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History of post-transfusion hepatitis

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Clinical Chemistry 43:8(B)
1487–1493 (1997)
History of posttransfusion hepatitis
Leslie H. Tobler1* and Michael P. Busch1,2
The risk of hepatitis virus transmission from transfu-
sions has declined dramatically from that of the 1940s
when posttransfusion hepatitis (PTH) was first appreci-
ated. Introduction of hepatitis B surface antigen screen-
ing and conversion to volunteer donors for whole-blood
donations in the late 1960s and early 1970s led to
substantial reduction in PTH cases. However, up to 10%
of the recipients continued to develop PTH, most cases
of which were attributed to an unknown non-A, non-B
viral agent. Implementation of surrogate marker testing
(i.e., alanine aminotransferase and anti-hepatitis B virus
core antigen) for residual non-A, non-B hepatitis in the
late 1980s reduced the per unit risk of PTH from 1 in 200
to about 1 in 400. Hepatitis C virus was discovered in
1989 and quickly was established as the causative agent
of >90% of non-A, non-B PTH. Introduction of progres-
sively improved antibody assays in the early 1990s
reduced the risk of PTH due to hepatitis C virus to about
1 in 100 000. Although additional hepatitis viruses exist
(e.g., hepatitis G virus), these appear to be minor con-
tributors to clinical PTH, which has been virtually
eradicated.
early history of posttransfusion hepatitis (pth3)
During World War II and the immediate postwar period
the demand for blood and blood components in the US
increased substantially. This resulted in the establishment
and growth of blood banks, transfusion services, and
other blood and laboratory support services. The technol-
ogy for collection, processing, and storage of whole blood
and blood components materialized rapidly. By 1971,
.5400 organizations were involved in the field of trans-
fusion medicine with .12 million annual whole-blood
1
Irwin Memorial Blood Centers, 270 Masonic Ave., San Francisco, CA
94118, and 2 Department of Laboratory Medicine, University of California, San
Francisco, CA 94143.
* Author for correspondence. Fax 415-775-3859; e-mail ltobler@ccnet.com.
3
Nonstandard abbreviations: PTH, posttransfusion hepatitis; HAV, hepa-
titis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HGV, hepatitis G
virus; GBV-C, GB virus C; HBsAg, hepatitis B virus surface antigen; EIA,
enzyme-linked immunoassay; NANBH, non-A, non-B hepatitis; ALT, alanine
aminotransferase; PCR, polymerase chain reaction.
Received March 3, 1997; revised April 18, 1997; accepted April 29, 1997.
1487
Beckman Conference
donations and a nearly equal number of plasmapheresis
donations (Table 1) [1].
PTH was first reported in the US by Beeson in 1943 [2].
Seven cases of PTH occurring 1– 4 months after transfu-
sion of blood or plasma were reported. The author com-
mented that the expanding number of blood and plasma
transfusions could lead to the occurrence of a consider-
able number of PTH cases. In 1964 Grady and Chalmers
[3] reported the results of a retrospective study of PTH in
nine Boston teaching hospitals, 1952–1962. In one of the
hospitals 29% of the blood transfused was from commer-
cial sources, while in the other eight hospitals blood only
from volunteer blood donors was transfused. The inci-
dence of clinically overt (i.e., symptomatic or icteric) PTH
in recipients of blood products from volunteer blood
donors was 0.6 cases/1000 units compared with 2.8 cas-
es/1000 units in recipients of blood products from a
mixture of volunteer and commercial blood donors.
In 1970 scientists at the NIH reported the results of a
prospective study to determine the incidence of icteric
and anicteric hepatitis in patients undergoing open-heart
surgery [4]. During surgery the patients were given blood
from either commercial or volunteer blood donors. Icteric
and anicteric hepatitis developed in 51% of the recipients
of commercial blood, whereas no hepatitis occurred in
patients who received blood from volunteer donors.
