Comparison of Nine Commercially
Available Assays for Quantification of
Antibody Response to Hepatitis B Virus
Surface Antigen
D. Huzly, T. Schenk, W. Jilg and D. Neumann-Haefelin
J. Clin. Microbiol. 2008, 46(4):1298. DOI:
10.1128/JCM.02430-07.
Published Ahead of Print 6 February 2008.
These include:
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JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2008, p. 1298–1306
0095-1137/08/$08.00⫹0 doi:10.1128/JCM.02430-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 4
Comparison of Nine Commercially Available Assays for Quantification
of Antibody Response to Hepatitis B Virus Surface Antigen䌤
D. Huzly,1* T. Schenk,1 W. Jilg,2 and D. Neumann-Haefelin1
Department of Virology, Freiburg University Medical Center, Freiburg,1 and Institute of Medical Microbiology and Hygiene,
University Hospital Regensburg, Regensburg,2 Germany
Received 18 December 2007/Returned for modification 24 January 2008/Accepted 29 January 2008
use, time to results, and general practicability of the systems as
requirements for integration into the workflow of modern
high-throughput medical laboratories. To our knowledge, this
report provides the first systematic and comprehensive comparison of currently marketed anti-HBs assays.
The antibody response to hepatitis B virus surface antigen
(anti-HBs) is an important serological marker for vaccineinduced immunity to hepatitis B virus (HBV). An adequate
vaccine response is defined as an anti-HBs level of ⱖ100 IU/
liter 4 to 8 weeks after the last of three or four vaccine injections. It is widely accepted that a sustained level of at least 10
IU/liter is protective against HBV infection. Vaccinees without
sufficient anti-HBs responses, so-called nonresponders or low
responders, undergo a special regimen of additional vaccine
doses. For liver transplant recipients, quantitative measurement of anti-HBs levels is used in the management of hepatitis
B immune globulin prophylaxis, which is initiated to maintain
anti-HBs levels of at least 100 or 200 IU/liter, according to
different guidelines. All these recommendations imply that the
measurement of anti-HBs levels by different assays is accurate
and consistent, yielding comparable quantitative results in various laboratories and countries. However, analysis of routine
clinical samples by different systems revealed significant discrepancies for a number of sera. This observation is in accordance with older reports comparing outdated methods such as
the radioimmunoassay and latex agglutination (9, 12, 17, 19).
The aims of this study were to determine whether the quantification of anti-HBs levels in routine clinical samples by
different test systems is comparable and accurate within
acceptable limits and to identify factors contributing to the
variability of test results. In addition, we evaluated the ease of
MATERIALS AND METHODS
Test samples. Two hundred serum samples from patients and health care
workers were taken from daily submissions for routine anti-HBs testing without
any preselection. A sample was included in the study if at least 2 ml of serum was
available. Retrospectively, 145 serum samples came from individuals with histories of vaccination, and 24 other samples were anti-HBc positive; 122 were from
health care professionals, and 78 were from patients, 20 of whom were immunosuppressed. A pool comprising 20 sera with anti-HBs levels measuring around
100 IU/liter by our routine anti-HBs assay was used for the evaluation of intraand interassay variability. The first reference preparation of hepatitis B immune
globulin, distributed by CLB (Amsterdam, The Netherlands), was measured in
different dilutions to check the calibration of the assays. The standard preparation was diluted in an anti-HBs negative-control serum (Dade Behring, Marburg,
Germany) to concentrations between 500 and 7.5 IU/liter. A pool of anti-HBsnegative lipemic sera and a pool of hemolytic sera were spiked with 100 IU/liter
of the standard preparation and tested by each system. Five selected serum
samples were serially diluted in negative-control serum for measurement of the
linearity of dilution.
Assay systems. Six different automated immunoassays (the Abbott Axsym
AUSAB and five chemiluminescence assays) and three enzyme immunoassays
(EIAs) were performed.
(i) Abbott Axsym AUSAB assay. The Abbott Axsym assay is a microparticle
EIA using recombinant HBsAg (ad/ay) on microparticles as the solid phase and
biotin coupled to recombinant HBsAg as the conjugate. In the next step, alkaline
phosphatase-conjugated anti-biotin is bound to the antigen sandwich. The reaction mixture is transferred to an inert glass fiber matrix to which the microparticles bind irreversibly. Methylumbelliferyl phosphate is used as a substrate, and
the fluorescence of the final product, methylumbelliferone, is read by the instrument.
(ii) Chemiluminescence assays. The following five chemiluminescence assays
were used: the Advia Centaur anti-HBs assay on the Advia Centaur system from
* Corresponding author. Mailing address: Institute for Medical Microbiology and Hygiene, Freiburg University Medical Center, HermannHerder-Str.11, 79104 Freiburg, Germany. Phone: 49-761-2036609. Fax:
49-761-2036608. E-mail: [email protected].
䌤
Published ahead of print on 6 February 2008.
1298
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Quantitative measurement of anti-HBs is used to evaluate the response to hepatitis B vaccination in health
care workers and to optimize postexposure management. The different guidelines for hepatitis B vaccination
and booster policy imply that the measurement of anti-HBs levels by different assays is accurate and consistent,
yielding comparable quantitative results. We measured anti-HBs levels in 200 serum samples from patients
and health care professionals by nine different anti-HBs assays and compared the quantitative results and the
performance characteristics of the different test systems. The assay specificity ranged between 96.8 and 100%
when sera from individuals without a vaccination history and with negative anti-HBc status were defined as
true negatives. Sensitivity ranged between 93.5 and 100%. A high number of sera showed discrepancies between
measurements by the different systems. The mean coefficient of variation between the different measurements
was 47.1% (range, 15.0 to 201.0%), and the factors of multiplication ranged from 2.8 to 105. Hemolysis or
lipemia did not seem to influence the measurement, and there was no difference between anti-HBc-positive and
-negative individuals. The classical enzyme immunoassays tend to find lower anti-HBs levels than the automated systems, with higher values by the Abbott AXSYM assay. The serial dilution of the international
standard preparation was measured accurately by most of the assays. In conclusion, the quantitative measurement of anti-HBs levels is not reliable, even though an international standard is used for the calibration
of the systems. Some systems showed specific problems that should be addressed by the manufacturers.
