Thyrogen) does not
seem to be a potent immunogen (23),
anti-bovine and anti-human TSH antibodies may both be spontaneously
present in the sera of thyroidal ill or
clinically healthy patients. Moreover,
it is worth noting that anti-TSH antibodies have been consistently reported to be a source of interference
in TRAb assays, yielding negative
TRAb values (5–7, 12, 13, 24). This interference frequently contributed to
the discovery of anti-TSH antibodies.
This work was supported by the
Hôpitaux Universitaires de Strasbourg. We thank H. Bornet for the
anti-human TSH antibody determinations and A.M. Madec for measuring the stimulating and blocking activities on thyroid cells. We also
thank N. Heider for carefully reviewing the manuscript.
9.
10.
11.
12.
13.
14.
15.
16.
17.
References
1. Després N, Grant AM. Antibody interference in
thyroid assays: a potential for clinical misinformation. Clin Chem 1998;44:440 –54.
2. Hays MT, Solomon DH, Beall GN. Suppression
of human thyroid function by antibodies to
bovine thyrotropin. J Clin Endocrinol Metab
1967;27:1540 –9.
3. Melmed S, Harada A, Hershman JM, Krishnamurthy GT, Blahd WH. Neutralizing antibodies to bovine thyrotropin in immunized patients
with thyroid cancer. J Clin Endocrinol Metab
1980;51:358 – 63.
4. Chaussain JL, Binet E, Job JC. Antibodies to
human thyrotropin in the serum of certain hypopituitary dwarfs. Rev Eur Etudes Clin Biol
1972;17:95–9.
5. Ochi Y, Nagamune T, Nakajima Y, Ishida M,
Kajita Y, Hachiya T, Ogura H. Anti-TSH antibodies in Graves’ disease and their failure to
interact with TSH receptor antibodies. Acta
Endocrinol (Copenhag) 1989;120:773–7.
6. Sakata S, Takuno H, Nagai K, Kimata Y,
Maekawa H, Yamamoto M, et al. Anti-bovine
thyrotropin autoantibodies in patients with
Hashimoto’s thyroiditis, subacute thyroiditis,
and systemic lupus erythematosus. J Endocrinol Investig 1991;14:123–30.
7. Yamamoto M, Fuwa Y, Chimori K, Yamakita N,
Sakata S. A case of progressive systemic sclerosis (PSS) with silent thyroiditis and anti-bovine thyrotropin antibodies. Endocrinol Jpn
1991;38:265–70.
8. Beall GN, Kruger SR. Binding of 125I-human
TSH by gamma globulins of sera containing
18.
19.
20.
21.
22.
23.
24.
thyroid-stimulating immunoglobulin (TSI). Life
Sci 1983;32:77– 83.
Raines KB, Baker JR Jr, Lukes YG, Wartofsky L,
Burman KD. Antithyrotropin antibodies in the
sera of Graves’ disease patients. J Clin Endocrinol Metab 1985;61:217–22.
Akamizu T, Mori T, Imura H, Noh J, Hamada N,
Ito K, et al. Clinical significance of anti-TSH
antibody in sera from patients with Graves’
disease and other thyroid disorders. J Endocrinol Investig 1989;12:483– 8.
Cho BY, Shong YK, Lee HK, Koh CS, Min HK.
Anti-bovine TSH antibodies in patients with
Graves’ disease and primary myxedema. Thyroidology 1989;1:31–7.
Noh J, Hamada N, Saito H, Oyanagi H, Ishikawa
N, Momotani N, et al. Evidence against the
importance in the disease process of antibodies to bovine thyroid-stimulating hormone
found in some patients with Graves’ disease.
J Clin Endocrinol Metab 1989;68:107–13.
Akamizu T, Mori T, Kasagi K, Kosugi S, Miyamoto M, Nishino K, et al. Anti-TSH antibody
with high specificity to human TSH in sera from
a patient with Graves’ disease: its isolation
from, and interaction with, TSH receptor antibodies. Clin Endocrinol 1987;26:311–20.
Ochi Y, Inui T, Hachiya T, Nakajima Y, Ishida M,
Kajita Y, et al. Coexistence of autoantibody to
human thyrotropin (TSH) and autoanti-idiotypic
antibody to antihuman TSH antibody in a case
with simple goiter. J Clin Endocrinol Metab
1990;71:1163–7.