These authors estimated the hepatitis carrier rate for
commercial blood donors to be 6.3% and for volunteer
donors to be ,0.6%. By 1971, the association between a
paid blood donor and an increased risk of PTH was
accepted by most, but not all, experts in the field. In early
1972, two states (California and Illinois) considered legis-
lation to specifically eliminate payment for blood. The
first law effective in eliminating paid blood donations was
passed on October 1, 1972 (i.e., The Blood Labeling Act of
Illinois). By the end of 1975, the Food and Drug Admin-
istration (FDA) mandated an all-voluntary blood donor
system in which blood donors could not receive monetary
payment for donations (Fig. 1) [1].
role of hepatitis b virus (hbv) in pth
A viral etiology for PTH was long suspected. In 1965,
Blumberg et al. [5] first described the Australia antigen
and stated that this antigen could be identified in the sera
1488 Tobler and Busch: History of PTH

Table 1. Events in understanding and minimizing PTH.

Date Event Reference
HBV and paid vs. nonpaid donors
1943 First US report of PTH 2
1964 Retrospective study: paid vs nonpaid donors on rate of PTH 3
1965 Discovery of HBV antigen (Australia antigen) 5
1970 Prospective study evaluating the contribution of paid vs nonpaid donors to the rate of PTH 4
1970 HBsAg as marker for PTH 6, 9
1972 Prospective study: effectiveness of exclusion of commercial and HBsAg-positive blood donors on 10
the incidence of PTH
1972 FDA mandates screening of all blood donations for HBsAg 11

NANBH and surrogate markers

1973 HAV discovered 12
HAV not causative agent of PTH; NANBH defined 14, 15
1975 FDA mandates an all-voluntary blood donor system 1
1975 FDA mandates use of a third generation HBsAg assay 11
1981 Transfusion-Transmitted Virus Study results suggest use of ALT as surrogate marker for PTH 16
1981 Results of NIH study confirm association between increased ALT concentration in donor blood 17
and the development of recipient PT-NANBH
1986–87 Implementation of surrogate marker testing 18, 19

HCV: primary cause of NANBH

1988 Discovery of HCV 22, 23
1989 Classification of HCV as a single-stranded positive-sense RNA virus belonging to the family 23
Flaviviridae
1990 Licensing and implementation of first-generation HCV screening
1992 Licensing and implementation of second-generation HCV screening
1993 Licensing of HCV supplemental testing
1995 Studies show declining value of surrogate markers 30, 37

Recent events post-HCV screening

1995 NIH Consensus Statement recommends discontinuation of ALT testing, continuation of anti-HBc 20
testing
1995 Discovery of GBV-C/HGV 38–41
1995 GBV-C/HGV transfusion-transmission established 40, 42, 43, 46
1996 Licensing and partial implementation of third-generation HCV screening

of many hemophiliacs who had received multiple trans-
fusions. Subsequently retrospective studies indicated that
the presence of the Australia antigen in donor blood
seemed to be clearly associated with the occurrence of
PTH [6]. During an NIH Conference in 1970, additional
evidence was presented that linked the presence of the
Australia antigen to the occurrence of PTH. Furthermore,
during this conference it was stated that the Australia
antigen was part of an infectious agent, presumably a
hepatitis virus [7]. Nevertheless, the screening of blood
donors was not recommended. In a position paper written
by Alter et al. [8], the authors recommended testing and
deferral of blood donors based on the presence of the
Australia antigen. (The Australia antigen is now referred
to as hepatitis B surface antigen, HBsAg; ultimately the
HBV was classified as a DNA virus in the Hepadnaviridae.)
These recommendations were not implemented because
of lack of consensus and concern over the general avail-
ability and variable detection limits of early-generation
HbsAg assays.
In 1970, Gocke et al. [9], using retrospective studies,
estimated that the exclusion of HBsAg-positive blood
donors through either first- or second-generation assays
(e.g., agar gel diffusion or counterelectrophoresis) would
decrease the rate of PTH by ;25%. The results of a
prospective study on the effectiveness of exclusion of
commercial and HBsAg-positive blood donors on the
incidence of PTH was reported in 1972 [10]. The exclusion
of HBsAg-positive blood resulted in a 25% reduction in
the PTH rate, as predicted, while the elimination of
commercial donors resulted in a 70% reduction in the
PTH rate. With the simultaneous exclusion of commercial
and HBsAg-positive donors, the PTH rate was reduced to
7.1% of the prior rate.
