Cancer Cell Culturing and Cytotoxicity Assays for Anticancer Screening at City of Hope
‘Noise’ in microbiological screening assays
Transcript of ‘Noise’ in microbiological screening assays
REVIEW ARTICLE
`Noise' in microbiological screening assays
B. C. Dow Scottish National Blood Transfusion Service Microbiology Reference Unit, Glasgow & West of Scotland Blood Transfusion
Service at Law Hospital, Carluke, Lanarkshire, UK
Received 15 November 1999; accepted for publication 16 March 2000
INTRODUCTION
At present, Transfusion Services can enjoy a choice of
several manufacturers' microbiological assays to screen
blood donations. However, the selection of assays is
based not only on the particular assay's capability of
detecting extremely low levels of the particular micro-
biological marker (sensitivity) but also on a low number
(or rate) of uncon®rmed repeatable reactivities (false
positives). Speci®city is usually measured as 100% minus
this rate of false positivity, so that a speci®city of 100% is
deemed to be perfection. In transfusion microbiology
testing laboratories a speci®city of less than 99´5% can
cause major logistical problems whilst speci®cities of
99´9% or greater are preferred ± these ®gures would
relate to 1 in 200 donations and less than 1 in 1000
donations, respectively.
Background prevalence of the (con®rmed positive)
marker in the donor population is essential in such
calculations. For example, where the true prevalence is
only 1 in 10 000, a test that has 99´9% speci®city will
produce 1 in 1000 reactives of which nine false positives
will occur for every true con®rmed positive. Thus the test
could be considered to have a positive predictive value of
10%.
The past 30 years has seen a dramatic increase in the
sensitivity of various microbiological assays such that
the blood supply has never been as safe from the agents
that are tested. The initial use of ®rst-generation tests for
the various markers was followed by numerous enhance-
ments in assay sensitivities. These increases in sensitivity
have resulted not only in the earlier detection of sero-
converting donors after infection but also the detection of
the various types and subtypes of the infective agent.
Unfortunately, these increases in sensitivity have not
resulted in comparable, or indeed any, improvements in
speci®city. Indeed, sensitivity of assays is now often set
at an acceptable level of `noise' or speci®city. The
sacri®ce of speci®city can lead to an extremely sensitive
assay whilst the sacri®ce of sensitivity can lead to
excellent speci®city.
The advent of automated sample processing equip-
ment has now meant that laboratories may be tied to one
manufacturer for their package of tests. Whilst one assay
may be highly sensitive and speci®c, other tests within
the system may well have barely acceptable speci®city or
sensitivity.
Several different test formats have or are currently in
use in transfusion microbiology laboratories. It is impor-
tant that their principles are understood. There are at least
six different assay formats (Fig. 1):
1 Indirect ELISA (enzyme linked immunosorbent assay)
(e.g. third-generation HCV ELISAs; Ortho (Raritan, NJ,
USA) and Biorad (Hemel Hempstead, UK) Anti-HBc
ELISAs). This utilizes solid-phase antigen to trap speci®c
antibody from the test sample. The speci®c antibody is
then detected using an enzyme-conjugated antiglobulin
that when bound will cause substrate to change colour.
2 Competitive ELISA (e.g. Omega (Alloa, UK) Patho-
zyme Syphilis ELISA, most anti-HBc ELISAs). Solid-
phase antigen is used similar to Indirect ELISA. However,
both enzyme-conjugated antibody and test antibody com-
pete for the sites on the solid phase, usually in a one-step
procedure. Negatives in such an assay cause substrate to
change colour, whereas positives are devoid of colour.
3 Antibody capture ELISA (e.g. Abbott/Murex (Delken-
heim, Germany) ICE HIV 1.0.2). This assay was ®rst
used as the basis of IgM-speci®c assays in clinical
diagnostics. One manufacturer developed this further to
capture any immunoglobulin type on the solid phase.
Addition of conjugated antigen for the agent and sub-
sequent substrate development determines the activity.