VOL. 46, 2008
COMPARISON OF ANTI-HBs ASSAYS
TABLE 1. Characteristics of chemiluminescence assays
Assay
Antigen
TABLE 3. Performance characteristics of EIAs
Luminogenic substrate
Amt of sample
(␮l)
EIA
Vitros ECI
Advia Centaur
Liaison
Roche Elecsys
Abbott Architect
Human (ad, ay)
Human(ad, ay)
Human (ad, ay)
Human(ad, ay)
Recombinant (ad, ay)
Luminol derivative
Acridinium ester
Isoluminol
Ruthenium complex
Acridinium ester
RESULTS
Performance of assays. The performance characteristics of
the six automated test systems are summarized in Table 2. The
hands-on-time for manual order entry is relatively high in most
of the systems, especially if the samples are not bar coded.
The system with the easiest and quickest sample order entry
is the DiaSorin Liaison; the most time-consuming systems are
the Advia Centaur and the Abbott Axsym. The Roche Elecsys,
Behring
Bio-Rad
DiaSorin
No. of wells for
controls and
standards
Time to
results
(min)
Linearity
(IU/liter)
3/12a
5b
5
100
165
230
8–ca. 200
10–150
10–1,000
100
100
100 (⫹100 ␮l
incubation
buffer)
a
Number of wells for alpha (one-point calibration) method/number of wells
for duplicate determinations for standard-curve results.
b
Samples are to be measured undiluted and 1:10 diluted in parallel.
Abbott Architect, and Ortho ECI systems are capable of diluting samples with ⬎1,000 IU/liter automatically, while the
DiaSorin Liaison system needs a special kit for automatic dilution (Anti-HBs Plus). For the Abbott Axsym and Advia Centaur assays, sera must be diluted manually in dilution buffer
(not included in the kit), but the results are calculated automatically. The systems require a minimum of 150 to 200 ␮l of
serum for test performance. Low sample volumes create annoying problems in some systems. The Advia Centaur and
Abbott Architect systems discard the serum when they aspirate
air with the fluid. Because the systems retry the aspiration,
there is often no sample left. The Liaison system performs the
assay even if a low volume is detected. The result will get an
error flag, or in some cases, there will be no result at the end
of the run. The Ortho ECI and Abbott Axsym systems measure
the level in the sample cup and will not perform the assay if the
level detected is too low.
The performance characteristics of the three EIAs are
shown in Table 3.
The DiaSorin EIA kit comes with a negative control and
four calibrators between 10 and 1,000 IU/liter. A 100-␮l volume of incubation buffer is pipetted into all wells before addition of samples and controls/calibrators. The manufacturer
recommends remeasuring sera with optical densities of ⬎3.000
at 405 nm and calculating corrected values if exact quantification is desired.
The Behring EIA kit comes with a negative control and one
standard using the alpha method for quantification. The highest anti-HBs levels measurable by this method are relatively
low (170 to 240 IU/liter in our runs), so many sera have to be
diluted further for exact quantification (70 of 200 sera of our
series had to be diluted). This results in higher test costs and a
longer time to results.
TABLE 2. Performance characteristics of automated test systems
Assay
Minimum amt
of sample (␮l)
Time to first
result (min)
Time for 100
samples (min)
Hands-on time
(min) for 100
samplesa
Linearity (IU/liter)/
autodilute function
Vitros ECI
Advia Centaur
Liaison
Roche Elecsys
Abbott Axsym
Abbott Architect
150
200
200
150
210
150
55
18
30
18
30
28
120
60
120
55
90
60
30/45
30/60
10/20
30/45
30/60
30/45
2–1,000/yes
2–1,000/no
5–1,000/yesb
2–1,000/yes
5–1,000/no
2–1,000/yes
a
b
Bar code reading/manual sample definition.
With the Anti-HBs Plus kit.
Downloaded from http://jcm.asm.org/ on March 4, 2014 by PENN STATE UNIV
Bayer Diagnostics (now part of Siemens Medical Solutions, Fernwald, Germany), the Vitros anti-HBs assay on the Vitros ECI Immunodiagnostic system
(Ortho Clinical Diagnostics, Raritan, NJ), the Roche Elecsys anti-HBs assay on
the Modular System (Roche Diagnostics, Basel, Switzerland), the Liaison antiHBs assay on the Liaison system (DiaSorin, Turin, Italy), and the Abbott Architect anti-HBs assay on the Architect i2000 system (Abbott, Chicago, IL). The
technical specifications of the assays are summarized in Table 1. Serum samples
with values beyond the linearity of the test system were diluted as recommended
by the manufacturers. Most of the automats have an autodilute function; in the
others, a special dilution buffer for these purposes is provided, and dilution
protocols are included in the software.
(iii) EIAs. The following three EIAs were used as recommended by the
manufacturers: the ETI-AB-AUK-3 assay (DiaSorin), the Enzygnost anti-HBs
assay (Dade Behring), and the Monolisa anti-HBs assay (Bio-Rad Laboratories,
Redmond, WA). The EIAs were performed on the Behring ELISA Processor III
using the protocol recommended by the manufacturer. Samples with results
beyond the linearity of the standard curve were diluted 1:20 and/or 1:100 in
negative-control serum. All the EIAs use human plasma-derived HBsAg (ad/ay)
and are sandwich immunoassays using a tetramethylbenzidine substrate. The
Behring assay uses a one-point calibration method (alpha method); the highest
values measurable without dilution ranged from 170 to 240 IU/liter. The Bio-Rad
and DiaSorin assays use four calibrators, from 10 to 150 IU/liter and from 10 to
1,000 IU/liter, respectively.