On the basis of these studies, screening of blood
donations for HBsAg began in 1971 and became a US
federal regulation in July 1972. The initial methods used
to detect HBsAg were relatively insensitive (i.e., immu-
nodiffusion and counterelectrophoresis), and transfusion-
associated HBV cases continued to occur. In 1975, federal
regulations were implemented requiring the screening of
all donor blood for HBsAg by one of the third-generation
 Clinical Chemistry 43, No. 8(B), 1997
tests [i.e., radioimmunoassay or enzyme-linked immuno-
assay (EIA)], as well as the labeling of blood components
with respect to the volunteer or paid status of the donor.
Since that time, virtually 100% of all blood used in
single-component transfusions has been collected from
volunteer donors. The transition to volunteer whole-
blood donors and routine third-generation HbsAg testing
of all blood donations resulted in a marked decrease in
HBV-PTH, although occasional cases continue to occur
[11]. In contrast to whole-blood donors, donors of plasma
for further manufacture into plasma derivatives continue
to be paid. The Cohen fractionation procedure used to
prepare plasma derivatives dramatically reduces viral
infectivity, and over the last decade, additional viral
inactivation procedures (e.g., heating and detergent treat-
ment) performed on these products render contemporary
plasma derivatives virtually risk-free.
non-a, non-b pth and surrogate markers
In 1973, the causative agent of hepatitis A was detected by
immune electron microscopy on stools from patients with
acute food-borne hepatitis [12]. The hepatitis A virus
(HAV) was later classified as a picornavirus closely re-
lated to the genus Enterovirus [13]. HAV is associated with
acute resolving hepatitis without a chronic carrier state.
Retrospective studies involving multiply transfused
thalassemia patients indicated that these patients were at
no greater risk of HAV infection than nontransfused
children [14]. Similarly, analysis of donor and recipient
samples from cases of PTH showed no evidence of
involvement by HAV [15].
After implementation of specific screening tests for
HBV and exclusion of HAV as a cause of PTH, it became
clear that a substantial proportion of PTH cases continued
to occur that were not caused by infections with HBV or
1489
Fig. 1. The decline in posttransfusion HCV infection.
Rates of PTH are expressed as percent of recipient devel-
oping PTH. The current risk of PTH because of HBV or HCV
is estimated at less than 1 in 10 000/unit transfused (see
text) (adapted from H. J. Alter).
other known viral agents. Termed non-A, non-B hepatitis
(NANBH), this entity represented 90% of residual PTH
cases in the US. In the late 1970s and early 1980s, rates of
NANBH in multiply transfused patients were reported to
be as high as 10%.
The results of the multicenter prospective Transfusion-
Transmitted Viruses Study were published in 1981 [16].
Two major goals of this study were to define the incidence
of PTH and to evaluate the factors influencing its occur-
rence. The data reported indicated a substantial associa-
tion between recipients with NANBH and donor alanine
aminotransferase (ALT) concentrations. Another indepen-
dent study conducted at NIH confirmed an important
association with an increased ALT concentration in donor
blood and the development of NANBH in recipients of
that blood [17]. Furthermore, in 1984 Stevens et al. [18],
representing the Transfusion-Transmitted Viruses Study,
reported an important association between the occurrence
of NANBH in recipients of blood that tested positive for
antibody to the core protein of HBV (anti-HBc). The
research group at NIH confirmed this observation [19].
