Clinical Chemistry 46: 1681-1685, 2000;
(Clinical Chemistry. 2000;46:1681-1685.)
© 2000 American Association for Clinical Chemistry, Inc.
Sensitivity and Specificity of Immunological Methods for the Detection of Anti-Topoisomerase I (Scl70) Autoantibodies: Results of a Multicenter Study
Nicola Bizzaro1,a,
Elio Tonutti2,
Danilo Villalta4,
Danila Bassetti5,
Renato Tozzoli6,
Fabio Manoni7,
Stefano Pirrone3,
Anna Piazza8,
Paolo Rizzotti8,
Marco Pradella9 and
for The Italian Society of Laboratory Medicine Study Group on the Diagnosis of Autoimmune Diseases
1
Laboratorio di Patologia Clinica, Ospedale Civile, 30027 S. Donà di Piave (VE), Italy.
2
Istituto di Chimica Clinica and
3
Divisione
4 Medica, Azienda Ospedaliera "S. Maria della Misericordia", 33100
Udine, Italy.
4
Servizio di Immunologia e Microbiologia, Azienda
Ospedaliera S. Maria degli Angeli, 33170 Pordenone, Italy.
5
Patologia Clinica II, Ospedale S. Chiara, 38100 Trento,
Italy.
6
Laboratorio Analisi Chimico-Cliniche e Microbiologia,
Ospedale Civile, 33053 Latisana (UD), Italy.
7
Dipartimento di Medicina di Laboratorio, Ospedale
Civile, 30015 Chioggia (VE), Italy.
8
Laboratorio Analisi Chimico-Cliniche, Ospedale
Geriatrico, 35100 Padova, Italy.
9
Laboratorio Analisi Chimico-Cliniche e Microbiologiche,
Ospedale Civile, 31033 Castelfranco, Veneto (TV), Italy.
a Author for correspondence. Fax 39-0421-227571; e-mail nbizzaro{at}dacos.it
 |
Abstract
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Background: The ability of immunometric methods to identify
anti-topoisomerase I (Scl70) antibodies is controversial. We
wished to quantify the performance of the currently available
commercial systems for the assay of anti-topoisomerase I antibodies in
a large multicenter study.
Methods: Fifty Italian clinical laboratories analyzed 36 serum
samples: 27 from individuals with scleroderma/systemic sclerosis, and 9
from a control group. The scleroderma/systemic sclerosis samples were
positive in our laboratories by both ELISA and immunoblot (IB), and the
control samples were negative. The laboratories used 42 immunoenzymatic
(ELISA), 21 IB, 3 counterimmunoelectrophoresis, and 2 dot-blot methods,
produced by 23 different manufacturers.
Results: We obtained 2389 results. The ELISA methods showed
99.2% specificity and 97.2% sensitivity for detection of anti-Scl70
antibodies. For IB methods, specificity was 97.6% and sensitivity was
96.1%. The Western-blot method had poor analytical specificity (27%
false positives for anti-extractable nuclear antigen antibodies other
than anti-Scl70).
Conclusions: Excluding Western blots, commercial ELISA and IB
reagents as used in clinical laboratories have a sensitivity and a
specificity >95% for determination of anti-Scl70 antibodies.
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Introduction
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The anti-topoisomerase I antibodies (anti-topo I and anti-Scl70)
are serological markers of diffuse systemic sclerosis (1)
and among its fundamental diagnostic-classification criteria
(2)(3). This rare but serious disease also
involves very high social and economic costs (4), and the
contribution of the autoimmunology laboratory to its diagnosis is
essential, thus requiring very high quality analysis. Studies conducted
by other workers and our group (5)(6)(7)(8)(9)(10)(11)(12) that addressed the
reliability of the immunometric methods in identifying anti-Scl70
antibodies have furnished discordant results, with very variable
sensitivities and specificities. Indeed, this phenomenon was confirmed
by a recent cooperative study (13) that evidenced remarkable
differences in sensitivity (range, 20–100%) and specificity (range,
85–100%) although CDC/WHO referral sera were used (14).