4 Sandwich assays (e.g. third-generation HIV assays;
HBsAg assays). Sandwich assays are generally more
sensitive than other assays. They can either be solid-
phase antigen-test antibody ± conjugated antigen as in
HIV assays; or solid phase antibody-test antigen ±
conjugated antibody as in HBsAg assays.
5 Agglutination (e.g. passive haemagglutination (PHA)
for Tetanus antibodies; Treponema pallidum haemag-
glutination assays (TPHA)). This uses the principle of
Transfusion Medicine, 2000, 10, 97±106
97q 2000 Blackwell Science Ltd
Correspondence: Dr B. C. Dow, SNBTS MRU, Glasgow & West of
Scotland BTS at Law Hospital, Carluke, Lanarkshire ML8 5ES, UK.
Tel.: �44 1698 360809; fax: �44 1698 359295.
98 B. C. Dow
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Fig. 1. Different format of assays used in transfusion microbiology.
red cell (e.g. PHA) or particle agglutination to detect
antibodies by the use of red cells or particles coated
with microbial antigens. Originally, test results were
subjectively read by eye but nowadays sophisticated
automated equipment can produce objective results.
Some of these agglutination assays were so sensitive
that soluble antigen had to be introduced as a diluent of
the red cells to decrease the optimal sensitivity of the
assay in order to detect high-titre antibodies (Barr et al.,
1975).
6 Combination assays. This category includes the detec-
tion of both antigen and antibody within the same test
procedure (e.g. combined HIV antigen/antibody tests). A
further test (Murex ICE Syphilis) is based on both anti-
body capture and sandwich principles for the detection of
antibodies to Treponema pallidum.
In the United States a ®gure of nearly 3% of donations
were shown to be repeatedly reactive over a 10-year
period when surrogate tests for ALT and anti-HBc were
performed in addition to HBsAg, Syphilis and anti-HIV
1/2 testing (McCullough, 1993). Busch (1997) has esti-
mated that less than 5% of these donors were probably
infected or infectious. Currently in the UK the mandatory
tests are for Treponema pallidum (Syphilis), HBsAg,
anti-HIV 1/2 and anti-HCV. Usually, less than 0´5% of
UK donations will be repeatedly reactive for any of these
markers. This will vary from centre to centre and is
obviously dependent on the chosen assay and also the
proportion of donors who have already been tested and
found negative by that same assay. Comparison of test
speci®cities on different populations should therefore be
limited to the repeat reactive rates amongst new donors
(Table 1). The Abbott Prism assay has the best speci®city
for HBsAg with only 0´01% (1 in 10 000) falsely reactive
whilst the Pasteur assay had 0´18% falsely reactive. HCV
tests were relatively comparable for speci®city although
Prism and Ortho test systems had almost a two-fold
difference in speci®city. Excluding the Murex VK85
test that has just been superseded by the GE95 assay,
the current HIV assays are also reasonably comparable.
Of the Syphilis assays, the Newmarket TPHA appears to
have the best speci®city whilst the Centocor ELISA has
slightly poorer speci®city compared with the other
TPHAs. These ®gures assume that variations in personal
donor interviews should not have an effect on the `noise'
of the test system used.
HEPATITIS B VIRUS
When hepatitis B surface antigen (HBsAg) donor testing
commenced in the early 1970s, the most common test in
use was counterimmunoelectrophoresis (CIEP). This test
was known to detect about 1 mg mLÿ1 HBsAg. As the test
was gel-based, result reading was very subjective. Two
or more people would read results with a democratic
decision being made for `grey-zone' samples, usually
erring on the side of caution. This led to a few units of
blood being excluded from the blood supply based on the
mere possibility of a line being present. It is quite ironic
that almost three decades later some polymerase chain
reaction (PCR) test results rely on similar gel-based
subjective readings. Thankfully, the next generation of
PCR tests rely on objective reading systems such as
enzyme linked immunosorbent assay (ELISA).
