African Journal of Microbiology Research Vol. 6(12), pp.

3024-3028, 30 March, 2012

Available online at http://www.academicjournals.org/AJMR
DOI: 10.5897/AJMR12.121
ISSN 1996-0808 2012 Academic Journals




Full Length Research Paper

Compare HBsAg cutoff index values of borderline or
initially weakly reactive samples with neutralization
results for electrochemiluminescence immunoassay in
China

Hongxia Ning, Dongdong Li, Xiaohui Bi, Tingting Wang, Xiaoqing Du, Kening Yan,
Liang Wang and Chuanmin Tao*

Department of Laboratory Medicine, West China Hospital, Sichuan University, P. R. China.

Accepted 8 March, 2012

To set up arbitrary cutoff values and to determine whether arbitrary cutoff values can be predictive of
confirmed positive results by compare cutoff index (COI) values of the borderline or initially reactive
samples (0.9COI<7.0) with HBsAg confirmation test. 238 HBsAg borderline or initially reactive sera or
plasma with 0.9COI<7.0 were collected from the clinical patients in West China Hospital of Sichuan
University. Each sample must be re-determined in duplicate with Elecsys HBsAg II assay according to
manufacturers instructions. All repeated reactive samples were confirmed with HBsAg neutralization
assay. HBV viral loads were tested if necessary. 116 out of 238 (48.7%) samples, deemed as weakly
reactive (WR) samples, were found COIs <2.0. 12.1% (11 out of 91) of WR samples did not pass
confirmation in repeated reactive samples. Statistical analysis indicated that much more WR samples
were found in HBsAg negative group (100%) than those in repeatedly reactive group (42.7%) (P<0.05). In
the 116 WR samples (COI<2.0), 25 samples were HBsAg negative and 11 did not pass the neutralization.
36 WR samples were discovered electrochemiluminescence immunoassay HBsAg negative or
confirmed negative. Furthermore, four HBsAg negative and one confirmed negative samples were
found HBV DNA existed. 36 WR samples harbored with anti-HBc and anti-Hbe. By setting up modified
cutoff values to 1.455 or 1.785, the positive predicted value (PPV) could be improved from 95.2 to 99.4%
or 100% in this study. Evaluation of HBsAg prevalence through Elecsys HBsAg II overestimated the true
positive, especially in WR samples. Adjusted COIs decreased the need of HBsAg neutralization test, to
same degree.

Key words: hepatitis B surface antigen (HBsAg), confirmation test, Electrochemiluminescence immunoassay
(ECLIA), weakly reactive.


INTRODUCTION

Although universal vaccination against HBV in infants has
been very successful in the control of chronic HBV
infection, the prevalence of hepatitis B surface antigen
(HBsAg) is still approximately 7% in China (Wang and
Jia, 2011). Accurate detection of HBsAg is an important
aid in the diagnosis of patients infected with the HBV.



*Corresponding author. E-mail: taochuanmin@sina.com. Tel:
86-28-85422618. Fax: 86-28-85423510.
Increasingly large numbers of routine specimens
(hospitalized patients, outpatients, pre-surgery, health
care workers and anonymous testing) are transmitted to
our laboratory with orders to perform an assay for
HBsAg. Electrochemiluminescence immunoassay
(ECLIA) intended for qualitative detection HBsAg in
human serum or plasma, was used to screen for HBV
infection. Most weakly reactive results, determined by
ECLIA, were negative when determined by enzyme-
linked immunosorbent assay (ELISA) (Xu et al., 2011).
We observed that a relatively high percentage of weakly




reactive samples (0.9 cutoff index <7.0) were false
positive or not in accordance with serological profiles in
clinical practice.
The usual criteria for analysis of HBsAg are detection
of HBsAg and result confirmation by antibody
neutralization. Chu et al. (2011) indicated that additional
neutralization tests might therefore become mandatory
for persons with a positive HBsAg test result. As the CDC
recommended supplemental testing algorithms of
antibody to hepatitis C virus for cutoff indexes (COI) (Alter
et al., 2003). We could improve the positive predicted
value (PPV) of the ECLIA assay by setting up an arbitrary
revised COI. O'Brien (2000) suggested that it is not
necessary to carry out neutralization test when the
S/N>6.0 for RIA assay. So in this study, we want to
compare COI values of the borderline (samples having a
cutoff index in the range 0.9 to < 1.0) or initially reactive
samples (1.0 COI<7.0) with HBsAg confirmation test to
set up modified cutoffs with an expectation of a decrease
in complexity, improved turnaround time, and decreased
expense.