Statistical analysis. Statistical analysis was performed with SPSS for Windows
(version 13.0) and Medcalc. Because there is no “gold standard” for the measurement of anti-HBs levels, we defined sera as true positive or true negative if
they were identified as positive (ⱖ10 IU/liter) or negative (⬍10 IU/liter) by ⱖ6
of 9 assays. We also calculated specificity by using sera from individuals without
vaccination histories that were negative for anti-HBc. Bland-Altman analysis was
used to compare the assays with each other and against the geometric mean value
for all assays. Inter-rater agreement was used to compare the rating of the assays.
Receiver operating characteristic (ROC) analysis was performed by postulating
that sera with anti-HBs levels of ⱖ10 IU/liter by ⱖ6 assays are true positives.
1299
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HUZLY ET AL.
J. CLIN. MICROBIOL.
TABLE 4. Measurement of the international standard using
calibration with Bio-Rad standards
International
standard (IU)
Value measured by
Bio-Rad EIA (IU)
7.5.........................................................................................⬍10
15.0.......................................................................................⬍10
31.25.....................................................................................⬍10
62.5....................................................................................... 15.6
125.0..................................................................................... 35.5
TABLE 5. Measurement of Bio-Rad standards with other
test systems
Specified
standard concn
(IU/liter)
Mean
measured value
(IU/liter)
ODa in Bio-Rad
EIA
10
50
100
150
37
177
343
518
0.346–0.354
1.298–1.358
2.169–2.259
2.689–2.741
a
OD, optical density.
No. of results
10.0–100
By all systems............................................................................... 9
By ⱖ6 systems .............................................................................29
100.1–1,000
By all systems...............................................................................29
By ⱖ6 systems .............................................................................58
⬎1,000
By all systems...............................................................................40
By ⱖ6 systems .............................................................................58
by the majority of test systems. When sensitivity was calculated
with these sera, five assays determined anti-HBs levels to be
ⱖ10 IU/liter in 100% of the sera, and the other four assays
found levels below 10 IU/liter in two to nine sera, as shown in
Tables 9 and 10.
Quantification. A high number of serum samples showed
large discrepancies between results from different systems. The
mean coefficient of variation (CV) between the different measurements was 47.1%. For 57.6% of the sera with positive
values in the majority of assays, the CV between the nine
measurements was greater than 47%; the factor of multiplication ranged from 2.8 to 105 for these sera. The range of
discrepant values for some of the sera with the highest CVs is
shown in Table 11. When only one or two assays showed
discrepant values, the measurement was repeated by these
assays, but all values were confirmed. When serum samples
were classified according to the anti-HBs levels determined by
the majority of assays (10 to 100, 101 to 1,000, and ⬎1,000
IU/liter), the mean CV was significantly lower for the group of
sera with anti-HBs levels between 101 and 1,000 IU/liter than
for sera with levels between 10 and 100 or above 1,000 IU/liter.
(36.2% versus 57.4% and 60.3%, respectively; P ⫽ 0.003).
However, there were serum samples with extreme differences
in all three groups.
The mean CV for anti-HBc-positive sera was not significantly different from that for anti-HBc-negative sera, and the
mean CV for the sera of patients did not differ significantly
from that for sera from health care professionals.
By assuming that all sera were taken 4 to 8 weeks after
TABLE 7. Specificities of test systems
Test system
Specificity (%) (no. of
false-positive sera/
total no. of sera)a
Abbott Architect............................................................... 97.9 (1/47)
Abbott Axsym ................................................................... 87.2 (6/47)
Bayer Centaur ................................................................... 97.9 (1/47)
Behring EIA...................................................................... 97.9 (1/47)
Bio-Rad EIAb ................................................................... 97.7 (1/47)
DiaSorin EIA ....................................................................100 (0/47)
DiaSorin Liaison............................................................... 89.4 (5/47)
Ortho ECI ......................................................................... 97.9 (1/47)
Roche Elecsys ................................................................... 93.6 (3/47)
a
Results were postulated to be true positives if ⱖ6 test systems found antiHBs levels of ⱖ10 IU/liter.
b
Calibrated with the international standard serum.
Downloaded from http://jcm.asm.org/ on March 4, 2014 by PENN STATE UNIV
The Bio-Rad EIA kit comes with a negative control and one
standard for qualitative measurement. For quantitative measurement, a standard kit has to be ordered separately (at extra
cost). The highest standard is 150 IU/liter, so Bio-Rad recommends measuring all sera both undiluted and at a dilution of
1:10. When the Bio-Rad EIA was used as recommended by the
manufacturer and the anti-HBs standard kit was used for quantification, the values obtained for all sera and for the dilutions
of the international standard were much lower than those
obtained by the other assays (Table 4). After excluding handling errors, we measured the standards of the standard kit by
three different systems and found out that the concentrations
of the standards were much higher than specified (Table 5).
We therefore calibrated the assay with the international standard in dilutions from 7.5 to 500 IU/liter. The results obtained
by this method seemed plausible and were therefore used for
further analysis.
Levels of anti-HBs in samples. For 36 serum samples, the
level of anti-HBs was determined to be ⬍10 IU/liter by all nine
assays. At least six of the nine assays determined anti-HBs
levels to be ⬍10 IU/liter in a total of 47 serum samples. The
distribution of positive values is given in Table 6.