Current HBsAg tests enjoy the ability to detect around
100 pg mLÿ1 or lower HBsAg ± an increase of 10 000-
fold in sensitivity over CIEP. Despite this huge increase
in sensitivity the assays do not detect 10 000 more donors
as HBsAg positive. In fact less HBsAg-positive dona-
tions are now detected each year when compared with
those early years. Several reasons may account for this.
Firstly, the introduction of HBsAg testing detected
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Table 1 Current British speci®city data on new donors (circa
1999)
Corrected false
positive rate
HBsAg test kits
Abbott Auszyme 0´04%
Abbott PRISM 0´01%
Launch Biokit 0´14%
Murex GE15 0´03%
Pasteur Monolisa 0´18%
Anti-HCV test kits
Abbott 3´0 0´13%
Abbott PRISM 0´19%
Ortho 3´0 0´11%
Pasteur Plus 0´17%
Anti-HIV 1� 2 test kits
Abbott 3 Plus 0´06%
Abbott PRISM 0´12%
Murex VK85 0´02%
Murex GE95 0´09%
Murex ICE 0´14%
Ortho 3´0 0´11%
Treponema pallidum antibody test kits
Centocor ELISA 0´08%
Murex TPHA VD35 0´05%
Newmarket TPHA 0´02%
Olympus 0´05%
Randox TPHA 0´05%
Data extracted from the unpublished NBS/PHLS monthly donation
testing report (K. Soldan, personal communication). NB. Kits are
continually being upgraded and improved so the false positive rate
shown may not re¯ect present rates.
HBsAg positives throughout the donor population (new
and regular) whereas current tests generally only detect
HBsAg in new donors or the few regular donors who
have seroconverted. Secondly, epidemiological follow-
up of HBsAg positives has led to the adoption of more
stringent donor selection procedures that tend to defer
donors in high-risk groups. Lastly, there is a ®nite pool of
infected individuals of whom only a few carry low levels
of the virus (Dow et al., 1980; Barbara, 1983).
One problem of the high sensitivity of current HBsAg
tests is that individuals who have recently received
hepatitis B vaccine may be repeatedly reactive (Dow
et al., 1998). Such donors will con®rm by speci®c neutra-
lization and will appear to be undergoing the early stage
of an acute HBV infection as no other HBV markers will
be present. Therefore, all donors with weak but neutra-
lizable HBsAg in the absence of other markers should be
counselled appropriately. Differentiation of vaccinees
from truly HBV-infected individuals is possible using
HBV DNA PCR detection methods.
Thankfully, the use of robotic sampling machines that
use separate pipette tips has eliminated the possibility of
weak HBsAg-positive donations due to `carry-over'.
Nevertheless, the possibility of carry-over may occur
amongst donor samples in long-term donation archives.
Whilst it is acknowledged that the early HBsAg
screening tests such as CIEP were comparatively insen-
sitive (detected only 57% new examples of HBsAg found
by radioimmunoassay (RIA) (Barr et al., 1979), the
subjective reversed passive haemagglutination (RPHA)
tests in use at the end of the 1970s could only detect 80%.
It should also be remembered that the speci®city of the
RPHA assays varied between 98´0% and 99´31% on
initial screening, causing a secondary testing procedure
involving titrations with test and control cells to clear
donations. Some modi®ed RPHA assays were shown to
be more sensitive than their standard counterparts
(Barbara et al., 1979, 1983). These modi®cations used
the principle of diluting the red cells (coated with anti-
body) to increase the sensitivity of the assay to detect
antigen. By comparison, the commercial RIA test had a
speci®city of 99´96% when the manufacturer's cut-off of
2´1 times the negative mean (corresponding to 7 standard
deviations from the negative mean) was strictly adhered
to. Lowering this cut-off to 1´5 times the negative mean
(3 standard deviations from the negative mean) resulted
in slightly poorer speci®city (99´88%) but had the cap-
ability of detecting very weak HBsAg-positive donations
that would have escaped detection using the standard cut-
off. A testing algorithm had to be introduced to cope with
the use of a lower cut-off. This involved repeating the
test in duplicate. If both results were below the 1´5
threshold then the unit was released for use. If any
result was equal to or greater than the 1´5 cut-off then
the unit was discarded and a con®rmatory neutralization
was performed. As this test involved prolonged incuba-
tion, true HBsAg positives ful®lled the greater than 50%
neutralization criterion and invariably exceeded the normal
2´1 standard cut-off. The comfort of HBV markers was not
available until around 1980.