This study showed that anti-HBc testing of donors, in
concert with ALT testing, might eliminate 30 –50% of
recipient NANBH. Primarily on the basis of these studies,
in 1986 – 87 blood collection agencies began screening
donated blood for surrogate markers of NANBH (anti-
HBc and ALT) [20]. The rate of PTH among recipients
dropped subsequently to as low as 2–3% [21].
hcv as major etiological agent of nanbh
Extensive research was conducted in the 1970s and 1980s
to identify the etiological agent(s) of NANBH. More than
25 preliminary reports of associated agents were deter-
mined to be false. Then, in 1988, the hepatitis C virus
(HCV) was identified with molecular biology techniques
1490 Tobler and Busch: History of PTH
by M. Houghton and associates at Chiron, in collaboration
with D. W. Bradley of the Hepatitis Branch of the Centers
for Disease Control [22]. The process of virus discovery
involved construction of a cDNA expression library in the
bacteriophage lgt11 by using high-titer plasma from a
chimpanzee inoculated with PTH plasma. The library was
then screened for rare clones expressing viral antigen with
serum from a chronic NANBH patient as a presumed
source of viral antibodies. Screening of this library led to
the identification of the positive cDNA clone 5-1-1 (Fig. 2)
[23].
HCV is a single-stranded positive-sense RNA virus
with a genome of ;9500 bases coding for ;3000 amino
acids. This small RNA lipid-envelope [24] virus has been
classified in the family Flaviviridae. The entire viral ge-
nome was sequenced within 1 year, and antigens were
expressed for development of antibody detection assays.
Early studies established that HCV was the etiological
agent of at least 80 –90% of residual NANBH [21].
hcv screening assays and residual risk of
hcv-pth
Blood collection agencies in the US implemented donor
screening immediately after licensure of the first-genera-
tion anti-HCV enzyme immunoassay (HCV 1.0 EIA) in
1990. This EIA detected antibodies to an antigenic protein
(c100-3) of HCV (Fig. 2). Even though this assay facilitated
the screening of blood donors for anti-HCV antibodies, it
did not detect all infectious blood donations [25] and had
a protracted window of infectivity ranging from 12 weeks
to .26 weeks postinfection [26]. Nevertheless, a prospec-
tive study of patients receiving transfusions before and
after mid-1990 found that the risk of transfusion-associ-
ated HCV infection per unit dropped from 0.36% (1 in
274) before anti-HCV screening to 0.07% (1 in 3300) for
donations screened with both surrogate markers and first-
generation anti-HCV tests [27, 28]. It has since been esti-
Fig. 2. Proposed genome structure of HCV.
The major open reading frames are shown in the upper box, with putative
properties of each generation shown above (deduced from parallels with other
flaviviruses). Major antigens used in antibody detection systems are shown
below (from H. J. Alter [21]).
mated that this test prevented transmission of HCV to
40 000 patients per year in the US [21].
A second-generation anti-HCV EIA (HCV 2.0 EIA) was
licensed and rapidly implemented in 1992. This test
incorporated two additional proteins, one structural
(c22-3) and one nonstructural (c33c) (Fig. 2). This test was
substantially more sensitive than HCV 1.0 EIA in detect-
ing acute and chronic HCV infections. Antibodies to these
proteins generally appear much earlier than those to
c100-3, so the seroconversion window period could be
shortened by 10 –20 days. In addition, parallel studies
comparing first- and second-generation tests showed that
HCV 2.0 EIA detected additional HCV chronically in-
fected donors at a rate of 1 in 1000 [25]. This increased
yield translated into prevention of an additional 13 000
HCV transmissions per year by transfusion [21]. Conflict-
ing estimates existed regarding the residual risk of HCV
after implementation of the second-generation assay. Pro-
jections based on incidence-window period models sug-
gested that the risk of HCV PTH had dropped to as low as
1 in 100 000 per unit [29], whereas extrapolation from
historical PTH studies suggested a risk of up to 1 in 5000
[30].
In 1996, a third-generation screening test (HCV 3.0
EIA) was licensed that detected antibodies to an even
greater number of HCV-encoded epitopes. This test dif-
fers in antigen content from the earlier-generation screen-
ing assays with deletion of the c-100 recombinant protein
and addition of the NS-5 recombinant protein (Fig. 2).