Sain A, Sham R, Singh A, Silver L. Erroneous
thyroid-stimulating hormone radioimmunoassay results due to interfering antibovine thyroid-stimulating hormone antibodies. Am J Clin
Pathol 1979;71:540 –2.
Frohman LA, Baron MA, Schneider AB. Plasma
immunoreactive TSH: spurious elevation due to
antibodies to bovine TSH which cross-react
with human TSH. Metabolism 1982;31:
834 – 40.
Lazarus JH, John R, Ginsberg J, Hughes IA,
Shewring G, Smith BR, et al. Transient neonatal hyperthyrotrophinemia: a serum abnormality due to transplacentally acquired antibody to
thyroid stimulating hormone. Br Med J 1983;
286:592– 4.
Sapin R, D’Herbomez M, Gasser F, Wemeau JL,
Schlienger JL. Analytical limitations of thyrotropin assays. J Clin Ligand Assay 1996;19:
198 –202.
D’Herbomez M, Sapin R, Gasser F, Schlienger
JL, Wemeau JL. Two centre evaluation of seven
thyrotropin kits using luminescent detection.
Eur J Clin Chem Clin Biochem 1997;35:
609 –15.
Bachelot I, Barbe G, Orgiazzi J, Halimi S. Autoanticorps anti-TSH: étude chez une mère et son
nouveau né [Abstract]. Ann Endocrinol (Paris)
1987;48:201.
Sapin R, d’Herbomez M, Gasser F, Schlienger
JL, Wemeau JL. Evaluation de sept trousses de
dosage immunométrique de TSH avec marqueur luminescent. Immunoanal Biol Spéc
1996;11:379 – 87.
Emerson CH, Colzani R, Braverman LE. Epithelial cell thyroid cancer and thyroid stimulating
hormone–when less is more [Editorial]. J Clin
Endocrinol Metab 1997;82:9 –10.
Ramirez L, Braverman LE, White B, Emerson
CH. Recombinant human thyrotropin is a potent stimulator of thyroid function in normal
subjects. J Clin Endocrinol Metab 1997;82:
2836 –9.
Akamizu T, Ishii H, Mori T, Ishihara T, Ikekubo
K, Imura H. Abnormal thyrotropin-binding immunoglobulins in two patients with Graves’ disease. J Clin Endocrinol Metab 1984;59:
240 –5.
2559
Clinical Chemistry 44, No. 12, 1998
Rémy Sapin1*
Michèle d’Herbomez2
Jean Louis Schlienger3
Jean Louis Wemeau4
1
Laboratoire Universitaire
de Biophysique
Unité d’Analyses Endocriniennes
CNRS UPRES-A 7004
Université Louis Pasteur
Faculté de Médecine
67085 Strasbourg Cedex, France
2
Service Central
de Médecine Nucléaire
Hôpital Salengro
CHRU
59037 Lille Cedex, France
3
Service de Médecine Interne
Hôpital de Hautepierre
67098 Strasbourg Cedex, France
4
Clinique Marc Linquette
Unité de Soins Normalisés A
CHRU
59037 Lille Cedex, France
*Address correspondence to this author at: Institut de Physique Biologique,
Faculté de Médecine, 67085 Strasbourg
Cedex, France. Fax 00 33 3 88 14 48 61;
e-mail [email protected].
Evaluation of Two Automated
Immunoassays for Measurement of
Free Deoxypyridinoline in Urine Using
Analytical Goals Derived from
Biological Variation
To the Editor:
Free deoxypyridinoline (fDPD) is increasingly used as a specific marker of
bone resorption (1). To date, the concentration of fDPD in urine has been
measured using cumbersome HPLC
or manual microtiter-based ELISA
procedures, which require ;4 h to
perform (2). Recently, two rapid, fully
automated chemiluminescent immunoassays were developed: by Chiron
Diagnostics for the ACS:180® analyzer
and by DPC® for the Immulite® analyzer. Both immunoassays use a competitive format involving the same
monoclonal anti-fDPD antibody from
Metra Biosystems (3, 4). If sufficiently
reliable, these assays can accommo-
date increasing testing demands with
both controlled operating costs and
dramatically reduced turnaround
time. This study assessed their analytical performance for the routine measurement of fDPD in our clinical laboratory. In particular, as suggested
previously (5), goals for precision and
accuracy were based on the biological
variation of fDPD excretion in urine
from healthy premenopausal women,
and the results obtained during the
evaluation were compared with these
to assess acceptability (6).