Current HBsAg ELISA tests now have remarkably low
cut-off levels that are often determined by an arbitrary
optical density above the mean of negative control
samples. These low cut-off points have resulted in
extremely sensitive assays that are surprisingly also
highly speci®c. Adjustment of a manufacturer's approved
instructions cannot now be mandated for transfusion
centres as any loss in speci®city must then be borne by
the transfusion centre. In addition, from a legal stand-
point, if it were known that different testing centres were
applying various modi®cations to cut-offs this could
result in considerable embarrassment to the national
transfusion service. The introduction of Standard Oper-
ating Procedures has therefore afforded the opportunities
to keep a tight control on the uniform performance and
interpretation of particular assays. This means that the
manufacturer's protocols are strictly followed.
All HBsAg screening tests should have complemen-
tary speci®c neutralization tests. Without such con®rma-
tory systems, too much reliance is based on the
development of other HBV markers. However, as
already described, individuals undergoing the early
stages of acute HBV infection will present as only
HBsAg reactive. Therefore, speci®c neutralization tests
should be deemed essential before a new HBsAg screen-
ing test is introduced. An exception to this has been the
HBsAg Prism assay (Abbott Laboratories, Delkenheim,
Germany) that was shown to have markedly enhanced
sensitivity compared with its competitors. Over 2 years
elapsed before its complementary neutralization test
procedure was launched. Recently, a microtitre ELISA
(Abbott/Murex GE34/36) has been developed with a
sensitivity approaching the HBsAg Prism assay.
The success of HBsAg screening has been measured
by the reduction of cases of post-transfusion hepatitis B
reported to transfusion centres. The more recent intro-
duction of anti-HCV donor screening in 1991 has seen a
further reduction in these cases but the recent Serious
Hazards of Transfusion (SHOT) report (Williamson et al.,
1999) does still include three cases of post-transfusion
hepatitis B in the entire UK over a 2-year period. Some
cases are due to early acute hepatitis B infection in
donors whilst some were due to `tail-end' carriers
responsible for other cases. Most current HBsAg tests
now rely on monoclonal antibodies to detect HBsAg.
Like good whiskies, a blend (of monoclonals) is neces-
sary to ensure satisfactory sensitivity. Unfortunately,
some donors react to murine antibodies and can cause
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a false positive result. Such individuals can fail to
con®rm on neutralization. Manufacturers can prevent
many of these reactions by inclusion of murine sera
within specimen diluent. Recently, mutant HBV variants
that evade detection with some assays have been
described (Wallace & Carman, 1997; Jongerius et al.,
1998; van Deursen et al., 1998). This has led to a rethink
of introducing donor anti-HBc screening to detect not
just the HBV variants but also the `low-level' chronic
HBsAg carriers that are often the apparent culprits of
these reported transfusion-transmitted hepatitis B infec-
tions. Indeed, from a cost-effectiveness point of view,
donor anti-HBc screening would be considerably more
advantageous than performing HCV NAT.
HUMAN IMMUNODEFICIENCY VIRUS
When commercial anti-HIV tests became available
during 1985, numerous problems were encountered.