Moreover, in addition to the differences in the designs of the above
studies, the variations encountered might also be explained by the
diverse characteristics and numbers of the patients examined as well as
differences in the type of laboratory involved and the reagents used.
In addition, most of these studies involved selected reference
laboratories that used home-made methods, whereas most clinical
laboratories use only commercial reagents.
In recent years, the use of antigenic substrates obtained with
molecular biology techniques and the optimization of methods for the
extraction and purification of native antigens have contributed to a
more precise characterization and identification of these and other
autoantibodies indicative of autoimmune diseases. Moreover, the
continuous development of analytical techniques has made possible the
use of new commercial tests for the determination of the various
autoantibody specificities in an ever increasing number of
laboratories, thus requiring continuous verification of quality. For
this reason, the Study Group on the Diagnosis of Autoimmune Diseases of
the Italian Society of Laboratory Medicine has performed an extensive
multicenter study involving hospital clinical laboratories to analyze
serum samples from patients affected by systemic sclerosis, using
numerous commercial methods and reagent set to define present-day
reliability in determining anti-topoisomerase I autoantibodies.
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Materials and Methods
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Fifty clinical laboratories in 14 Italian regions participated in
this study that included the determination of anti-extractable nuclear
antigen
(anti-ENA)1
antibodies in 36 serum samples, 27 of which were obtained
from patients with a clinical diagnosis of scleroderma/systemic
sclerosis (SSc), according to the criteria of the American College of
Rheumatology (15). Sixteen of these samples were obtained
from subjects with diffuse cutaneous SSc (anti-Scl70 positive and
anti-centromere negative), and 11 were from patients with limited
cutaneous SSc (anti-Scl70 negative and anti-centromere
positive). Two diffuse cutaneous SSc sera also displayed a
simultaneous reactivity for Ro/SSA. The expected antibody specificity
was defined not only in reference to the clinical diagnosis, but also
on the basis of preliminary results obtained with two different methods
[immunoenzymatic (ELISA) and immunoblot (IB) assays] in the four
laboratories that collected the test samples; only the samples that
were positive with both methods were included in the study. The control
group consisted of sera from three patients with systemic lupus
erythematosus (one with anti-RNP and anti-Sm, one with anti-RNP, and
one with anti-Ro/SSA), three patients with Epstein-Barr virus
infection, and three healthy subjects. The aliquoted sera were stored
at -85 °C until they were shipped. The participating laboratories
were not given any clinical information and were asked to use their
usual reagents and analytical systems to determine anti-ENA antibodies.
The 50 participating laboratories conducted 68 series of antibody
determinations for a total of 2389 results. The number of series
exceeds that of the participants because several laboratories used more
than one method: 42 laboratories used ELISA methods (61.8% of the
tests conducted); 21 used IB (30.9%), 3 used
counterimmunoelectrophoresis (CIE; 4.4%), and 2 used the dot-blot
method (2.9%). The reagents were purchased from 23 different
manufacturers (listed in Table 1
). Only one laboratory used an in-house CIE.
Statistical analyses were conducted by calculating the sensitivity of
each method and reagent set in detecting anti-Scl70 antibodies in the
16 diffuse cutaneous SSc samples, and the specificity in the other 20
samples in which this antibody was absent. We also evaluated analytical
specificity, i.e., the number of false positives that were recorded for
other anti-ENA antibodies (e.g., anti-RNP, anti-Sm, anti-Ro, anti-La,
and anti-Jo1) in sera in which such positivities were not expected.
Moreover, in reference to the IB methods, data were elaborated by
separately evaluating the two different types of commercially available
reagents: the simplified IB (for which we prefer to use the terms
line-blot or bar-blot) (16), in which optimal quantities of
ENAs are deposited on the nitrocellulose supports and in predefined
positions to facilitate the reading and interpretation of the results,
and the classical Western blot, in which the entire electrophoretic
course is instead present, with all the extractable antigens from the
cell material used.