All measurements on the two instruments were performed according
to the recommendations of the manufacturers by the same trained technician. Linearity in the working ranges
of the tests (ACS:180, 2–350 nmol/L;
Immulite, 7–300 nmol/L) was good
(r 5 0.999 for the ACS:180; r 5 0.998
for the Immulite). The data for the
imprecision study, which used two
pooled human urines, are summarized in Table 1. Eighty-one urine samples (first morning void) with fDPD
concentrations ranging from 13 to 184
nmol/L were assayed, using the
(Metra
Biosystems)
Pyrilinks®-D
method as the reference (x) and the
two automated procedures (y) in a
correlation study. The following results were obtained: ACS:180 5 0.96
(60.03)x 2 0.8 (63.0); Syux 5 12.9
nmol/L; r 5 0.9552; and Immulite 5
0.99 (60.03)x 1 9.8 (62.8); Syux 5 12.1
nmol/L; r 5 0.9629.
When biology is used to set analytical goals, desirable imprecision (CV)
is less than or equal to one-half of the
average within-subject biological variation (i.e., for urinary fDPD, total CV
#6.7%), and desirable inaccuracy
(bias) is less than or equal to onequarter of the group (within-subject
Table 1. Imprecision of the evaluated
immunoassays.
Mean fDPD
ACS:180
82 nmol/L
304 nmol/L
Immulite
78 nmol/L
289 nmol/L
Within-run CV
(n 5 20)
Between-day
CV (n 5 10)
4.1%
2.6%
9.0%
4.2%
5.6%
4.7%
3.9%
3.2%
plus between-subject) biological variation (i.e., for urinary fDPD, average
bias #5.5%) (6). From our experimental results, we conclude that the ACS:
180 assay is probably accurate but is
too imprecise for the between-day
evaluation and that the Immulite assay
shows good precision but also has a
significant, constant positive bias. The
final considerations depend on medical needs (7). Because the bone markers are useful adjuncts in monitoring
patients and not in screening for bone
disorders, low imprecision (at least as
good as the goal) is required, whereas
some degree of inaccuracy is probably
less important (8).
We acknowledge the expert technical assistance of Cristina Serena.
We also thank Chiron Diagnostics
(Cassina de’ Pecchi, Milano, Italy)
and Medical Systems (Genova, Italy)
for the generous loan of instruments
and reagents to carry out the study.
References
1. Knott L, Bailey AJ. Collagen cross-links in mineralizing tissues: a review of their chemistry,
function, and clinical relevance [Review]. Bone
1998;22:181–7.
2. James IT, Walne AJ, Perrett D. The measurement of pyridinium crosslinks: a methodological overview [Review]. Ann Clin Biochem 1996;
33:397– 420.
3. Chen SY, Sickel M, Kline S, Corkery J, BerardAubin J, Hesley R. An automated chemiluminescent immunoassay for deoxypyridinoline: a specific urinary marker for bone resorption
[Abstract]. Clin Chem 1995;41:S40.
4. Barkley J, Lei JD, El Shami AS. Immulite
Pyrilinks-D: a random-access immunoassay for
deoxypyridinoline in the monitoring of bone resorption [Abstract]. Clin Chem 1997;43: S276.
5. Fraser CG. Data on biological variation: essential
prerequisites for introducing new procedures?
[Editorial]. Clin Chem 1994;40: 1671–3.
6. Panteghini M, Pagani F. Biological variation in
urinary excretion of deoxypyridinoline [Abstract]. Clin Chem 1995;41:S200.
7. Fraser CG, Hyltoft Petersen P. Desirable standards for laboratory tests if they are to fulfill
medical needs. Clin Chem 1993;39:1447–55.
8. Beck Jensen JE, Kollerup G, Sorensen HA, Pors
Nielsen S, Sorensen OH. A single measurement of biochemical markers of bone turnover
has limited utility in the individual person.
Scand J Clin Lab Investig 1997;57:351– 60.
Mauro Panteghini*
Franca Pagani
1st Laboratorio Analisi
Chimico-Cliniche
Spedali Civili
25123 Brescia, Italy
*Author for correspondence. Fax 39 030
3995369; e-mail [email protected].