International Journal of Epidemiology Vol. 25, No.

3
O International Epidemlological Association 1996 Printed in Great Britain

False Positive Tests for

Anti-Hepatitis C Antibodies and the
Problem of Notifying Blood Donors
SHULAMtTH BAR-SHANY,* MANFRED S GREEN" AND EILAT SWNAR*

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Bar-Shany S (Blood Services, Magen David Adorn, Tel Hashomer, Israel), Green M S and Shlnar E. False positive tests
for anti-hepatitis C antibodies and the problem of notifying blood donors. International Journal of Epidemiology 1996;
25: 674-678.
Background. All donated blood in Israel is tested for anti-hepatitis C virus (HCV) antibodies by enzyme immunoassay
(EIA) and donors are notified of the result. There Is evidence that at low antibody titres, the percentage of false positives
may be high, with consequent labelling of healthy people as being infected with HCV.
Aim. In this study we examined the correlation between anti-HCV antibody titres determined by a second generation EIA
test with supplemental EIA tests and evidence of abnormal liver function.
Methods. Blood samples of 201 Israeli civilians who donated blood during 1992 and were repeat reactive for antl-HCV
antibody based on second generation EIA, were tested by a supplemental test. Follow-up data were obtained from the
donors and their family physicians.
Results. Results of anti-HCV EIA tests on two separate occasions of blood donation were highly correlated with each
other (r = 0.86). Positive supplemental tests and abnormal liver function tests were found only in those subjects with high
antibody titres. Furthermore low antibody titres were more prevalent during the winter months, suggesting that seasonal
intercurrent infections may increase the percentage of false positives.
Conclusions. A high proportion of blood donors labelled as anti-HCV antibody positive based on low antibody titres, may
not be at increased risk of carrying HCV. Since labelling would result in creating unnecessary anxiety among blood
donors, It is important to confirm such results with tests such as radioimmunoblot assay (RIBA).
Keywords: blood donors, supplemental EIA tests, liver enzymes, anti-HCV antibody levels