PATIENTS AND METHODS

Patients

The observational study received ethical approval from West China
Hospital of Sichuan University, and included patients with
borderline or initially reactive HBsAg (0.9COIs<7). All sera or
plasma from those patients were collected and stored at -80C for
later analysis.


HBV serological tests

All HBV serological makers, including HBsAg, anti-HBs, HBeAg,
anti-HBe and anti-HBc, were measured with ECLIA (Elecsys HBsAg
II assay) (Roche, German) on Modular E170. Regard to HBsAg
testing, borderline or initially reactive samples must be re-
determined in duplicate with Elecsys HBsAg II assay according to
manufacture instruction. If these samples yield mean COI values of
<0.9 upon redetermination, they are considered negative for
HBsAg. Initially reactive or borderline samples giving COIs of >0.9
in either of the redeterminations are considered repeatedly reactive.
Repeatedly reactive samples must be investigated using an
independent neutralization test.


Elecsys HBsAg confirmation test

270 L repeatedly HBsAg-reactive samples were pre-incubated
with 30 L confirmatory regent during 60 min at room temperature
in order to confirm the presence of HBsAg by specific antibody
neutralization. In parallel, the same volume sample was incubated
with control reagent. Prior to evaluation and interpretation of the
results, the validity of the test has been verified. COIs of Elecsys
PreciControl HBsAg II (Roche, German) in the test with
confirmatory reagent must be <50% of that with control reagent.


HBV viral loads

Plasma HBV DNA levels were measured with the COBAS
R

Ning et al. 3025



AmpliPrep/COBAS
R
TaqMan
R
HBV test

according to manufacture
instruction

(Roche, German) by COBAS
R
AmpliPrep/COBAS
R
TaqMan
R
instrument (Roche, German). The quantitative detection
limit is>=12 IU/ml.


Statistical analysis

Frequency variables were compared by
2
test. All statistic analyses
were carried out using SPSS 13.0 software (SPSS Inc, Chicago,
USA). Results were considered statistically significant at P<0.05.


RESULTS

Our initial results were shown in Table 1. 48.7% (116 out
of 238) samples, deemed as weakly reactive (WR)
samples, were found 0.9 COIs < 2.0 (Table 1). After we
performed neutralization test, up to 12.1% (11 out of 91)
WR samples did not pass confirmation. Statistical
analysis indicated that more (100%) initially WR samples
were found in HBsAg negative group than those (42.7%)
in repeatedly reactive group (P<0.05).
The performance of this screening test was evaluated
by HBsAg confirmatory test. The area under the
summary receiver operator characteristic (ROC) curve
was 0.905 (0.845, 0.966), and the optimal coordinate
point of the ROC curve is 1.455 with 90.9% specificity.
The false positive rate of Elecsys HBsAg II assay in
repeated reactive samples was 4.8%. There were 25
HBsAg negative samples and 11 samples that did not
pass the neutralization, suggesting the current cutoff
values is problematic. In another word, even with high
sensitivity, a lower PPV was problematic in situations of
higher HBV prevalence. Furthermore, four HBsAg nega-
tive and one confirmed negative samples were found
HBV DNA existed. In our study, we could improve the
PPV from 95.2 to 99.4% and even to 100% by setting up
modified cutoff values of 1.455 and 1.785 in repeated
reactive sera (Table 2), as similar studies showed (Kim et
al., 2010).
The high initially false positivity rate (36 out of 238,
15.1%) and the subsequent failure at the confirmation
step for a significant portion of the initially reactive led us
to question the clinical significance of these results and
led to further investigation. To get a better understanding
of the true nature of these WR samples, we combined
HBV viral loads with other HBV serologic markers to
judge the HBV infectious status. All these WR samples
were found with anti-HBc and anti-HBe, suggesting the
chronic infection phase.


DISCUSSION

The article by Chen and Kaplan (2006) indicates that
laboratories need to be aware of the performance of their
HBsAg assays. Louisirirotchanakul et al. (2010) compared
3026 Afr. J. Microbiol. Res.



Table 1. Distribution of samples in the Roche Elecsys HBsAg II assay (0.9COI<7).

Parameter
Gender HBsAg(COI)
Male (n=159) (%) Female (n=79) (%) 0.9~2(%) 2.1~3.0(%) 3.1~4(%) 4.1~5(%) 5.1~7(%)
HBsAg negative (n=25) 14 (54.5) 11 (45.5) 25(100) 0(0) 0(0) 0(0) 0(0)
Repeated reactive(n=213) 145(67.1) 68(31.9) 91(42.7) 42(19.7) 38(17.8) 22(10.3) 20(9.4)
Positive(n=202) 138(68.3) 64(31.7) 80(39.6) 42(20.8) 38(18.8) 22(11.0) 20(9.9)
Negative (n=11) 7(63.6) 4(36.4) 11(100) 0(0) 0(0) 0(0) 0(0)



Table 2. Performance Roche Elecsys HBsAg II assay in repeatedly reactive samples according to cutoff index.