Firstly, as anti-HIV was associated with the killer disease,
acquired immunode®ciency syndrome (AIDS), labora-
tories reintroduced the serum inactivation procedure of
56 8C for 30 min to render samples safer for laboratory
personnel. Unfortunately, this inactivation process caused
one particular commercial assay to have an unaccep-
tablly high number of false positive samples (Mortimer
et al., 1985). In addition, one of the two commercial
assays selected for use by the UK transfusion services
developed problems within weeks of commencing blood
donor screening. This particular assay depended on
competition between enzyme-labelled anti-HIV and
anti-HIV in the test sample for HIV antigen sites on
the solid phase. This assay was selected by all but one
UK RTC because of its markedly superior speci®city
(99´97%), simplicity (one less manipulation) and no
requirement for predilution (Table 2). The cut-off point
was determined by using a weak positive sample that the
manufacturers declared should be within 33% and 66%
of the negative mean. Unfortunately, owing to a supply
problem of the weak control sample, a more dilute
sample was issued in kits that resulted in this cut-off
often being in excess of 66% of the negative mean. Plates
with such a result were invalid and repeated tests were
often also invalid. Methods to overcome this problem
were published (Barclay et al., 1986; Barr et al., 1986,
1987; Challis et al., 1988) but the manufacturer never
condoned their use. The assay utilized a cut-off of 10%
above the weak control sample and many laboratories
pushed the test sensitivity by utilizing a grey-zone to
include all results between 10% and 20% above the weak
control. This had the effect of quadrupling the initial
reactives from 0´05% to 0´2% in blood donors. Using
statistical packages, negative test samples on the plate
could be meaned and a 3´2 standard deviation cut-off
could be calculated that added a further 0´18% of
samples requiring repeat testing. Whilst this is probably
considered excessive today, 0´4% was the approximate
false positive rate of many HBsAg assays in use in the
mid 1980s. The added sensitivity gained by sacri®cing
some of the speci®city was real as judged by some
archive `high-risk' samples being reactive, but no con-
®rmed HIV-positive donations were identi®ed through
the routine use of this statistical package.
This competitive assay also had a further problem owing
to `a rheumatoid factor-like' reaction whereby high optical
densities over 1´5 times the negative mean were obtained
with certain samples. To ensure these samples were not
masking anti-HIV, absorption with polymerized human
immunoglobulin and re-testing produced acceptable
results. Experience with other competitive assays for
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Table 2. Comparison of HIV tests used
routinely to screen British blood donors
in 1987 and 1991New Repeat Con®rmed Corrected false
Test kits donors reactive positive positive rate
1987
Anti-HIV
Wellcozyme (polyclonal) HIV 359 193 114 7 0´03%
Dupont HIV 39 473 80 0 0´20%
Organon HIV 1388 4 0 0´28%
Ortho HIV 10 027 21 0 0´21%
January±May 1991
Anti-HIV 1� 2
Wellcozyme HIV 1� 2 193 544 325 1 0´16%
Abbott HIV 1� 2 36 209 38 1 0´10%
Behring HIV 1� 2 10 358 12 0 0´11%
Extracted from the UK BTS monthly donation testing report (V. Rawlinson, personal
communication).
other microbiological speci®cities has shown that samples
from certain individuals can cause weak positive tests, yet
after absorption to eliminate any anti-immunoglobulin
activity, results are clearly negative.
Most early anti-HIV assays relied heavily on viral
lysate as their source of HIV antigens for their solid-
phase component. These viral lysate preparations were
often contaminated with other cellular proteins, includ-
ing HLA antigens. It was therefore not surprising that
some HLA antibody-reactive samples were also reactive
in these tests (Kuhnl et al., 1985).
HIV Western blots have consistently used viral lysate
for their source of antigenic material, more recently
supplemented with viral speci®c recombinant proteins
or synthetic peptides. Thankfully, nonviral proteins tend
to aggregate on these blots away from the viral envelope
high-molecular-weight bands of gp120 and gp160. The
speci®city of Western blots varies between manufac-
turers but it is widely accepted that as much as 60% of
HIV screen negative samples will exhibit an indetermi-
nate banding pattern in some HIV Western blots (Genesca
et al., 1989). Follow-up specimens from repeat reactive
donors with indeterminate levels in Western blot invari-
ably show identical reactivity indicating no evolution of
HIV infection (Courouce et al., 1986). Despite this
speci®city problem, Western blot remains the best con-
®rmatory method to con®rm true HIV infection.