There is evidence that a high percentage of patients who
are positive for anti-hepatitis C virus (HCV) antibody
have hepatitis C viraemia and are infectious.1'2 How-
ever, the relatively low specificity of the enzyme im-
munoassay (EIA) tests for anti-HCV antibodies could
result in a large number of healthy donors being falsely
labelled as carriers of HCV. The second generation EIA
is more specific than the earlier tests,3'4 but there is
evidence of cross-reaction of the test with antibodies
against other viruses.5'6 This cross-reaction is usually
seen at low levels of positivity on EIA, resulting in a
low specificity for HCV infection at these levels.4'7
Furthermore, Zhang el al* found that using the second
generation EIA, higher antibody titres reflected by an
S/C ratio of > 5 discriminated well between infective
and non-infective subjects.
In this study we analysed the serological and
biochemical findings in a sample of anti-HCV positive
• Blood Services, Magen David Adorn, Tel Hashomer, Israel.
*• Israel Center for Disease Control, PO Box 559, Petach Tikva 49202.
Israel and Faculty of Medicine. Tel Aviv Universily, Israel.
674
volunteer Israeli blood donors. The aim of the study
was to estimate the extent of possible false positive
tests. We determined the correlation between the sec-
ond generation EIA screening test and supplementary
EIA tests, and examined results of liver enzymes at
different strengths of positive reactions on screening
EIA.
MATERIALS AND METHODS
All blood collected by the Magen David Adorn blood
services in Israel is obtained from voluntary donors. In
addition to testing for hepatitis B surface antigen and
anti-HIV antibody, anti-HCV antibody titres are deter-
mined by means of a second generation EIA (Abbott,
Chicago, Illinois). The test antigen is based on the
antigens HC34, HC31 and c 100-3.
This is followed by supplemental tests in initially
positive subjects. The supplemental assay (Abbott HCV
EIA supplemental assay) uses two different recombin-
ant antigens, separately coated onto two different beads.
The structural antigen is derived from the core region of
 FALSE POSITIVE TESTS FOR ANTI-HCV ANTIBODIES
TABLE 1 Risk groups of Israeli blood donors positive for anti-hepatitis C antibodies by ALT results
Risk group Normal ALT*
N %
History of blood
transfusion 30 20.4
Intravenous drug use 3 2.0
Medical/paramedical
occupations 14 9.5
Tattoos 2 1.4
History of
hospital ization 31 21.1
None 67 45.6
Total 147 100.0
" Chi-square = 28.1, P < 0.001, for difference in distribution of risk groups between subjects with normal and abnormal ALT.
the putative HCV genome and the non-structural anti-
gen from sequences in the NS3 and NS4 regions. The
results are expressed as the ratio of the actual EIA test
reading (sample = S) to the cutoff (C) value for the test
(S/C ratio). During 1992, of 161 813 blood units (from
130 122 donors) tested at the Tel Hashomer blood
service facility, 896 (0.55%) were anti-HCV repeat
reactive. The S/C ratios of the positive tests were distri-
buted as follows (only the lower bound is inclusive):
42% were between 1 and 2, 14% between 2 and 3, 6%
were between 3 and 4 and 37% were s=4. Of 97 965
units donated by civilians, 585 (0.6%) were found to be
anti-HCV positive. During the first half of 1992, only
subjects whose blood tested strongly positive for anti-
HCV antibodies (S/C > 3) were notified of their results
and subsequently all subjects testing positive were
notified. During the whole year, a total of 303 positive
subjects were notified and given a referral letter to their
family physicians, which recommended that they
undergo a general medical check-up and liver function
tests. The first author (S.B-S.) attempted to contact by
telephone all these donors for risk factor history. The
subjects were classified by risk groups according to
possible previous exposure to contaminated blood or
blood products. Those who fell into more than one risk
category were classified by what was considered by the
interviewer to be the more likely exposure according
to the following hierarchy, previous blood tranfusion
being the most important exposure to previous hospital-
ization as being the least important. Attempts were made
to contact the physicians of all subjects. The family
physicians of 201 subjects were contacted, the findings
on the patients discussed and liver enzyme test results
(aspartate aminotransferase) recorded. For technical
reasons, the physicians of the remainder of the subjects
675
Elevated ALT* Total
N % N %
20 37.0 50 24.9
6 11.1 9 4.5
3 5.6 17 8.5
1 1.9 3 1.5
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8 14 8 39 19.4
16 29.6 83 41.3
54 100.0 201 100.0
could not be located, but there was no reason to suspect
any bias resulting from the failure to obtain data on
these subjects. In order to test the consistency of the test
results, an additional study was made of anti-HCV posi-
tive donors who had previously donated blood since
HCV antibody testing was first implemented and the
test results on the two occasions were compared. Fin-
ally, for the entire study group, the prevalence of anti-
HCV antibodies was also analysed by month of blood
donation. Correlations between different tests were
estimated using Pearson correlation coefficients. The
Mantel-Haenszel extension test was used to test for
significance of trend in the seasonality of the preval-
ence of positive results. An approximate randomization
test was used for comparing several percentages where
some cells have very small numbers.
RESULTS
Of the 201 subjects in the study, 47 (23%) were women.
This proportion is similar to that in the whole popu-
lation of blood donors. The average age was 43.5 years
(range 18-65 years). When examined by region of
origin, 40% were born in Israel, 18% were from the
previous USSR, 10% were from Eastern Europe, 9%
from Western Europe, and less than 5% were from any
other specific country. The characteristics of the anti-
HCV antibody positive subjects by risk category are
given in Table 1. Those with a history of blood trans-
fusion or intravenous drug use were more likely to have
elevated serum alanine aminotransferase (ALT). The
graph of the EIA anti-HCV antibody results of the first
and subsequent tests in a group of 45 initially antibody
positive subjects is shown in Figure 1. The correlation
coefficient was 0.86.
676 INTERNATIONAL JOURNAL OF EPIDEMIOLOGY

TABLE 2 Elevation of liver enzymes in HCV positive donors

EIA S/C ratio No. with elevated

liver enzymes (%)

1 < S/C <2 52 0(0)

2 < S/C < 3 22 0(0
3 < S/C < 4 19 2(10.5)
>4 108 52(48.1)
Total 201 54 (26.9)

P = 0.01 for difference in percentages (by approximate randomization

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test for small samples).

2 3 4 5 6 7
1 st Donation S/C
Prevalence (%)
FIGURE 1 Scattergram of the results of 45 anti-HCV positive
subjects, repealed within a period of 6 months S/C ratio
(1.5(1