This allows detection of antibodies to a greater number of
HCV-encoded epitopes. HCV 3.0 EIA has consequently
narrowed the seroconversion window by about 10 days
relative to the HCV 2.0 EIA [31, 32]. This window period
reduction is projected to detect 1–2 additional serocon-
verting donors per million units screened. Although the
impact of the HCV 3.0 EIA on residual HCV risk has not
been prospectively quantified, the ongoing NIH prospec-
tive study of PTH has failed to detect any HCV transmis-
sions among .650 patients transfused with .2500 units
of blood screened by second-generation HCV EIA since
late 1992 (H. Alter, personal communication).
hcv confirmatory assays
Given the low prevalence of viral infection in preselected
volunteer donors, it is important that confirmatory assays
are developed and used to discriminate between true and
falsely positive EIA-reactive donors. The Chiron RIBATM
HCV 2.0 strip immunoassay (RIBA 2.0) was licensed on
June 27, 1993. The RIBA 2.0 is an immunoblot assay in
which four recombinant HCV-encoded antigens fused to
human superoxide dismutase are immobilized on nitro-
cellulose strips. A negative, indeterminate, or positive
interpretation is based on the reaction pattern present on
the strip. About 89% of RIBA 2.0-positive specimens are
HCV RNA-positive by PCR tests, while ;19% of RIBA
2.0-indeterminate specimens are HCV RNA-positive by
PCR tests [25].
 Clinical Chemistry 43, No. 8(B), 1997
The HCV 3.0 EIA companion supplemental test, Chi-
ron RIBA HCV 3.0 SIA (RIBA 3.0), is now pending
licensure from the FDA. This assay uses a mixture of
peptides, rather than recombinant antigens, for the 5-1-1/
c-100 and c22-3 regions of the HCV genome. The use of
peptides, when appropriate, eliminates those amino acid
sequences that are a source of nonspecific cross-reactivity
with other antigens [33, 34]. On the basis of data from
Europe [35], these changes have substantially improved
this assay’s lowest detection limits and specificity.
The prevalence of anti-HCV antibody in US blood
donors, as determined with HCV 2.0 and 3.0 EIA con-
firmed by RIBA 2.0 and 3.0, respectively, ranges from
0.2% to 0.4% [21]. At least 70% of seropositive donors are
viremic [25]. Similar rates have been observed in Europe,
and higher rates in Japan and other Asian countries.
Importantly, these rates are in highly selected blood
donors [26]. The prevalence of HCV infection in the
general population of the US is currently estimated to be
between 1% and 2% [36].
retention of surrogate tests
With virtual elimination of NANBH by HCV screening,
the issue of whether surrogate tests should be retained
has surfaced. Although the direct cost of ALT testing is
low, the indirect cost is very high because .1% of units
test positive and are discarded. Anti-HBc screening has a
higher direct cost and results in ;0.5% unit loss. Further-
more, the donors of these units are either temporarily or
permanently deferred. The preponderance of available
data indicates that, in the presence of anti-HCV testing,
retention of ALT testing prevents few if any residual PTH
cases [30, 37]. Therefore, a 1995 NIH Consensus Statement
recommended that ALT testing of volunteer blood donors
be discontinued. On the other hand, the consensus con-
ference recommended that anti-HBc testing be retained, at
least temporarily, for the following reasons: possible
prevention of some PTH HBV cases and possible preven-
tion of some cases of transfusion-transmitted HIV from
donors who test negative for anti-HIV during the window
phase of infection [20]. Recent studies suggest that these
applications of anti-HBc have a very low predictive value
and very poor cost effectiveness [37]. Therefore, discon-
tinuation of anti-HBc is being actively reconsidered.
hepatitis g virus (hgv) and other putative pth
agents
Evidence indicates that additional viruses that explain
rare residual PTH cases may exist. Indeed, before HCV
was identified there was strong suspicion that more than
one blood-borne viral agent was causing NANBH [26].
