‘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

q 2000 Blackwell Science Ltd, Transfusion Medicine, 10, 97±106

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

`Noise' in microbiological screening tests 99


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

100 B. C. Dow


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



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

102 B. C. Dow


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



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!

REFERENCES

Barbara, J.A.J. (1983) Microbiology in Blood Transfusion. John

Wright.

104 B. C. Dow


Barbara, J.A.J., Harrison, P.J., Howell, D.R., Cleghorn, T.E.,

Dane, D.S., Briggs, M. & Cameron, C.H. (1979) A sensitive

single reverse passive haemagglutination test for detecting

both HBsAg and anti-HBs. Journal of Clinical Pathology,

32, 1180±1183.

Barbara, J.A.J., Howell, D.R. & Mochnatz, P.Z. (1983) HBsAg

detection rate in the blood donor population: RIA and RPHA

compared by parallel screening. In: Proceedings of Interna-

tional Hepatitis Workshop (eds Hopkins, R. & Field, S.), pp.

123±125. Nuclear Enterprises, Edinburgh.

Barclay, G.R., Hopcroft, W. & McClelland, D.B.L. (1986)

What is an `equivocal' negative HTLV-III antibody test in

blood donors? Lancet, 372, 912±913.

Barr, A., Dow, B.C., Arnott, J., Crawford, R.J. & Mitchell, R.

(1986) Anti-HTLV III screening speci®city and sensitivity.

Lancet, 372, 1032.

Barr, A., Dow, B.C. & Macvarish, I. (1979) HBsAg detection ±

results of comparative large scale testing of blood donations.

Medical Laboratory Science, 36, 109±114.

Barr, A., Dow, B.C., Watson, W.C. & Hunter, E. (1975)

Detection and quantitation of tetanus antitoxin in blood

donations. Journal of Clinical Pathology, 28, 969±971.

Barr, A., Muir, W., Dow, B.C., Arnott, J. & Macvarish, I.P.

(1987) Detection of anti-HTLV III: modifcation of a com-

mercial enzyme immunoassay. Medical Laboratory Science,

44, 97±99.

Blomberg, J., Vincic, E., Jonsson, C., Medstrand, P. &

Pipkorn, R. (1990) Identi®cation of regions of HIV-1 p24

reactive with sera which give `indeterminate' results in

electrophoretic immunoblots with the help of long syn-

thetic peptides. Aids Research and Human Retroviruses, 6,

1363±1372.

Burin des Roziers, N., Coste, J., Courouce, A.M., Bibollet-

Ruche, F., Guillard, A. & Nasr, O. (1998) Detection of HIV-1

RNA in two consecutive blood donations screened negative

for HIV antibodies. Vox Sanguinis, 75 298±302.

Busch, M.P. (1997) To thy (reactive) donors be true! Transfu-

sion, 37 117±120.

Challis, P., Dow, B.C., Mitchell, R., Barr, A. & Barbara, J.A.J.

(1988) Sequential ELISA for anti-HIV should not replace the

standard competitive method. Vox Sanguinis, 55 244±245.

Chan, S.W., Simmonds, P., McOmish, F., Yap, P.L., Mitchell, R.,

Dow, B. & Follett, E. (1991) Serological responses to

infection with three different types of hepatitis C virus.

Lancet, 338 1391.

Chuansumrit, A., Varavithya, W., Isarangkura, P., Sirinavin, S.,

Chiewsilp, P., Tanprasert, S. & Hathirat, P. (1996) Transfu-

sion-transmitted AIDS with blood negative for anti-HIV and

HIV-antigen. Vox Sanguinis, 71 64±65.

Courouce, A.M., Muller, J.Y. & Richard, D. (1986) False-

positive western blot reaction to human immunode®ciency

virus in blood donors. Lancet, 373 921±922.

Courouce, A.M., Noel, L., Barin, F., Elghouzzi, M.H., Lusel,

F., North, M.L. & Smilovici, W. (1998) A comparative

evaluation of the sensitivity of ®ve anti-hepatitis C virus

immunoblot assays. Vox Sanguinis, 74 217±224.

Crawford, R.J., Mitchell, R., Burnett, A.K. & Follett, E.A.C.

(1987) Who may give blood? British Medical Journal, 294

572.

van Deursen, F.J., Hino, K., Wyatt, D., Molyneaux, P., Yates,

P., Wallace, L.A., Dow, B.C. & Carman, W.F. (1998) Use of

PCR in resolving diagnostic dif®culties potentially caused by

genetic variation of hepatitis B virus. Journal of Clinical

Pathology, 51 149±153.

