Journal of Virological Methods 285 (2020) 113962

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Journal of Virological Methods

journal homepage: www.elsevier.com/locate/jviromet

Head-to-head comparison of Enzyme Linked Immunosorbent Assay (ELISA)

and Enhanced Chemiluminescence Immunoassay (ECLIA) for the detection
of Transfusion Transmitted Disease (TTD) Markers; HIV, HCV and HBV in
blood donors, in India
Aseem Kumar Tiwari , Anand Prakash Upadhyay *, Dinesh Arora , Tina Wadhwa ,
Geet Aggarwal , Swati Pabbi , Aanchal Sunil Luthra , Sunder Singh Rawat
Medanta- The Medicity Hospital, Department of Transfusion Medicine, Sector-38, Gurgaon, India

A R T I C L E I N F O A B S T R A C T

Keywords: Safe blood transfusion being the cornerstone of any Blood Transfusion Services requires meticulous testing for
ELISA Transfusion Transmitted Disease (TTD) markers in donated blood. We performed head-to-head comparison of
ECLIA ELISA and ECLIA for detection of TTD markers for Human Immunodeficiency Virus (HIV), Hepatitis C Virus
Chemiluminescence
(HCV) and Hepatitis B Virus (HBV) in 10,164 Indian blood donors. All concordant reactive, discordant reactive
TTD markers
Blood donors screening
and concordant non-reactive samples were re-confirmed using Individual Donor-Nucleic Acid Amplification Test
(ID-NAT) as the ‘gold standard’ test. 223 samples were found reactive; out of which 93 (four HIV, 34 HCV and 55
HBV) samples were concordant reactive and tested positive by both methods while 130 discordant reactive
samples were reactive either by ELISA or ECLIA. Out of 130 discordant reactive samples ELISA-reactive and
ECLIA-non-reactive samples for HIV, HCV and HBV were 15, eight, and 29 respectively while ECLIA-reactive
ELISA-non-reactive samples for HIV, HCV and HBV were 39, 36 and three respectively. Sensitivity of ECLIA
and ELISA was 100 % for all three TTD markers, while specificity was 99.62 % and 99.85 % for HIV; 99.64 % and
99.84 % for HCV and 99.97 % and 99.70 % for HBV respectively. Strength of agreement and Kappa Statistics for
ECLIA and ELISA compared to the gold standard test was poor and fair for HIV (k = 0.169 and 0.347), moderate
and good for HCV (k = 0.539 and 0.763), and very good and good for HBV (k = 0.973 and 0.783). According to
this study, it can be concluded both the testing techniques; ELISA and ECLIA have 100 % sensitivity for the
detection for HBV, HCV and HIV in blood donors and therefore, either can be used for TTD screening in blood
banks in India.

1. Introduction
Safe and adequate blood transfusion is cornerstone of any Blood
Transfusion Services (BTS). Several ways by which the risk of Trans­
fusion Transmitted Infections (TTIs) can be reduced are careful selection
of donors, diligently administrating medical history questionnaire and
brief and meticulous physical examination of blood donors, testing for
TTD markers, removal of specific blood component known to harbor
infectious agents (leukocyte filtration), by physical or chemical inacti­
vation of microbes (pathogen inactivation) and rational use of blood
components in patients, etc. The cornerstone of blood safety remains
immaculate testing of the donated blood unit for TTD markers and
discarding all the units which are tested reactive. Units tested non-
reactive are used for transfusion in patients. Screening tests applied in
BTS should be highly sensitive so that none of unit remains undetected
for transmissible pathogen and simultaneously it should be highly spe­
cific to avoid unnecessary high discard rate due to false positivity.
Immuno-Chromatographic Assay (ICA) or rapid tests, Enzyme Linked
Immuno-Sorbent Assay (ELISA), Chemiluminescence Immuno-Assay
(CLIA) are routinely used for TTI screening of donated blood in India.
CLIA is a relatively newer technology and is gradually replacing ELISA
due to shorter Turn-Around-Time (TAT), higher reproducibility and
random access; especially at medium (10,000–20000 units/annum) to
large size (more than 20,000 units/annum) BTS. However, there is

* Corresponding author.
E-mail address: email.upadhyay@gmail.com (A.P. Upadhyay).