A considerable proportion of the indeterminates
obtained in HIV Western blots are related to the p24
(core) antigen. Indeed, many samples react particularly
strongly to this solitary component. One explanation for
this reactivity is a common core antigen for other retro-
viruses that are hopefully nonpathogenic to humans
(Blomberg et al., 1990).
Most con®rmatory laboratories utilize a con®rmatory
algorithm based on several manufacturers' HIV antibody
assays (Dow, 1999). Unfortunately, many of these assays
utilize similar recombinant proteins or synthetic peptides
that can result in two or three manufacturers' assays
producing strongly reactive results. Western blot has
often shown that these reactions are nonspeci®c and
therefore this assay should be an essential component
of blood donor HIV con®rmation.
The process of HIV lookback has uncovered a few
cases of HIV transmission from HIV antibody screened
negative blood (Crawford et al., 1987; Burin des Roziers
et al., 1998). The use of HIV p24 antigen screening or
alternatively HIV RNA detection by PCR should reduce
these few cases even further but a report from Thailand
would suggest that cases may still occur infrequently
from HIV antigen and antibody-negative blood
(Chuansumrit et al., 1996).
The realization of a new strain of HIV-1, designated
type O, resulted in a number of assays being deselected in
many European countries owing to their failure to detect
most examples of this new strain. Manufacturers were
quick to respond by ®rstly manipulating their current
assays to detect these rare examples of HIV-1 (by cross
reactivity) (Jongerius et al., 1997) and eventually producing
assays that included speci®c HIV-1 type O components
to ensure more complete detection of this rare type.
The recent advent of combination anti-HIV and HIV
p24 antigen tests have realized a further speci®city issue.
Do we accept a cumulative speci®city for both anti-HIV
and HIV p24 antigen components? Alternatively, do we
accept a speci®city similar to the currently used anti-HIV
tests as the HIV p24 antigen component is really only a
`bonus' in this test system? The introduction of such
`combi' assays raises con®rmatory test issues that are
resolvable. On testing a repeat reactive in this assay,
independent tests will need to be performed for both
components. Any reactivity with the p24 antigen test
should then be subject to a neutralization test (similar to
HBsAg) or even HIV RNA testing.
HEPATITIS C VIRUS
Hepatitis C virus (HCV) has been circulating in the
world's population for centuries prior to a patent being
®led by the Chiron Corporation. This has revolutionized
the ®eld of diagnostics with the payment of royalties and
licensing of assays now being as lucrative as marketing
the leader in the ®eld. There are now considered to be six
major HCV genotypes, although it should be realized that
most HCV assays are still based on HCV genotype 1.
In 1990, ®rst-generation anti-HCV assays were trialled
in the UK and were shown to be relatively unsuccessful
in identifying HCV infection amongst cases of post-
transfusion hepatitis non-A, non-B. In retrospect, this is
not surprising as these assays were based on one HCV
antigenic component, c100, from the nonstructural 4
(NS4) region. Similarly, the ®rst-generation recombinant
immunoblot assay (RIBA) (Chiron, Emeryville, CA,
USA, and Ortho) con®rmatory test utilized this c100
and a further 5-1-1 antigen from the same (NS4) region.
Thus, con®rmation was based on reactivity to basically
the same component but in two different assay formats.