Specificity and sensitivity. Thirty-one serum samples were
from individuals who had never received hepatitis B vaccine
and who tested negative for anti-HBc. By taking these sera as
true negatives for the calculation of specificity, five assays
showed a specificity of 100% and four assays yielded a lowpositive value for one serum sample each (specificity, 96.8%;
range of false-positive values, 10.3 to 14.7 IU/liter). At least six
of the nine assays found anti-HBs levels below 10 IU/liter in a
total of 47 sera. The specificities calculated with these sera and
the range of anti-HBs levels determined for each false-positive
serum sample are shown in Tables 7 and 8, respectively. Three
of the serum samples with positive values by single systems
were anti-HBc positive, and eight were from individuals with
histories of vaccination.
For 139 sera, anti-HBs levels of ⱖ10 IU/liter were obtained
TABLE 6. Distribution of positive values
Result (IU/liter)
VOL. 46, 2008
COMPARISON OF ANTI-HBs ASSAYS
1301
TABLE 8. Properties of false-positive samples
Mean result for
all systems
(IU/liter)
Positive measurement (IU/liter)
(test system)
5
15
42
45
61
81
94
101
144
166
178
5.5
4.8
8.0
5.0
6.5
8.3
5.9
1.9
7.2
20.5
4.9
11.2 (Liaison)
13.5 (Axsym)
13.4–14.3 (Architect, Axsym, Roche)
11.0 (Liaison)
12.1–15.6 (Liaison, Roche)
13.5–19.1 (Liaison, Axsym, Roche)
10.1–10.8 (Liaison, Axsym)
10.1 (Behring)
10.3–14.7 (ECI, Bio-Rad, Axsym)
170 (not dilutable) (Centaur)
11.4 (Axsym)
vaccination and by using the actual definitions of low antibody
response as levels between 10 and 100 IU/liter and of good
response as levels above 100 IU/liter, 27.5% of the vaccinees
would have been sorted into different groups by different assays. Twenty-nine sera showed anti-HBs levels below 10 IU/
liter by some assays and above 10 IU/liter by others.
When the anti-HBs levels obtained by the assays were compared with each other and with the geometric mean titers for
all assays by using Bland-Altman analysis (2), the Abbott
Axsym assay in most cases showed higher levels and the EIAs
showed lower levels than the automated chemiluminescence
assays. The mean bias for the Axsym assay and the DiaSorin
EIA, for example, was 56.8% (Fig. 1). It can be seen from Fig.
1 that results for many serum samples differed by more than
100%. The bias was also high when systems with recombinant
antigen but different test platforms were compared with each
other. A lower bias was seen when systems using similar platforms (e.g., the Bayer Centaur and Abbott Architect systems;
the Behring, DiaSorin, and Bio-Rad EIAs) were compared,
but results for many serum samples showed very high discrepancies among these test systems as well. The calculated biases
are shown in Table 12.
When the assay systems were compared not by analyzing the
absolute values but by inter-rater agreement classifying the
sera as either negative, ⱖ10 IU/liter, ⬎100 IU/liter, or ⬎1,000
IU/liter, the strength of agreement (kappa) ranged from 0.650
to 0.879 (lower 95% confidence interval, ⬍0.610) (Table 13).
TABLE 9. Sensitivities of test systems
Test system
Sensitivity (%) (no. of
false-negative sera/
total no. of sera)a
Abbott Architect ............................................................. 100 (0/139)
Abbott Axsym.................................................................. 100 (0/139)
Bayer Centaur ................................................................. 100 (0/139)
Behring EIA .................................................................... 98.5 (2/139)
Bio-Rad EIAb .................................................................. 94.2 (8/139)
DiaSorin EIA................................................................... 93.5 (9/139)
DiaSorin Liaison ............................................................. 100 (0/139)
Ortho ECI........................................................................ 97.1 (4/139)
Roche Elecsys.................................................................. 100 (0/139)
a
Results were postulated to be true positives if ⱖ6 test systems found antiHBs levels of ⱖ10 IU/liter; negative results were defined as ⬍10 IU/liter.
b
Calibrated with the international standard serum.
No. of other systems with
the following result:
⬍5 IU/liter
⬎5 IU/liter
4
6
3
4
4
3
4
8
3
8
5
4
2
3
4
3
3
3
0
3
0
3
Patient information
History of vaccination;
Anti-HBc positive
History of vaccination;
Anti-HBc positive
History of vaccination;
History of vaccination;
History of vaccination;
History of vaccination;
History of vaccination;
Anti-HBc positive
History of vaccination
renal transplant patient
health care professional
health care professional
health care professional
renal transplant patient
health care professional
health care professional
Kappa values of 0.610 and lower are interpreted as moderate
agreement, which means that the reliability of rating is relatively low.
The serial dilution of the international standard preparation
was measured accurately by most of the systems (except for the
Bio-Rad EIA, as mentioned above), but some of the assays
tended to measure the 500-IU/liter standard too high (Fig. 2).
Interassay variability was within an acceptable range for all
of the assay systems, with values between 0.7% (Ortho ECI)
and 13.5% (DiaSorin EIA). The different systems found different anti-HBs levels for the serum pool (Fig. 3).
Anti-HBs levels obtained for the lipemic and hemolytic serum pools spiked with 100 IU/liter of the international standard were 80 to 120 IU/liter each; thus, no influence of lipemia
or hemolysis on the quantification could be shown in this
analysis.
To find out whether interferences such as matrix effects (21)
could be the cause of discrepant results, we measured anti-HBs
levels in serial dilutions of three sera with highly discrepant
results and, for comparison, in two sera with low CVs. The
results of these measurements were highly confusing, since
there were three different effects of dilution: for one serum
sample, the variance decreased with higher dilutions; for the
second, the difference remained stable; and for the third, the
difference actually increased with higher dilutions. The dilution of one of two sera with low CVs also showed higher
differences with higher dilutions.
ROC analysis. To find out whether the cutoff of 10 IU/liter
is valid for all assay systems, we performed a ROC analysis,
presuming that all sera determined to be positive by at least six
systems were true positives and all sera determined to be
negative by at least six systems were true negatives. The resulting cutoff levels for the different systems are given in Table 14.