https://doi.org/10.1016/j.jviromet.2020.113962
Received 30 March 2020; Received in revised form 12 August 2020; Accepted 23 August 2020
Available online 26 August 2020
0166-0934/© 2020 Elsevier B.V. All rights reserved.
A.K. Tiwari et al. Journal of Virological Methods 285 (2020) 113962

paucity of literature on head-to-head comparison between the two
technologies. We, therefore, compared ELISA and ECLIA in term of
various statistical parameters like sensitivity, specificity, accuracy,
Youden’s index and kappa agreement.
2. Materials and methods
2.1. Settings
The antigens (or antibodies) coated on the solid-phase of ELISA and
ECLIA are also detailed in this table.
2.3.1. Quality Assurance
All equipments used were calibrated. Kit controls were used daily
and Levy Jennings graph was maintained and monitored. The laboratory
participates in EQAS (External Quality Assurance Scheme) from three
different PT (Proficiency testing) providers.

A prospective, observational study was performed in department of
Transfusion Medicine at a large tertiary care hospital in North India on
10,164 consecutive whole blood donors coming from North Indian
states and union territories including Rajasthan, Madhya Pradesh,
Chhattisgarh, Jharkhand, Bihar, Uttar Pradesh, Uttaranchal, Himachal
Pradesh, Punjab, Haryana, Laddakh, Jammu and Kashmir, Chandigarh
and Delhi. Head-to-head comparison of ECLIA (VITROS 3600; Ortho
Clinical Diagnostics, USA) and ELISA (EVOLIS, Bio-Rad, France) was
done on the donor samples for the presence of markers of HBV, HCV and
HIV. Study population included consented donors qualifying the donor
selection criteria for whole blood donation mentioned in Drugs and
Cosmetic Act, 1940 (Malik, 2003) and Directorate General of Health
Services (DGHS) Technical Manual, 2003 (Saran, 2003). The study was
conducted over a period of 10 months (April 2018- January 2019).
2.2. Sample size calculation
2.3.2. Concordance/discordance and confirmation by gold standard test
All concordant reactive (donor samples which were found reactive
by both ELISA and ECLIA), discordant reactive (donor samples which
were found reactive by only one method; either by ELISA or ECLIA) and
concordant non-reactive (samples which were found non-reactive by
both ELISA and ECLIA) samples were reconfirmed by the gold standard
Individual Donor-Nucleic Acid Testing (ID-NAT) using Procleix Ultrio
Elite Assay on Procleix Panther System (Hologic and Grifols, Spain). The
Procleix Ultrio Elite Assay involves three main steps which take place in
a single tube on the Procleix Panther System: 1) Sample preparation/
target capture 2) HIV RNA, HCV RNA, and HBV DNA target amplifica­
tion by Transcription-Mediated Amplification (TMA) and 3) Detection of
the amplification products (amplicon) by the Hybridization Protection
Assay (HPA). The Procleix assays incorporate an Internal Control for
monitoring assay performance in each individual reaction tube. All
reactive units were discarded to ensure patient safety (Fig. 1).