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Results
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For all of the methods used, the sensitivity and specificity for
anti-Scl70 were 96.9% and 98.6%, respectively (Table 2
). Detailed analyses of the performances of the single reagent
set, subdivided according to method, are shown in Tables
3-T4-T5,
4, and
5 for ELISA, IB, and CIE methods, respectively.
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Table 2. False positives and false negatives for anti-Scl70
antibody, according to the method used by the participating
laboratories.
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elisa method (1481 results)
For anti-Scl70 detection, the specificity was excellent (99.2%)
and the sensitivity was good (97.2%). Although it was reported that
the systems using recombinant antigens have a better sensitivity than
those using native antigens (17), this observation was not
confirmed in this study; the mean sensitivity of the 30 laboratories
that used native antigens was better than that obtained by the 12
laboratories that used recombinant antigens (98.3% vs 89.4%).
Specificity data obtained with the two types of antigen preparations
overlapped (99.4% vs 98.3%).
ib method (729 results)
The performance of the reagents utilized in this method was
slightly lower than that of the ELISA methods: specificity was 97.6%,
and sensitivity was 96.1% for anti-Scl70. Bar-blot and Western-blot
methods did not demonstrate different specificities (false positives,
2.4% vs 2.5%) or sensitivities (false negatives, 4.3% vs 3.5%) for
anti-Scl70 (Table 4
); however, remarkable differences emerged regarding
analytical specificity because of the high number of false positives
for anti-ENA antibodies other than anti-Scl70 obtained with the
Western-blot method (23%) compared with the bar-blot method (7.8%;
Table 6
).
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[in this window]
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Table 6. Analytical specificity (false-positive reactions for
anti-ENA antibodies other than anti-Scl70) according to the methods
used by the participating
laboratories.
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cie method (108 results)
Although specificity was quite good (96.7%), sensitivity was poor
(72.9%) because of the single test conducted with in-house reagents
whose sensitivity was seen to be entirely insufficient (68.7% false
negatives; Table 5
). When this in-house method was excluded and only
the commercial ones considered, sensitivity and specificity (93.8% and
95%, respectively) were comparable to other methods.
dot-blot method (71 results)
The best performance was obtained with this method, which showed
100% sensitivity and specificity. However, this finding should be
considered with caution because of the small number of laboratories and
observations involved.
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Discussion
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Anti-topoisomerase I antibody is specific for patients affected by
scleroderma (18). The prevalence is 
40%, but the range
is very wide (3–75%) (19)(20), and highest
values are observed in patients with the diffuse form for which it is
the diagnostic antibody (21)(22). Its presence
has been associated with a poorer prognosis and increased pulmonary
involvement (23)(24). The early and correct
identification of this antibody is thus crucial in both the diagnostic
and prognostic phases of the disease and requires very accurate data
from the autoimmunology laboratory. The objective of this study,
therefore, was to evaluate the sensitivities and specificities of the
various methods/reagent sets now on the market and the variability
among autoimmunology laboratories in assaying the anti-topoisomerase I
antibody.
Unlike some previous studies that had difficulty in identifying this
type of antibody
(6)(10)(12)(13), the
excellent results in terms of both sensitivity and specificity obtained
with all the methods/reagent sets used in this study allow us to state
that, in general, the commercial systems for determining anti-Scl70
antibodies are reliable. The immunoenzymatic methods, which constituted
60% of the methods/reagent sets used, were determinant in reaching
such high efficiency and produced the best results.
It is worth mentioning that the reagents made up of native antigens
(obtained from either HEp-2 human cells or bovine or rabbit thymus)
yielded better results in terms of sensitivity than reagents containing
recombinant antigens (obtained from both eukaryotic and prokaryotic
systems), whereas no substantial differences in terms of specificity
were observed. This means that the consistent improvement in the
techniques of purification of the native antigens, at least as far as
Scl70 antigen is concerned, has allowed the production of sufficiently
pure preparations, devoid of significant antigenic contamination, while
maintaining the characteristics of molecular identity unchanged. The
sensitivity and specificity thus obtained are comparable, if not
superior, to those of preparations using proteins expressed by
recombinant DNA technology.