Some individuals with isolated cross-reactivity to the
NS4 proteins may have been mislabelled as HCV posi-
tive. Eventually, a second-generation RIBA (RIBA-2)
test incorporating further speci®c HCV proteins was
marketed in early 1991 and shortly after the ELISA
based on similar proteins was also launched. Second-
generation HCV ELISAs were soon adopted by the UK
transfusion services and examination of the ®rst 100
con®rmed HCV antibody reactives amongst Scottish
blood donors showed that only 61% were reactive on
the ®rst-generation ELISA tests (Dow, 1999). A
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considerable number of these donations were shown to
be nonreactive with the c100 and 5-1-1 NS4 bands on
the second-generation recombinant immunoblot assay
(RIBA-2) yet they were shown to be PCR reactive,
indicating the presence of HCV. Sequencing of such
samples showed them to be of alternative genotype ±
either type 2 (for those only reactive to NS3 and Core
bands) or type 3 (for those only reactive to Core bands)
(Chan et al., 1991). When these nongenotype 1 speci-
mens were tested using the c100-based ®rst-generation
HCV ELISA, only 30% were reactive compared with
90% of genotype 1. This important observation led to the
development of an HCV serotyping assay based on the
use of genotype-speci®c NS4 components (Simmonds
et al., 1993).
The discovery of three further main HCV genotypes,
types 4, 5 and 6 in Northern Africa, South Africa and
South East Asia, respectively, together with the realiza-
tion that the Indian subcontinent was the source of
genotype 3, led virologists to contemplate whether they
should use assays based on their geographically preva-
lent genotypes rather than rely on the genotype 1 based
assays produced by the main diagnostic companies
(Dhaliwal et al., 1996).
The advent of third-generation HCV ELISAs that
included an NS5 protein was found to be highly acceptable
for microtitre-based systems but of dubious acceptability
for bead-based assays (Table 3). The poorer speci®city
obtained with the third-generation bead-based system
included 15´2% that exhibited solitary or indeterminate
reactivity to NS5 in the third-generation RIBA (RIBA-3).
The NS5 component in RIBA-3 was extremely large and
when smaller peptides were constructed and used as solid-
phase antigens on microtitre plates, two particular peptides
(numbers 3 and 4) appeared to be highly speci®c whilst the
remaining peptides were nonspeci®c (Table 4). Leon et al.
(1998) used a similar technique for the HCV core antigen.
The technique of epitope mapping identi®es areas of the
genome that may show good cross reactivity amongst
different genotypes or alternatively certain areas respon-
sible for speci®city problems.
A comparison of ®ve anti-HCV immunoblot assays
(Courouce et al., 1998) showed that RIBA-3 and Dec-
iscan (Sano® Pasteur, Marnes-la-Coquette, France) were
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Table 3. Comparison of Abbott HCV second- and third-generation assays
Abbott HCV 2´0 Abbott HCV 3´0
Period of testing October 1992 ± January 1994 January 1994 ± December 1995
Total donations tested 334 683 480 165
RIBA-3 Positive 86 112
RIBA-3 Indeterminate 133 565
c100p 27 55
c33c 51 94
c22p 48 82
NS5 7 344
RIBA-3 Negative 619 1695
Total nonspeci®c results 752 2260
(0´22%) (0´47%)
Table 4. HCV NS5 peptide analysis on samples with 3� or 4� NS5 reactivity on RIBA-3
Percentage reactive with peptides
No. tested 1 2 3 4 5 6 7
Anti-HCV
positive 86 68% 65% 43% 87% 87% 52% 73%
Anti-HCV
indeterminate (NS5 only) 90 34% 7% 1% 2% 5% 61% 12%
Peptides 4 and 5 were the more sensitive peptides whilst peptides 3 and 4 were the more speci®c.
more sensitive than Innolia (Innogenetics, Zwijndrecht,
Belgium), Western Blot (Murex, Dartford, UK) and
Matrix (Abbott) for NS3 antibodies. This is signi®cant
as NS3 (or core) is often the ®rst antibody to appear after
seroconversion. The Innolia test interpretation is unlike
other con®rmatory immunobots. The Innolia test is
deemed positive for any specimen exhibiting isolated
2� reactivity or indeed 1� reactivity to both core
peptides. Other con®rmatory immunoblots require the
demonstration of reactivity to at least two independent
components of the virus. Experience with the Innolia test
on 840 RIBA-3 indeterminate samples has shown that
10´2% would be Innolia positive using the manufac-
turer's criteria whereas 3´9% would remain positive if
two genome products are used (Dow, 1999). Not only are
potential false positives a problem with the Innolia test,
but also the more widely used RIBA-3 assay can also
occasionally exhibit two-band positivity with samples
negative in other HCV assays (Dow et al., 1996). Thus
the speci®city of screening assays is somewhat depen-
dent on the choice of con®rmatory procedures performed
on repeat reactives. The use of highly speci®c con®rma-
tory assays will obviously increase the proportion of false
positivity amongst screening assay reactives.