The prevalence of HIV (0.23 %) is relatively least in comparison to
HBV and HCV [30, 46 47, 48] and was therefore taken as the variable to
calculate the study sample size. Assuming 95 % confidence level and ±
10 % margin of error sample size was calculated by applying formula n =
[Zα 2 P Q]/[d2*Prevalence]; Where; Zα = 1.96 (Value of standard normal
variate corresponding to α level of significance corresponding to 95 %
confidence interval, P = 0.95 (Likely value of parameter), Q = 0.05 ( = 1
– P) and d = 0.10 (Margin of errors which is a measure of precision).
With these assumptions the minimum sample size worked out as 8260.
The actual sample size in this study was 10,164 blood donors.
2.3. Sample collection and TTD testing
Three milliliter samples were collected from each consecutive whole
blood donors in EDTA vacutainer from diversion pouch of blood
collection bags using strict aseptic techniques. Specimens were centri­
fuged at 5000 rpm for 10 min. Routine testing for the presence TTD
markers of HBV, HCV, and HIV 1 and 2 were performed on VITROS 3600
(Ortho Clinical Diagnostics, Johnson and Johnson, USA) using VITROS
HBsAg test, VITROS aHCV test and VITROS HIV Combo test assays
strictly following manufacturer’s instructions and departmental stan­
dard operating procedures (SOP). Same samples were tested on the
EVOLIS (Bio-Rad, France) using Monolisa™ HBsAg ULTRA, Monolisa™
HCV ULTRA V2, Genscreen™ ULTRA HIV Ag-Ab assay to detect HBV,
HCV and HIV respectively. Sensitivity and specificity of all ELISA and
ECLIA assays (anti-HIV, anti-HCV and HBsAg) are depicted in Table 1.
2.4. Statistical analysis
The analysis included profiling of donors on demographic blood
group and findings of ELSIA, ECLIA and ID-NAT. Screening test perfor­
mance of ELISA and ECLIA were presented in terms of Sensitivity,
specificity, accuracy, Youden’s index and kappa agreement with respect
to ‘gold standard’ ID-NAT. Cross tables were generated based on ELISA,
CLIA and confirmatory test (gold standard). This was done separately for
HIV, HBV and HCV. SPSS software, version 24.0 was used for analysis.
2.5. Ethical approval
Institutional Review Board (IRB) approved this study. (Reference no:
MICR-798/2017)
3. Results
3.1. Donors demographic profile
Total number of donors included in this study was 10,164. Out of
these 94.99 % (n = 9655) were male and 5.01 % (n = 509) were female
donors. First time and repeat donors were 94.95 % (n = 9651) and 5.05
% (n = 513) respectively. Replacement and voluntary donors were 91 %
(n = 9250) and 9% (n = 914) respectively. Mean age of distribution was
32.64 years. Frequency of ABO group donors was 21.03 % (n = 2138),
36.74 % (n = 3735), 8.60 % (n = 871) and 33.64 % (n = 3420) for A, B,

Table 1
Sensitivity, specificity, antigens and antibodies coated on solid phase of assays used in ELISA and ECLIA for detection of TTD markers (as per manufacture’s kit insert).
TTD HIV HCV HBV

Testing Method ELISA ECLIA ELISA ECLIA ELISA ECLIA

Assay Genscreen™ ULTRA HIV Ag-Ab assay VITROS HIVc Monolisa™ VITROS Monolisa™ VITROS HBsAg
HCV ULTRA aHCV HBsAg ULTRA
V2
Sensitivity % 100 100 100 100 100 100
Specificity % 99.95 99.84 99.94 99.76 99.94 99.98
Antigens and Monoclonal anti-HIV p24 Ab, purified Antigens Biotinylated anti-HIV p24 Ab, Capsid peptide C22− 3, Monoclonal Monoclonal
antibodies gp160 (envelop) recombinant protein, HIV-1 HIV–1 envelope (env 13), HIV-1 NS3, NS4 NS3, NS4, anti-HBs anti-HBs
coated on solid group O-specific epitope and peptide mimicking group O envelope (env 70− 3) NS5 antibodies antibodies
phase immunodominant epitope of HIV-2 and HIV–2 envelope (env 31).