100* 040

030
75%
0.20

• S+ ANDNS+
50% 0 10
DS- AND NS+
QS+ ANDNS-
SS-ANDNS- 0.00
1 2 3 4 5 6 7 8 9 10 II 12
25%
Month

FIGURE 3 Prevalence of anti-HVC antibodies at high and low

1-S/C<2 2-S/CO 3-S/C<4 S/C-4+
S/C ratio cut-off
FIGURE 2 Distribution of 201 blood donors positive for anti-HCV
antibodies by second generation EIA, at different S/C cut-points
according to the presence of antibodies to the structural (S) and
non-structural (NS) antigens on a supplemental EIA
The prevalence of positivity of the supplemental
anti-HCV antibody tests by level of positivity of EIA is
shown in Figure 2. Only at levels of S/C > 4 were most
of the tests positive for both the structural and non-
structural antigens and at levels of S/C < 3, only 1 out
of 71 was positive for both antigens. The association
of the level of the EIA tests with elevated liver enzymes
in anti-HCV antibody positive blood donors is shown
in Table 2. At levels equal to or exceeding an S/C ratio
of 4, nearly 50% had elevated enzymes whereas none
with an S/C ratio < 3 had elevated enzymes. Thus for an
S/C ratio 3= 3, 83.6% (102/122) were positive on both
the structural and non-structural antigens, and 42.5%
(54/127) had elevated liver enzymes. The prevalence of
litres by month of blood donation among Israeli donors
positive EIA antibody titres at two levels, by calendar
month, is shown in Figure 3. For the lower titres there
is a significant trend for increased prevalence of sero-
positivity during November and December (P < 0.001
for trend).
DISCUSSION
We found a very high correlation between second
generation EIA tests on repeated blood donations,
indicating stability of the results over a period of
months. Thus a finding of either high or low titre can be
regarded as a consistent finding over a short period. The
Abbott EIA supplemental test was positive almost
exclusively at second generation EIA S/C ratios * 3,
suggesting that at S/C ratios < 2, the specificity of the
standard EIA test may be very low. The findings in this
study indicate that evidence of active hepatitis (as
reflected by abnormal enzymes) is only present in blood
 FALSE POSITIVE TESTS FOR ANTI-HCV ANTIBODIES
donors with high anti-HCV antibody titres. This is
consistent with the findings of Kurosaki et al.9 who
observed that patients with increased liver enzymes had
higher levels of HCV RNA by PCR than those with
normal enzymes and concluded that active liver disease
was correlated with the level of viraemia. Romeo
et al.10 found that in anti-HCV positive blood donors
with normal liver enzymes, in only a minority was HCV
viraemia detectable.
The more specific radioimmunoblot assay (RIBA)
has been licensed as a confirmatory anti-HCV test, but
is not universally available because of its high cost.
Another limitation of the RIBA is the high per cent of
indeterminate results. Sayers et al.11 found that 34%
(52/153) of repeat reactive samples of blood donors
were RIBA indeterminate. Yun et al.12 found that RIBA
was positive in about 46% of those with anti-HCV EIA
antibodies. HCV RNA was found in 95% of the RIBA
positive patients, in 21% of the RIBA indeterminate,
and in none of the RIBA negative patients. In addition,
HCV RNA was found in the serum of all but one of
those with chronic active or persistent hepatitis. They
observed that HCV viraemia can be present in patients
who have antibodies to only one HCV antigen, particu-
larly the C22 epitope. In another study, HCV EIA-2
correctly identified all RIBA-confirmed specimens.13
However, 80% of HCV EIA-1 positive who were RIBA
negative or had an S/C ratio < 2 in EIA-1 were nega-
tive by EIA-2 and they suggested that in such cases
donors can be reassured.
The higher prevalence of anti-HCV positives at low
EIA titres observed during the winter months, suggests
that there is cross-reactivity with other antibodies,
possibly to respiratory viruses which are prevalent in
winter. This phenomenon has also been observed in
African subjects by Hyams et al.1 In two studies of
blood donors who received influenza vaccine, multiple
false-positive EL1SA were obtained for HIV, HTLV-1
and HCV.14'15 This phenomenon appears to persist
longer for HCV.14 In the present study, the higher
prevalence of anti-HCV antibodies in winter is
explained mainly by an excess of subjects with S/C
ratios < 3, which strengthens the impression that the
specificity of the EIA tests for anti-HCV antibodies
may be much reduced at low antibody titres.
In summary, blood donors who are identified as
positive for anti-HCV antibodies on second generation
EIA are much more likely to be true positive if they
have high antibody titres and consequently at increased
risk for chronic liver disease. Low antibody titres may
be non-specific and could result from cross-reaction
with other antigens. Consequently, a more reassuring
notification can be sent to donors found to be positive
677
at S/C ratios < 3, and a limited deferral time before
repeat donation of blood should be considered. It is
well-established that RIBA is the standard confirmatory
test and should be used whenever possible. However,
we suggest that where RIBA confirmatory assays are
not readily available, the strength of the EIA anti-HCV
test (S/C ratio) should be included in donor notification.
Donors with high ratios (S/C > 3) should be urged to
see their physicians for long-term follow-up, and be
advised not to donate blood in the future. However,
donors with low antibody titres should be notified that
the result may be non-specific and they should be
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followed-up by their physicians for repeat testing.
ACKNOWLEDGEMENT
This work was supported by an ARMDI fellowship
grant.
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