HCV antigen serological testing has now become a
realistic alternative to performing HCV NAT. Prelimin-
ary data suggest that the HCV antigen assay lags behind
HCV PCR by only 1 or 2 days in most HCV seroconver-
sion series. Furthermore, the assay has a con®rmatory
procedure (similar to HBsAg) to discriminate between
real and false positives.
POLYMERASE CHAIN REACTION
European legislation has imposed HCV NAT on our trans-
fusion services to provide HCV PCR tested frozen plasma
components (Flanagan & Snape, 1998). The use of mini-
pools was originally validated for a pilot study on parvo-
virus B19 NAT screening. The idea was subsequently
utilized for the introduction of HCV NAT screening.
Whilst pool sizes have varied from 512 to 24 according to
geographical location and sensitivity claims, through time
the pool size will eventually diminish to single donation
level. Based on negative primary screening results, all pool
constituents are cleared for issue. However, the ®nding of a
primary screening reactive result cascades an algorithm
involving repeat testing and identi®cation of the `culprit'
sample using secondary `cross' pools (Mortimer, 1997).
Whilst initial positive rates for HCV PCR mini-pool
screening can range from 0´25% to 4´5%, testing of
secondary pools and individual donations is generally
fruitless. Indeed, current ®gures suggest that only 1 in 2
million North European blood donations will contain
HCV RNA in the absence of HCV antibodies.
Speci®city in ELISA tests is judged on repeat reactiv-
ity. Donations are acceptable from samples that are
initially reactive but fail to react on repeat testing. The
initial reactivity is often associated with a technical
problem, e.g. poor washing. Similarly, initial PCR reac-
tivity that fails to repeat is usually accepted as a technical
problem. PCR involves more manipulations and steps
compared with ELISA and therefore cannot immediately
attain the speci®city levels of a robust ELISA. Increasing
use of automation will remove some of the technical
variables. Contamination in PCR can cause havoc in
routine laboratories so it is essential that procedures are
used to minimize that possibility and have planned back-
up (Kwok & Higuchi, 1989).
Con®dence in manual and automated PCR procedures
has been provided by the incorporation of internal con-
trols to validate that nucleic acid has been extracted and
ampli®ed. Internal control failures will result in repeat
testing and repeated failure would require testing of
secondary pools to provide valid test results.
CONCLUSION
Manufacturers who push their assay's sensitivity to the
extreme often end up with unmarketable assays due to
poor speci®city. Transfusion laboratories are best equipped
to ascertain the speci®city of manufacturers' assays
particularly if only new donors are tested with these
assays. Arti®cially low repeat reactive rates will be
attained should regular donors (known to be negative
to previous tests) be included in such trials.
The ideal microbiological screening test will detect all
examples of a particular agent with no false or nonspe-
ci®c results. Unfortunately, such a test has not been
developed although currently transfusion services have
lists of `approved' assays that have demonstrable excel-
lent sensitivity and acceptable speci®city. As transfusion
scientists, we have a duty to our recipients to use the most
sensitive assays whilst remembering that we also have a
duty to our donors to use those tests that minimize the
possibility of false positive results due to `noise'.
Finally, in recent years it has been noted that many
manufacturers have reintroduced the use of a `grey-
zone', particularly in protocols written in German (as
demanded by the Paul Ehrlich Institute). Reported spe-
ci®cities can therefore vary amongst users dependent on
whether `grey-zone' samples are included or not. Perhaps
the EC will eventually seek conformity amongst its
member states to avoid a national embarrassment!
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