However, it has now become clear that reinfection by
different HCV strains is not uncommon, which explains
many of the cases of recurrent PTH that led to speculation
regarding multiple agents. In addition, the rate of non-
ABC hepatitis in recipients is now comparable with that
in nontransfusion controls (i.e., ,0.8% in both US and
1491
Canadian studies, similar to background values) [20].
Nevertheless, the search for additional agents has contin-
ued.
In 1995, researchers at Abbott Laboratories reported
the isolation of three viral agents [GB viruses (GBV) A, B,
and C] from tamarins inoculated with serum from a
surgeon “GB” who had contracted acute icteric hepatitis
20 years earlier [38, 39]. Only one of the isolates (GBV-C)
proved to be a human viral hepatitis candidate; the other
two were tamarin agents incidentally infecting the ani-
mals at the time of the experiments. A second indepen-
dent group of researchers from GeneLabs performed
molecular cloning with plasma from a patient designated
PNF2161, who was originally identified by the CDC as
having NANBH. The virus cloned by these researchers
was designated HGV [40]. Given the amino acid sequence
matching of 95% between GBV-C and HGV, these viruses
are therefore considered to be different strains of the same
virus, tentatively termed GBV-C/HGV [41]. GBV-C/HGV
is a positive-stranded RNA virus classified in the Flavi-
viridae, the same family as HCV. Only 32% amino acid
homology exists between GBV-C/HGV and HCV. There-
fore, although GBV-C/HGV is distantly related to HCV,
this agent is not a different serotype of HCV.
GBV-C/HGV RNA has been detected at a high prev-
alence (2–25%) among various high-risk groups, e.g.,
intravenous drug users, hemophiliacs, and hemodialysis
patients. About 1–2% of blood donors have also been
found to be RNA-positive [42]. A recent study, using PCR
technology, clarified the relationship of GBV-C/HGV and
the occurrence of hepatitis [43]. PCR was performed on
selected patients from a surveillance study of acute viral
hepatitis in four US counties. The patients included in this
study were: 45 patients with a diagnosis of non-A-E
hepatitis, 116 patients with hepatitis C, 100 patients with
hepatitis A, and 100 patients with hepatitis B. The results
of this study indicated that HGV was implicated as a
potential etiological agent in only 0.3% of cases. These
HGV cases had only mild liver enzyme increases. Further-
more, coinfection by HGV did not affect the clinical
course in patients with hepatitis A, B, or C.
Initial studies involving the prevalence of GBV-C/
HGV infection were hampered by the absence of an
antibody detection system; therefore, the diagnosis of
GBV-C/HGV infection depended on the use of PCR to
detect GBV-C/HGV RNA. Recently an antibody test has
been developed that has demonstrated seroconversion
associated with viral RNA clearance in about two-thirds
of infected persons [44, 45].
GBV-C/HGV is unequivocally a highly prevalent
transfusion-transmissible agent [46]. However, a causal
relationship between HGV infection and hepatitis or other
diseases has not been established [41, 43]. GBV-C/HGV
appears to explain only a small fraction of either transfu-
sion- or community-acquired hepatitis cases, and these
cases are very mild [43, 46]. Thus at present, it is unclear
1492 Tobler and Busch: History of PTH
whether screening of blood donors for GBV-C/HGV will
be recommended.
The overall risk of PTH has declined markedly over the
past 2 decades with virtual elimination of transmission of
HBV and HCV infections. The advances in prevention of
PTH have been not only extraordinary with regard to
recipient safety, but also highly cost-effective. Indeed, the
cost of screening the blood donor population for HCV is
more than offset by the reduced healthcare costs achieved
by prevention of PTH cases [30]. Unfortunately, many
infections did occur before the availability of screening
assays. The tragedy of the transfusion AIDS epidemic also
echos loudly. Thus, the current priority of infectious
disease blood banks is further enhancement of blood
safety, through elimination of rare residual cases of virally
transmitted hepatitis by implementation of nucleic acid
screening assays [47] and avoidance of complacency
through proactive surveillance of the blood supply for
new or emerging infectious agents.
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