2
A.K. Tiwari et al.
Fig. 1. Algorithm for TTD markers testing.
AB and O blood groups respectively. 92.98 % (n = 9451) donors were
RhD positive and 7.02 % (n = 713) were RhD negative.
3.2. Comparison of ELISA, ECLIA for TTD markers
Out of 10,164 donors 223 samples were reactive. Ninety three
samples were concordant reactive while 130 samples were discordant
reactive. Out of 93 concordant reactive there were four HIV, 34 HCV and
55 HBV reactive sample. Out of 130 discordant reactive samples ELISA-
reactive and CLIA-nonreactive samples for HIV, HCV and HBV were 15,
eight, and 29 respectively while CLIA-reactive ELISA-nonreactive sam­
ples for HIV, HCV and HBV were 39, 36 and three respectively. All
concordant reactive (n = 93), discordant reactive (n = 130) and all
concordant nonreactive samples for all three TTD markers were further
tested by the ID-NAT as the gold standard.
3.3. Prevalence
In this study overall prevalence of HIV, HCV and HBV was 0.85 %.
True prevalence (confirmed positive samples with gold standard) of
HIV, HCV and HBV among whole blood donors was found to be 0.04 %,
0.26 % and 0.54 % respectively.
3.4. Statistical parameters of ELISA and CLIA for detection of HBV, HCV
and HIV
Sensitivity of ELISA and ECLIA for detection of HIV was 100 % while
specificity was 99.85 % and 99.62 % respectively. Accuracy was found
to be 99.85 % for ELISA and 99.62 % for CLIA. Youden’s index for both
ELISA and CLIA was 0.99. Cohen’s kappa statistics; k = 0.347 ± 0.127
3
Journal of Virological Methods 285 (2020) 113962
for ELISA shows a fair strength of agreement with gold standard test
while Cohen’s kappa statistics; k = 0.169 ± 0.074 shows a poor strength
of agreement of ECLIA with the gold standard (Table 3) (Fig. 2).
Sensitivity of ELISA and ECLIA for detection of HCV was 100 % while
specificity was 99.84 % and 99.64 % respectively. Accuracy of ELISA
and CLIA was 99.84 % and 99.64 % respectively. Youden’s index for
both ELISA and CLIA was 0.99. Cohen’s kappa value; k = 0.764 ± 0.573
for ELISA shows good strength of agreement with gold standard test,
while kappa value; k = 0.539 ± 0.061 moderate agreement of ECLIA
with the gold standard test (Table 3) (Fig. 2).
Sensitivity of ELISA and ECLIA for detection of HBsAg was found to
be 100 % while specificity was 99.70 % and 99.97 % respectively. Ac­
curacy for ELISA and CLIA was 99.70 % and 99.97 % while Youden’s
index was 0.99 for both methods. Strength of agreement with the gold
standard for ELISA was good (k = 0.783 ± 0.037 while it was very good
(k = 0.973 ± 0.015for ECLIA (Table 2) (Fig. 2).
4. Discussion
The study was conducted on 10,164 blood donors, which was large
enough to determine any statistical difference between ELISA and
ECLIA. This study was probably first head-to-head comparison of ELISA
and ECLIA for screening TTD markers in blood donors on a large sample
size. Most of the published studies had smaller sample size
(n = 21–2000) which compared different types of CLIA and/or ELISA
with each other and/or with gold standard (Bisseye et al., 2017;
Majumder and Shetty, 2017; Madiyal et al., 2016; Sommese et al., 2014
and Xu et al., 2012,). Most of the published studies compared single TTD
marker with known positive and/or negative samples (Majumder and
Shetty, 2017; Madiyal et al., 2016; Xu et al., 2012 and Huh et al., 2007).
A.K. Tiwari et al. Journal of Virological Methods 285 (2020) 113962

Fig. 2. Comparison of sensitivity, specificity and accuracy of ELISA and CLIA for HIV, HCV and HBV.

(n = 9655) donors were male and 5.01 % (n = 509) were female donors.
Table 2
Out of 223 reactive donors by two methods for HIV, HCV and HBV 99.10
Summary of Reactive and Non-reactive samples with ELISA, CLIA and Confir­
% (n = 221) were male donors and 0.9 % (n = 2) were female donors.
matory test.
Both female donors were reactive for HBsAg. Donors between ages of
Concordant Non-reactive samples 18–65 year were included in this study. Mean age of donation was 32.64
Concordant Non-reactive (Non-reactive by ELISA and ECLIA Confirmatory Test years. Maximum donors were from 18− 30-year (48 %) followed by
for HIV, HBV and HCV) Positive Negative 31− 40-year (34.22 %) and least in the 50–65 (17.78 %) year age group.
Highest seroreactivity was found in 18− 30-year age group (45.16 %)
HIV (n = 10,106) 0 10,106
HCV (n = 10,086) 0 10,086 followed by 31− 40-year age group (37.63 %) and 41–65 age group (17.2
HBV (n = 10,077) 0 10,077 %). This was in concordance with study done by Karmakar et al. (2014)
Concordant reactive
and Dobariya et al. (2016). Nine percent (n = 914) donors were volun­
(n = 93) tary while 91 % (n = 9250) donors were replacement donors. Replace­
ment donors outnumber the voluntary donors and this finding is similar
Concordant Reactive Confirmatory Test
to studies published by Makroo et al. (2015) and Pahuja et al. (2007).
TTD Markers (n = 93) Positive Negative
(Reactive by both ELISA and ECLIA) (n = 85) (n = 8)