DISCUSSION
Current recommendations for hepatitis B immunization of
health care professionals and other groups at risk of HBV
exposure include anti-HBs testing after completion of vaccination and different management of individuals with negative or
low-positive (10 to 100 IU/liter) and high-positive (ⱖ100 IU/
liter) titers. Similarly, guidelines for postexposure prophylaxis
require quantitative anti-HBs testing in certain cases. The rec-
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Serum
sample no.
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HUZLY ET AL.
J. CLIN. MICROBIOL.
TABLE 10. Properties of false-negative samples
Mean result for
all systems
(IU/liter)
7
12
55
113
114
29.3
11.9
14.5
29.4
37.5
131
132
133
175
194
26.6
20.6
45.7
46.2
12.7
Negative measurement (IU/liter)
(test system)
Range of results from
other systems (IU/liter)
Patient information
9.7 (Bio-Rad)
7.8–9.0 (Sorin, Behring, Bio-Rad)
0.0 (Bio-Rad)
0.0–9.1 (Sorin, ECI, Bio-Rad)
8.6 (test definition, borderline)
(ECI)
4.0 (Sorin, Bio-Rad)
9.0 (Sorin, Bio-Rad)
7.3 (Sorin)
8.0 (Sorin)
8.0–9.9 (Sorin, Bio-Rad, Behring)
11.1–53.3
10.1–17.8
10.1–23.0
11.0–63.0
24.7–63.3
Anti-HBc-positive patient
History of vaccination; health care professional
History of vaccination; health care professional
History of vaccination; health care professional
History of vaccination; transplant patient
11.5–69.7
11.5–44.4
13.0–84.5
11.8–135.0
13.0–17.3
History of vaccination; health care professional
Anti-HBc-positive patient
Anti-HBc-positive patient
History of vaccination; health care professional
No documentation of vaccination; anti-HBcnegative patient
ommendations of several countries are shown in Table 15 (3–5,
7, 11, 13, 15, 18, 20). Most recommendations are based on the
assumption that the measurements by different assay systems
are sufficiently standardized by the use of an international
standard preparation. However, as shown by our study, this is
obviously not the case.
The standard preparation was produced in 1977 for the
quantification of anti-HBs in immune globulin preparations.
The assays available at that time differ completely from those
used nowadays. The study group involved in the production of
this preparation noted high discrepancies in measurements by
some test systems even in these first analyses, and they decided
to exclude discrepant values from further evaluation (1).
In several studies comparing different assay systems, similar
discrepancies were found. The authors postulated diversity of
antigens, problems with low-avidity antibodies, and different
vaccine antigens to explain the discrepancies (8, 10, 12, 17, 19,
22). Different calculation methods have also been shown to
cause problems for standardized quantification (22). However,
not all sera showed discrepant values, and some sera showed
extremely high discrepancies. In our study, sera with extremely
TABLE 11. Examples of sera with very high discrepancies
between measurements
Serum
sample no.
Range of measured values (IU/liter)
(factor of multiplication)
CV between
measurements (%)
8
11
17
57
79
81
102
113
114
126
127
133
134
148
175
183
185
200
68.3–354 (5.2)
150–1,664 (11.1)
190–2,230 (11.7)
210–5,114 (24.4)
33.7–170 (5.0)
1.0–19.1 (19.0)
17.0–189.0 (11.1)
1.0–82.4 (82.4)
8.6–63.3 (7.4)
65.0–331.0 (5.1)
1.0–105.0 (105)
7.3–84.5 (11.6)
90.0–509.0 (5.7)
44.0–228.0 (5.2)
7.9–135.0 (17)
68.9–2,280.0 (33.1)
57.1–455.8 (8.0)
28.2–227 (8.1)
60.6
90.6
86.0
63.0
47.3
92.8
63.2
99.4
47.7
44.3
97.1
65.5
56.3
57.7
91.6
201.0
75.7
73.4
high discrepancies were mainly from vaccinated healthy individuals, most of whom had been vaccinated with the same
antigen preparation. Thus, different vaccine antigens and different calculation formulas did not seem to be essential for
these differences. The antigens used in the assay systems did
not seem to be responsible, either, since systems using the
same antigen source also showed large differences for these
sera. However, antigens may be produced in different ways, so
the possibility that antigen preparation is one of the reasons
for the differences cannot be excluded. Diversity in the individual immune response (proportion of low-avidity antibodies,
immunoglobulin G [IgG] subclasses, etc.) and interference by
endogenous proteins or other substances in the individual samples are other possible explanations for the phenomenon
(21, 23).
Twenty-nine of the 200 randomly chosen serum samples
showed negative values for anti-HBs in some assays and positive values in others. The range of positive values was 10.1 to
171 IU/liter. In one case (171 IU/liter by the Advia Centaur),
dilution did not result in a linear curve; the values remained
high. A nonspecific result is therefore probable in this case. In
a second case, there was a wide range of different values between 7.8 and 135 IU/liter; the serum sample was from a
vaccinated health care professional who had acquired a needle
FIG. 1. Bland-Altman (difference [expressed as a percentage] versus average [expressed in IU per liter]) plot of Abbott Axsym and
DiaSorin EIA results (values of ⬍1,000 IU/liter).
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Serum
sample no.