Dhaliwal, S.K., Prescott, L.E., Dow, B.C., Davidson, F.,

Brown, H., Yap, P.L., Follett, E.A.C. & Simmonds, P.

(1996) In¯uence of viraemia and genotype upon serological

reactivity in screening assays for antibody to hepatitis C

virus. Journal of Medical Virology, 48 184±190.

Dow, B.C. (1999) Microbiology con®rmatory tests for blood

donors. Blood Reviews, 13 91±104.

Dow, B.C., Buchanan, I., Munro, H., Follett, E.A.C., Davidson, F.,

Prescott, L.E., Yap, P.L. & Simmonds, P. (1996) Relevance

of RIBA-3 supplementary test to HCV PCR positivity and

genotoypes for HCV con®rmation of blood donors. Journal

of Medical Virology, 49 132±136.

Dow, B.C., Macvarish, I., Barr, A., Crawford, R.J. & Mitchell, R.

(1980) Signi®cance of tests for HBeAg and anti-HBe in

HBsAg positive blood donors. Journal of Clinical Pathology,

33 1106±1109.

Dow, B.C., Munro, H., Frame, D. & Yates, P. (1998) Several

assays show hepatitis B positivity soon after vaccination.

British Medical Journal, 316 311.

Flanagan, P. & Snape, T. (1998) Nucleic acid technology

(NAT) testing and the transfusion service: a rational for the

implementation of mini-pool testing. Transfusion Medicine,

8 9±13.

Genesca, J., Shih, J.W.K., Jett, B.W., Hewlett, I.K., Epstein,

J.S. & Alter, H.J. (1989) What do Western blot indeterminate

patterns for human immunode®ciency virus mean in EIA ±

negative blood donors? Lancet, 334 1023±1025.

Jongerius, J.C.M., van der Poel, C.L., van Loon, A.M., van den

Akker, R., Schaasberg, W. & van Leeuwen, E.F. (1997)

Human immunode®ciency virus (HIV) antibodies detected

by new assays that are enhanced for HIV-1 subtype O.

Transfusion, 37 841±844.

Jongerius, J.M., Wester, M., Cuyper, H.T.M., van Oostendorp,

W.R., Lelie, P.N., van der Poel, C.L. & van Leeuwen, E.F.

(1998) New hepatitis B virus mutant form in a blood donor

that is undetectable in several hepatitis B surface antigen

screening assays. Transfusion, 38 56±59.

Kuhnl, P., Seidl, S. & Holzberger, G. (1985) HLA DR4

antibodies cause positive HTLV-III antibody ELISA results.

Lancet, 370 1222±1223.

Kwok, S. & Higuchi, R. (1989) Avoiding false positives with

PCR. Nature, 339 237±238.

Leon, P., Lopez, J.A., Elola, C., Lee, S.R., Calmann, M. &

Echevarria, J.M. (1998) Use of overlapping synthetic pep-

tides to characterize samples from blood donors with inde-

terminate results to hepatitis C virus core antigen. Vox

Sanguinis, 75 32±36.

McCullough, J. (1993) The nations changing blood supply



system. Journal of the American Medical Association, 269

2239±2245.

Mortimer, J. (1997) Intersecting pools and their potential

application in testing donated blood for viral genomes. Vox

Sanguinis, 73 93±96.

Mortimer, P.P., Parry, J.V. & Mortimer, J.Y. (1985) Which anti

HTLV III/LAV assays for screening and con®rmatory test-

ing? Lancet, 371 873±877.

Simmonds, P., Rose, K.A., Graham, S., Chan, S.W., McOmish,

F., Dow, B.C., Follett, E.A.C., Yap, P.L. & Marsden, H.

(1993) Mapping of serotype-speci®c, immunodominant

epitopes on the NS-4 region of hepatitis C virus (HCV):

use of type-speci®c peptides to serologically differentiate

infections with HCV types 1, 2 and 3. Journal of Clinical

Microbiology, 31 1493±1503.

Wallace, L.A. & Carman, W.F. (1997) Surface gene variation

of HBV; scienti®c and medical relevance. Viral Hepatitis

Reviews, 3 5±16.

Williamson, L.E., Lowe, S., Love, E.M., Cohen, H., Soldan, K.,

McClelland, D.B.L., Skacel, P. & Barbara, J.A.J. (1999) Serious

hazard of transfusion (SHOT) initiative: analysis of the ®rst two

annual reports. British Medical Journal, 319 16±18.

106 B. C. Dow


‘Noise’ in microbiological screening assays

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