HIV 04 04 0 4.2. Prevalence

HCV 34 26 8
HBV 55 55 0 Overall seroprevalence for the presence three TTD markers in this
Discordant reactive study was 0.85 %. Seroprevalence of TTD markers on the basis of single
(n = 130) test varied between 0.19− 0.42 %, 0.42− 0.72% and 0.58− 0.84 % for
Discordant Reactive Confirmatory Test HIV, HCV and HBV respectively and findings were similar to previous
TTD publications by Makroo et al. (2015); Pallavi et al. (2011); Chandra et al.
Reactive by Reactive by Positive Negative
Markers ELISA only ECLIA only (n1+n2 = 130) (2009) and Ismail et al. (2004).
(n1 = 52) (n2 = 78)

HIV 15 39 0 54 4.3. Comparison of ELISA and ECLIA for TTD markers

HCV 08 36 0 44
HBV 29 03 0 32
4.3.1. Comparison of ELISA and CLIA for detection of HIV
Four concordant reactive samples found confirmed positive by ID-
Very few studies compared all three viral markers with smaller sample NAT while all concordant non-reactive samples by ELISA and CLIA
size (Bisseye et al., 2017, Sommese et al., 2014 and Maity et al., 2012). were found negative by ID-NAT. Out of 54 discordant HIV reactive
samples, 15 samples were reactive by ELISA alone while 39 samples
were reactive by ECLIA only. All 54 discordant reactive samples were
4.1. Donor demographics found negative when confirmed by the gold standard test. Sensitivity of
ELISA and ECLIA was 100 % (CI 39.76%–100%) while specificity was
In this study 10,164 donors participated out of which 94.99 % 99.85 (CI 99.76%–99.92 %) and 99.62 % (CI 99.48–99.73 %)

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A.K. Tiwari et al. Journal of Virological Methods 285 (2020) 113962

Table 3
Comparison of different statistical parameters for ELISA and CLIA for HIV, HCV and HBV.
STASTISTICAL HIV Values (CI)* HCV Values (CI) HBV Values (CI) STASTISTICAL HIV Values (CI)* HCV Values (CI)
PARAMETER PARAMETER
ELISA ECLIA ELISA ECLIA ELISA ECLIA

SENSITIVITY (Sn) 100 % 100 % 100 % 100 % 100% 100%

(39.76 %–100 %) (39.76 %–100 %) (86.77 %–100 %) (86.77 %–100 %) (93.62 %–1 00%) (93.62 %–100 %)
SPECIFICITY (Sp) 99.85 (99.76 %– 99.62 (99.48 %– 99.84 (99.74 %– 99.64 (99.51 %–99.75 99.70 (99.58 %–99.80 99.97 (99.51 %–99.75
99.92 %) 99.73 %) 99.91 %) %) %) %)
ACCURACY 99.85 (99.76 %– 99.62 (99.48 %– 99.84 (99.84 %– 99.65 (99.51 %–99.75 99.70 (99.58 %–99.80 99.97 (99.51 %–99.75
99.92 %) 99.73 %) 99.97 %) %) %) %)
YOUDEN’S INDEX 0.99 0.99 0.99 0.99 0.99 0.99
KAPPA STATISTICS 0.347 ± 0.127 (0.097 0.169 ± 0.074 (0.024 0.764 ± 0.573 (0.651 0.539 ± 0.061 (0.419 0.787 ± 0.037 (0.71306 0.9737 ± 0.015
(k ± S.E) to 0.597) to 0.314) to 0.876) to 0.660) to 0.86157) (0.94409–1.0)
STRENGTH OF Fair Poor Good Moderate Good Very Good
AGREEMENT
*
CI; confidence interval.