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COMPARISON OF ANTI-HBs ASSAYS
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TABLE 12. Bland-Altman analysis of assay systems compared with each other and with geometric mean titers
Mean bias (difference 关%兴 vs avg) of the following test system:
Test system
Architect
Architect
Axsym
Behring
Bio-Rad
Centaur
DiaSorin
ECI
Liaison
Roche
⫺29.4
15.8
25.9
⫺12.0
27.2
2.9
⫺24.2
⫺20.8
Behring
Bio-Rad
Centaur
DiaSorin
ECI
Liaison
Roche
29.4
⫺15.8
⫺46.1
⫺25.9
⫺57.2
⫺21.7
12.0
⫺18.8
34.0
51.3
⫺27.2
⫺56.8
4.5
22.5
⫺29.6
29.6
22.7
⫺0.1
⫺2.5
⫺7.5
⫺26.4
⫺31.1
⫺2.9
⫺35.8
11.0
33.8
⫺22.7
7.5
24.2
⫺7.1
31.7
53.9
0.1
26.4
21.6
20.8
⫺10.5
34.6
55.1
2.5
31.1
25.5
3.5
18.8
⫺12.8
46.1
57.2
18.8
56.8
35.8
7.1
10.5
21.7
⫺34.0
⫺4.5
⫺11.0
⫺31.7
⫺34.6
⫺51.3
⫺22.5
⫺33.8
⫺53.9
⫺55.1
31.5
⫺16.6
⫺39.6
stick injury from an unknown donor. Dilution of this serum
sample resulted in values between 10 and 60 IU/liter. The
values declined with dilution in systems with a higher initial
measurement and increased in those with a lower or negative
initial measurement. Some sort of matrix effect might be instrumental in this behavior, and it seems probable that the
serum is true positive.
Four of the assay systems tested in our study showed specific
problems that should be addressed by the manufacturers. The
standard lot of the Bio-Rad EIA was obviously not produced
with the usual quality control measurements. The concentrations of the standards were much higher than those specified
on the vials. The DiaSorin EIA found anti-HBs levels below 10
IU/liter for nine sera, while most of the other systems found
positive values. ROC analysis resulted in a cutoff of 5 IU/liter
for this system, and Bland-Altman analysis showed that this
system yielded significantly lower values than most of the other
assay systems. Additionally, dilution of one of the serum samples with an initial negative value led to a positive result. The
Abbott Axsym assay found anti-HBs levels above 10 IU/liter
for six sera, while most of the other systems found negative
values. ROC analysis resulted in a cutoff of 19.1 IU/liter for
this system, and Bland-Altman analysis showed a clear tendency of this system to find higher anti-HBs levels than other
assay systems. The Advia Centaur assay had a clearly nonspecific result of 171 IU/liter for one serum sample. Dilution of
this sample did not result in a linear curve; instead, the values
remained high. None of the other systems found anti-HBs
⫺21.6
⫺25.5
⫺5.4
⫺3.5
18.0
21.4
levels above 5 IU/liter for this sample. This high nonspecific
value raises concerns about the specificity of the system.
Thus, our study shows that levels of anti-HBs determined by
one assay system cannot be compared with those determined
by other systems, although all the assays are calibrated with the
same international standard. Because there is no gold standard
assay, the question which test is right and which is wrong
remains open.
The reasons for this are manifold. The international standard preparations, initially, were not produced for the standardization of antibody assays but for the measurement of
specific antibody content in immune globulin preparations (1,
16). Most of the actual standards are themselves immune globulin products and not sera. Since the 1980s, the manufacturers
of quantitative immunoassays have used such products in establishing standard curves to achieve comparability of quantitative values in different assays. However, from the beginnings
of quantitative measurements, discrepancies have been noticed
with different assay systems. We have seen similar problems
with rubella IgG and varicella-zoster IgG (unpublished data).
Regarding the user friendliness of the test systems, all automated assays are easy to use and fast, if samples are bar
coded and the order is entered by the laboratory computer
system. Manual sample identification and order entry are relatively time consuming and complicated in all systems. The
manufacturers should improve the software for easier handling
of such samples. Better management of low-volume samples
would also be desirable.
TABLE 13. Inter-rater agreement between the assay systemsa
Inter-rater agreement between the indicated systems
Test
system
Architect
Architect
Axsym
Behring
Bio-Rad
Centaur
DiaSorin
ECI
Liaison
Roche
0.730
0.812
0.684
0.879
0.805
0.784
0.791
0.838
a
Axsym
Behring
Bio-Rad
Centaur
DiaSorin
ECI
Liaison
Modular
0.730
0.812
0.650
0.684
0.636
0.778
0.879
0.755
0.758
0.683
0.805
0.682
0.832
0.744
0.791
0.784
0.756
0.839
0.798
0.797
0.831
0.791
0.815
0.684
0.609
0.844
0.703
0.743
0.838
0.809
0.718
0.630
0.892
0.751
0.757
0.898
0.650
0.636
0.755
0.682
0.756
0.815
0.809
0.778
0.758
0.832
0.839
0.684
0.718
0.683
0.744
0.798
0.609
0.630
0.791
0.797
0.844
0.892
0.831
0.703
0.751
0.743
0.757
Based on classification of the results as follows: class 1, negative; class 2, 10 to 100 IU; class 3, ⬎100 to 1,000 IU; class 4, ⬎1,000 IU.
0.898
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⫺0.8
Geometric mean
Axsym
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HUZLY ET AL.
J. CLIN. MICROBIOL.
TABLE 14. ROC analysis
FIG. 2. Measurement of the international standard preparation.
Calculated cutoff
(IU/liter)
Sensitivity
(%)
Specificity
(%)
Architect
Axsym
Behring
Bio-Rad
Centaur
DiaSorin EIA
ECI
Liaison
Roche
13.4
19.1
9.7
12.0
9.9
4.5
8.3
13.5
15.6
97.1
93.2
97.3
94.0
99.3
98.0
98.7
99.3
98.0
100
100
100
100
97.9
100
97.9
100
97.9
dose. Another possibility would be regular administration of a
fourth vaccine dose at month 12, which may result in a higher
proportion of vaccine responders (6). Several countries recommend a fourth vaccine dose in high-risk settings, as well as a
booster dose after 5 years (4, 7, 11).