Concerning analytical specificity, good performance was obtained with
all of the methods evaluated with the exception of Western blot, which
demonstrated poor specificity, suggesting that interpretative
difficulties remain the main problem in assigning antibody positivity.
That this difficulty in interpretation is attributable not only to the
capacity and experience of the operator, but also to an intrinsic
problem in the method appears evident from the following observation.
One of the four producers of Western-blot methods who furnished the
material to the laboratories has a computerized system for reading the
strips by means of a scanner and a software application for the
interpretation of the strips. Of the four laboratories that used this
reagent, two read the strips with a manual method and two used an
automated system, and no substantial differences in terms of
specificity emerged; indeed, in both situations there was a high number
of false positives (27%; data not shown). This means that even the use
of automated systems cannot resolve the problem inherent in this
methodology, which undoubtedly is an extremely valid tool for the
identification of rare or atypical antibody patterns but has a lower
specificity than other methods.
The data for the CIE and dot-blot methods do not permit a reliable
judgment given the small number of laboratories that used these
methods. Although CIE together with double immunodiffusion has
represented the most widespread method for years, its further use seems
unlikely mostly because of its poor sensitivity for some
autoantibodies; on the other hand, the dot-blot method offers ease of
performance as well as direct interpretation, and produced excellent
results, features that predict its much wider use in clinical
laboratories.
In agreement with the recommendations of the European Consensus Group
(6) and the guidelines proposed by our group
(25), which support the need to use two different methods to
identify anti-ENA antibodies, the use of two methods in series should
have increased the sensitivity, reducing by one-half the number of
false negatives. Indeed, almost one-half of the false negatives
recorded in this study occurred with only one of the four methods used.
From a careful examination of the results of this study, it is also
possible to draw other conclusions that are helpful for a correct
interpretation of the findings, as well as applicable to all external
quality assessment studies of this type. First, the expected
unreliability of an isolated laboratory that furnished results that
were clearly anomalous compared with the general mean, and especially
with other laboratories that used the same reagent, confirms that the
variance related to the operator is an important part of the total
variability. Second, despite the high number of observations, the data
are not distributed homogeneously among the various reagent sets, and
great caution must be taken in interpreting results obtained with
reagent sets for which few data are available. Finally, the lack
of a reference method for the anti-ENA antibodies that would allow
assignment of a reliable value is one of the main problems that must be
faced in the planning and interpretation of results of external quality
assessment studies; only the use of sure positive samples with a
medium-high antibody titer can obviate this important methodological
bias, even if the use of test sera with low antibody titer and values
distributed near the threshold of positivity of the methods is more
advantageous for evaluating the accuracy of the analytical systems.
In conclusion, this study demonstrated that the commercial reagents
used in clinical laboratories for the determination of
anti-topoisomerase I antibody have acceptable reliability. In
particular, specificity was excellent (98.6%) and sensitivity was good
(96.9%). Among the various methods, ELISA presents the best
characteristics of accuracy; among the blot methods, Western blot lacks
sufficient analytical specificity. It is likely that the realization of
national external quality assessment programs will lead to a further
improvement in analytical quality, evidencing and correcting the few
cases of unreliability related to reagents and/or laboratories.
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Footnotes
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A preliminary report of this work, entitled "Evaluation of commercial
ELISA, CIE and IB assays for the detection of anti-topoisomerase I
antibodies: results of a multicentric study" [Clin
Chem 1999;45(Suppl S6):A152], was presented as Abstract 538 at
the 51st AACC Annual Meeting, July 25–29, 1999, New Orleans, LA.
1 Nonstandard abbreviations: ENA, extractable nuclear antigen; SSc, systemic sclerosis; IB, immunoblot; and CIE, counterimmunoelectrophoresis. 
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Abstract
Introduction