Parameter Values based on cutoff Index
Value of cutoff index 1.0 1.455 1.785
False positive 10 1 0
Specificity 9.1% (0.2%, 41.3%) 90.9% (58.7%, 99.8%) 100% (71.5%, 100%)
Positive predictive value 95.2 (91.3%, 97.7%) 99.4% (99.7%, 100%) 100% (97.8%, 100%)



the Elecsys HBsAg II assay with the Architect,
AxSYM, and Advia Centaur HBsAg assays for
HBsAg screening, and demonstrated that it is
suitable for routine HBsAg screening in Asia. Jia
et al. (2009) also indicated that it is suitable in
China. As with all infectious assays, nonspecific
binding can occur, and false positive results can
be found, especially in weakly positive samples
(Dufour, 2006). In the case of HBsAg, we need a
neutralization test that can be used to confirm the
true positivity of results (Orgel et al., 1996;
Fletcher et al., 2010). All initially reactive or
borderline must be re-determined in duplicate with
Elecsys HBsAg II test before HBsAg confirmatory
test. However, in developing countries, such as
China, it was hard for a laboratory to afford the
burden for so many HBsAg initially or borderline
samples. Previous studies (Kiely et al., 2010; Alter
et al., 2003) showed that high S/CO ratio were
predictive of confirmed positive results, therefore
the aim of this study was to analyze the extent of
COIs overlap between false positive and
confirmed positive results to determine the optimal
cutoff which is reliable predictive of the final status
of HBV infection.
As shown in Table 1, the false positive rate of
initially reactive or borderline samples was 15.1%
(36 out of 238), which seemed lower than that of
the results for Immulite HBsAg assay (Chen and
Kaplan, 2006). All 36 false positive samples were
initially weakly reactive or borderline. In another
word, 31% (36 of 116) initially weakly reactive or
borderline results were false positive. However,
this finding can be acceptable because the impact
of inherent high internal CV (~10%) and the non-
specific signal (Chen et al., 2005). Only 5.2% (11
out of 213 samples) were false positive in HBsAg
repeatedly reactive samples. Nearly 95%
repeated reactive samples were true HBsAg
positive. Therefore, it was important to carry out
redetermination, especially in WR samples. In this
study, 90% (213 of 238) initially reactive or
borderline samples were considered reactive only
by the criteria that the COI of at least 1 of the 2
replicates is>=0.9 (Figure 1). Therefore, not all
initially reactive or borderline samples need
further duplicate testing. To some degree, it can
avoid the complexity to HBsAg test. We retro-
spectively analyzed the performance of ECLIA in
repeatedly reactive samples with 0.9COI<7.0.
However, the PPV could increase to 90.99 and
99.40%, even to 100% using an arbitrary cutoff
value with an expectation of a decrease in
complexity, improved turnaround time, and
decreased expense.
36 WR samples were found HBsAg negative or
confirmed negative. In addition, five of them were
found HBV DNA existed. Chen and Kaplan (2006)
indicated 41.2% HBsAg confirmed positive and
7% confirmed negative samples were found HBV
Ning et al. 3027





Figure 1. Flow chart of the study (the result are in brackets).



DNA, when ROCHE COBAS Amplicor HBV Monitor test
(Ver. 2.0) was used, the detection limit of which goes to
zero. Occult HBV is defined as the presence of HBV DNA
in the liver (with detectable or undetectable HBV DNA in
the serum) of individuals testing HBsAg negative by
currently available assays (Said, 2011). Ocana et al.
(2011) indicated that blood detection of occult hepatitis B
requires assays of the highest sensitivity and specificity
with a lower limit of detection of <10IU/ml for HBV DNA
and <0.1 ng/ml for HBsAg. Therefore, our data indicated
that WR samples, which did not pass neutralization
criteria, might have evidence of HBV infection.
In conclusion, it was the first report of evaluation of
ECLIA with HBsAg confirmatory test in lower COIs sam-
ples. Evaluation of HBsAg prevalence through Elecsys
HBsAg II overestimated the true positive, especially in
WR samples. Besides, when interpreting WR samples for
diagnostic purpose, the results should always be
assessed in conjunction with the patients medical
history, clinical examination and other findings.
ACKNOWLEDGEMENT

This study was supported by grants from National Natural
Science Foundation of China (#81072432/H1005).


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