The problem of false positives still remains in countries
lacking such recommendations (e.g., the United States) and in
all situations where actions to be taken (e.g., giving a booster
dose) depend on whether the anti-HBs level determined is
below or equal to/above 10 IU/liter. This might be the case for
vaccinated individuals at high risk for infection who had never
been tested after vaccination, and for whom it is necessary to
determine whether they are immune or not, e.g., after exposure to HBV. In such a case, an anti-HBs level of only 10
IU/liter or higher would be proof of successful vaccination and
probable protection. However, in view of the fact that in our
study the range of positive values for sera testing positive in ⬍4
assays (i.e., possibly false positive) was 10.1 to 19.1 IU/liter, a
result of 10 IU/liter is likely to be inaccurate with the tests
presently available. The ROC analysis resulted in cutoffs between 5 and 19.1 IU/liter for the different assays. Thus, the
introduction of a gray zone between 5 and 20 IU/liter should be
considered. This would lead to a cutoff value of 20 IU/liter for
proven seropositivity. Vaccinated individuals with anti-HBs
FIG. 3. Interassay variability, determined with pooled sera. A serum pool was analyzed five times in repeat tests on different days.
Downloaded from http://jcm.asm.org/ on March 4, 2014 by PENN STATE UNIV
In conclusion, the immune response to specific antigens is a
complex system with high variability between individuals, since
the antibodies are directed against a variety of epitopes in
variable concentrations. It is probably impossible to standardize the quantification using completely different assay systems.
What does this mean for measuring anti-HBs levels after immunization against hepatitis B? An anti-HBs concentration of
ⱖ10 IU/liter is assumed to be protective against both acute and
chronic disease (14). Thus, a level of at least 10 IU/liter determined 4 to 8 weeks after the last injection of the basic course
of immunization (usually the third dose) is regarded as proof
of response to vaccination. However, this value is generally the
lower limit of assay accuracy in the different systems; in fact,
according to our results, five of nine tests show a calculated
cutoff value above 10 IU/liter (as high as 19.1 IU/liter in one
case). Thus, the risk of false-positive as well as false-negative
results is relatively high. A false-negative result is more or less
harmless, because it only leads to (unnecessary) revaccination.
A false-positive result, on the other hand—meaning that a
nonresponder is, by mistake, considered a responder—is dangerous, since the necessary revaccination will not be performed. Therefore, some countries set the lower limit of the
anti-HBs level proving a good response to vaccination at 100
IU/liter (Table 15); individuals with lower levels determined 4
to 8 weeks after the third vaccination receive an additional
Assay system
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COMPARISON OF ANTI-HBs ASSAYS
1305
TABLE 15. Recommendations for hepatitis B revaccination of persons at increased risk in different countries
Country
No. of vaccine
doses
recommended
Australia
3
Belgium
France
Time of postimmunization
serology
Booster management of
responders (postvaccination
titer, ⱖ10 IU/liter)
3 mo after last dose
No booster
3 or 4
1–3 mo after last dose
No booster
3 or 4
4–8 wk after booster dose
⬍25 yr, no booster; ⬎25 yr,
booster after 5 yr
3
4 wk after 3rd dose
For postvaccination titer of
⬍100 IU/liter, immediate
booster and serology; for
⬎100 IU/liter, booster
after 10 yr
India
3
4–8 wk after last dose
No booster
3 or 4
4–8 wk after last dose
United Kingdom
4
1–4 mo after last dose
United States and
Canada
3
4 wk after last dose
For postvaccination titer of
ⱕ100 IU/liter, booster
every 6–12 mo; for ⬎100
IU/liter, no booster
For postvaccination titer of
⬍100 IU/liter, immediate
booster; for ⬎100 IU/
liter, a single booster
after 5 yr
No booster
Switzerland
a
Serology; booster when titer is ⬍10
IU/liter ⫹ HBIG when there is no
documentation of vaccine response
Serology; booster when titer is ⬍10
IU/liter; when postvaccination titer
is ⬍10 IU/liter (defined as
nonresponder), HBIG
Serology when no documentation of
response; booster and HBIG when
titer is ⬍10 IU/liter
Serology only when immunization was
more than 5 yr earlier or when
postvaccination titer is ⬍100
IU/liter; booster when actual titer is
⬍100 IU/liter, ⫹HBIG when ⬍10
IU/liter
Serology when no documentation of
response; booster ⫹ HBIG when
titer is ⬍10 IU/liter
Serology when postvaccination titer is
⬍100 IU/liter; booster when ⬍100
IU/liter, ⫹HBIG when ⬍10 IU/liter
For responder (titer, ⬎10 IU/liter),
consider booster; for nonresponder,
⫹ HBIG
Serology when no documentation of
response; booster ⫹ HBIG when
titer is ⬍10 IU/liter
HBIG, hepatitis B immune globulin.
levels below 20 IU/liter should receive a booster dose and be
tested 4 weeks later in order to find out whether they are true
responders. The impact of quantitative anti-HBs testing in
other, different contexts (e.g., following liver transplantation
for HBV-infected recipients) has to be discussed separately.
REFERENCES
1. Barker, L. F., D. Lorenz, S. C. Rastogi, J. S. Finlayson, and E. B. Seligmann.
1977. Study of a proposed international reference preparation for antihepatitis B immunoglobulin. WHO Expert Committee on Biological Standardization technical report series. World Health Organization, Geneva,
Switzerland.
2. Bland, J. M. 1999. Measuring agreement in method comparison studies.
Statistical Methods Med. Res. 8:135–160.
3. British Columbia Centre for Disease Control. 2006. Immunization manual.
Section II. Immunization schedules, p. 1–20. Communicable Disease Control
Immunization Program, BCCDC, Vancouver, British Columbia, Canada.
http://www.bccdc.org/content.php?item⫽193.