respectively. Both testing methods show false positive results for
detection HIV markers which were similar to the findings published by
Sommese et al. (2014). False positivity for HIV is commonly encoun­
tered in healthy donors. Few possible reasons are: high sensitivity of
ELISA and ECLIA, and very low prevalence of disease in healthy donor
population (Lequin, 2005; Sommese et al., 2014; and Maity et al., 2014).
4.3.2. Comparison of ELISA and CLIA for detection of HCV
Out of 34 concordant samples 26 samples were confirmed positive by
confirmatory test and eight samples found negative. All concordant non-
reactive samples (n = 10,086) were confirmed negative by confirmatory
test. Out of 44 discordant samples, eight reactive donor samples by
ELISA were non-reactive by ECLIA, while 36 samples reactive by ECLIA
were non-reactive by ELISA. All 44 discordant reactive samples were
confirmed negative by confirmatory test. Sensitivity of both methods;
ELISA and CLIA were 100 % while specificity of ELISA and CLIA was
99.84 %and 99.64 % respectively. Cohen’s kappa value;
k = 0.764+0.573 for ELISA shows good strength of agreement with gold
standard test, while kappa value; k = 0.539 ± 0.061 shows moderate
agreement of ECLIA with gold standard test. Sensitivity, specificity and
Cohen’s kappa values were similar to the findings published by
Sommese et al. (2014) and Ismail et al. (2004). Possible explanation to
52 false positive anti-HCV results include high sensitivity of ELISA and
ECLIA, clearing of viremia before antibodies form circulation, natural
recovery from previous infection (Tulsiani et al., 2012), viremia below
detectable lavel (Busch et al., 2006), early testing during acute infection
(before complete seroconversion) and waning antibody due to resolved
infection (Ismail et al., 2004).
4.3.3. Comparison of ELISA and CLIA for detection of HBV
All concordant samples (n = 55) were confirmed positive with the
gold standard ID-NAT. 10,077 concordant non-reactive samples by
ECLIA and ELISA were confirmed negative by gold standard test. Out of
32 discordant samples, 29 samples reactive by ELISA and three samples
reactive by ECLIA were tested negative with gold standard test. Sensi­
tivity of ELISA and ECLIA was 100 % while specificity was 99.70 % and
99.97 % respectively. Higher sensitivity and specificity of both methods
make them better choice for the screening of HBsAg. Strength of
agreement with gold standard for ELISA was good (k = 0.787 ± 0.037;
CI 0.713 to 0.861) while it was very good (k = 0.973 ± 0.015; CI
0.944–1.000) for ECLIA. This study had similar findings as published by
Bisseye et al. (2017) and Madiyal et al. (2016) on ECLIA and ELISA. Both
ECLIA and ELISA showed a similar performance for the detection of
HBsAg. Both ELISA and CLIA detected false positive results due to high
sensitivity (Sommese et al., 2014 and Maity et al., 2012).
4.3.4. Overall comparison of ELISA and CLIA for detection of TTD
ELISA and ECLIA had 100 % sensitivity in this study; however,
specificity varied for the three TTD markers due to false positivity. In
case of HIV and HCV false positivity was higher with ECLIA than ELISA
(39 vs. 15 for HIV and 44 vs. 16 for HCV) while false positivity for HBV
was more with ELISA than ECLIA (29 vs. 3). Both testing methods; ELISA
and ECLIA were capable of detecting all true positive cases. Therefore,
these two screening technologies can be suitably used in blood trans­
fusion services for donor screening. For donor notification, BTS could
then choose sequential serological testing as per WHO HIV testing
strategies for HIV reactive donor notification, counseling and referral
(Barbara et al., 2008, NACO guidelines, 2018). Similar strategies have
been published for HBsAg reactive donors by Tiwari et al. (2019).
Youden’s Index (YI) of ELISA and ECLIA was 0.99, detection of HIV,
HCV and HBV. In this study ELISA and ECLIA both had higher YI which
indicates a better ability to avoid failure. YI is one of the globally
accepted methods of diagnostic measure of overall performance of test
and used to compare the two diagnostic test procedures for the same
disease in question. This index is not affected by disease prevalence.
5. Conclusion
According to this study, it can be concluded both the testing tech­
niques; ELISA and ECLIA have 100 % sensitivity for the detection for
HBV, HCV and HIV in blood donors and therefore, either can be used for
TTD screening in blood banks in India.
CRediT authorship contribution statement
Aseem Kumar Tiwari: Conceptualization, Methodology, Project
administration, Writing - original draft. Anand Prakash Upadhyay:
Conceptualization, Writing - original draft, Writing - review & editing,
Data curation, Visualization, Formal analysis. Dinesh Arora: Method­
ology, Resources, Validation. Tina Wadhwa: Investigation. Geet
Aggarwal: Data curation. Swati Pabbi: Formal analysis. Aanchal Sunil
Luthra: Formal analysis. Sunder Singh Rawat: Supervision,
Investigation.
Declaration of Competing Interest
The authors declare that they have no competing financial interests
or personal relationships that could have appeared to influence the work
reported in this paper.
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