4. Bundesamt für Gesundheit, Schweizerische Kommission für Impffragen,
and Schweizerische Expertengruppe für virale Hepatitis. December 1997.
Empfehlungen zur Hepatitis-B-Impfung. Bundesamt für Gesundheit, Bern,
Switzerland. http://www.bag.admin.ch/themen/medizin/00682/00684/02535
/index.html?lang⫽de.
5. Centers for Disease Control and Prevention. 2001. Updated U.S. Public
Health Service guidelines for the management of occupational exposures to
HBV, HCV, and HIV and recommendations for postexposure prophylaxis.
MMWR Recomm. Rep. 50(RR-11):1–22.
6. Das, K., R. K. Gupta, V. Kumar, and P. Kar. 2003. Immunogenicity and
reactogenicity of a recombinant hepatitis B vaccine in subjects over age of
forty years and response of a booster dose among nonresponders. World J.
Gastroenterol. 9:1132–1134.
7. Department of Health, United Kingdom. 2006. Immunisation against infectious disease—the Green Book, p. 161–184. Department of Health, London,
United Kingdom. http://www.dh.gov.uk/en/Publichealth/Healthprotection
/Immunisation/Greenbook/DH_4097254.
8. Galanti, L. M., C. Cornu, P. L. Masson, A. R. Robert, D. Becheanu, M. E.
Lamy, and C. L. Cambiaso. 1991. Assay of anti-HBs antibodies using a
recombinant antigen and latex particle counting: comparison with five commercial tests. J. Virol. Methods 32:221–231.
9. Haas, J., and G. Hotz. 1980. Rapid detection of HBsAg and anti-HBs by
enzyme-immunoassay. J. Virol. Methods 2:63–69.
10. Heijtink, R. A., P. M. Schneeberger, B. Postma, and W. Crombach. 2002.
Anti-HBs levels after hepatitis B immunisation depend on test reagents:
routinely determined 10 and 100 IU/l seroprotection levels unreliable. Vaccine 20:2899–2905.
11. Institut de Veille Sanitaire. 2004. Calendrier vaccinal 2004. Bull. Epidemiol.
Hebdom. 28-29:122–124.
12. Kruining, J., P. G. Mulder, and R. A. Heijtink. 1988. Immune response to
HBsAg differently interpreted by RIA and EIA. J. Virol. Methods 22:89–98.
13. Laboratory Centre for Disease Control, Health Canada. 1998. Canada communicable disease report, vol. 24, suppl. 4. Proceedings of the Consensus
Conference on Infected Health Care Workers: risk for transmission of
bloodborne pathogens. Public Health Agency of Canada, Ottawa, Ontario,
Canada. http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/98vol24/24s4/index
.html.
14. Mast, E. E., C. M. Weinbaum, A. E. Fiore, M. J. Alter, B. P. Bell, L. Finelli,
L. E. Rodewald, J. M. Douglas, Jr., R. S. Janssen, and J. W. Ward. 2006. A
comprehensive immunization strategy to eliminate transmission of hepatitis
B virus infection in the United States: recommendations of the Advisory
Committee on Immunization Practices (ACIP) Part II: immunization of
adults. MMWR Recomm. Rep. 55(RR-16):1–33.
15. Moniteur Belge. 7 October 1999. Arreté royal du 29.4.1999 modifiant l’arreté
royal du 4 Aout 1996 concernant la protection des travailleurs contre les
risques liés à l’exposition à des agents biologiques au travail (M.B.
1.10.1996), p. 37917.
16. National Institute for Biological Standards. 16 December 2003. The 1st
international standard for anti-rubella immunoglobulin, human. Code
RUBI-1-94, version 06. National Institute for Biological Standards, Potters
Bar, Hertfordshire, United Kingdom. http://www.nibsc.ac.uk/documents/ifu
/RUBI-1-94.pdf.
Downloaded from http://jcm.asm.org/ on March 4, 2014 by PENN STATE UNIV
Germany
Management after exposurea
1306
HUZLY ET AL.
17. Ostrow, D. H., B. Edwards, D. Kimes, J. Macioszek, H. Irace, L. Nelson, K.
Bartko, J. Neva, C. Krenc, and L. Mimms. 1991. Quantitation of hepatitis B
surface antibody by an automated microparticle enzyme immunoassay. J. Virol. Methods 32:265–276.
18. Queensland Health. 2001. Queensland Health infection control guidelines,
p. 286–290. Communicable Diseases Unit, Queensland Health, Brisbane,
Queensland, Australia.
19. Racela, L. S., G. E. Tegtmeier, G. R. Hodges, and J. S. Reed. 1986. Comparison of two testing methods to determine hepatitis B surface antibody
response to hepatitis B vaccine among health-care workers. Am. J. Clin.
Pathol. 86:527–529.
J. CLIN. MICROBIOL.
20. Robert Koch-Institut. 28 July 2006. Epidemiologisches Bulletin, no. 30.
Empfehlungen der Ständigen Impfkommission (STIKO) am Robert KochInstitut, p. 242, 252. Robert Koch-Institut, Berlin, Germany.
21. Selby, C. 1999. Interference in immunoassays. Ann. Clin. Biochem. 36:704–
721.
22. Vranckx, R. P. 1989. Evaluation of different calculation methods for antiHBs titration. Zentralbl. Bakteriol. 272:47–57.
23. Wang, L., S. J. Lin, J. H. Tsai, C. H. Tsai, C. C. Tsai, and C. C. Yang. 2005.
Anti-hepatitis B surface antigen IgG1 subclass is predominant in individuals
who have recovered from hepatitis B virus infection, chronic carriers, and
vaccinees. Med. Microbiol. Immunol. 194:33–38.
Downloaded from http://jcm.asm.org/ on March 4, 2014 by PENN STATE UNIV