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MAJOR ARTICLE
Higher Human T Lymphotropic Virus (HTLV)
Provirus Load Is Associated with HTLV-I
versus HTLV-II, with HTLV-II Subtype A
versus B, and with Male Sex and a History
of Blood Transfusion
Edward L. Murphy,1,2 Tzong-Hae Lee,2 Daniel Chafets,2 Catharie C. Nass,3,a Baoguang Wang,4 Katharine Loughlin,4
and Donna Smith,4 for the HTLV Outcomes Study Investigatorsb
1
Departments of Laboratory Medicine and Epidemiology/Biostatistics, University of California, San Francisco, and 2Blood Systems Research
Institute, San Francisco; 3American Red Cross Blood Services, Chesapeake and Potomac Region, Baltimore, and 4Westat, Rockville, Maryland
Background. High human T lymphotropic virus (HTLV)–I provirus load (VL) has been associated with an
increased risk of HTLV-associated myelopathy, but little is known about variation in HTLV-I or -II VLs by demographic characteristics and risk behaviors.
Methods. We measured HTLV-I and HTLV-II VLs in a large cohort of 127 HTLV-I–seropositive and 328
HTLV-II–seropositive former blood donors, by use of real-time polymerase chain reaction using tax primers.
Multivariable linear regression was used to control for confounding by relevant covariates.
Results. The mean VLs were 3.28 log10 copies/106 peripheral blood mononuclear cells (PBMCs) (range, 0.5–
5.3 log10 copies/106 PBMCs) for HTLV-I and 2.60 log10 copies/106 PBMCs (range, 0.05–5.95 log10 copies/106 PBMCs)
for HTLV-II (P ! .0001). HTLV-II VLs were higher in those subjects with subtype A infection (mean, 2.82 log10
copies/106 PBMCs) than in those with subtype B infection (mean, 2.29 log10 copies/106 PBMCs) (P p .005). Higher
HTLV-I VL was associated with previous receipt of a blood transfusion (P p .04 ), and lower HTLV-II VL was
associated with female sex (P p .007 ). These associations persisted in virus-specific multivariate linear regression
models controlling for potential confounding variables.
Conclusions. VL was significantly higher in HTLV-I than in HTLV-II infection and was higher in HTLV-II
subtype A than in HTLV-II subtype B infection. Chronic HTLV VLs may be related to the infectious dose acquired
at the time of infection, with higher VLs following acquisition by blood transfusion and lower VLs following
sexual acquisition.
Human T lymphotropic virus (HTLV) types I and II
are human type C retroviruses derived most likely from
simian-to-human transmission thousands of years ago
[1, 2]. The 2 HTLV types share ∼60% nucleotide ho-
Received 24 December 2003; accepted 18 February 2004; electronically published
6 July 2004.
Financial support: National Heart, Lung, and Blood Institute Retrovirus Epidemiology Donor Study (research contracts N01-HB-97077 [superseded by N01-HB47114], N01-HB-97078, N01-HB-97079, N01-HB-97080, N01-HB-97081, N01-HB97082, and R01-HL-62235 [through the HTLV Outcomes Study]).
a
Present affiliation: C.C.N. is retired from the American Red Cross Blood Services,
Chesapeake and Potomac Region.
b
HOST investigators are listed after the text.
Reprints or correspondence: Dr. Edward L. Murphy, c/o Blood Centers of the Pacific,
UCSF, 270 Masonic Ave., San Francisco, CA 94118 ([email protected]).
The Journal of Infectious Diseases 2004; 190:504–10
2004 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2004/19003-0012$15.00
504 • JID 2004:190 (1 August) • Murphy et al.
mology but differ in their epidemiology and disease
associations. HTLV-I is prevalent in Africa, the Caribbean, Brazil, and southern Japan and causes adult T
cell leukemia (ATL) and HTLV-associated myelopathy
(HAM) [3]. HTLV-II is prevalent among Amerindians,
African pygmies, and injection drug users (IDUs) in the
Americas and Europe and has been linked to HAM, as
well as to a higher occurrence of pneumonia and bronchitis [4, 5]. HTLV-II subtype A is more prevalent among
North American and European IDUs and their sex partners, whereas subtype B is more prevalent among Native
Americans [4].
HTLV-I and -II viral RNA is low to undetectable in
human serum, and viral propagation is thought to occur via the clonal expansion of lymphocytes with integrated proviral DNA [6] or the transfer of viral RNA
via direct contact between infected and uninfected cells [7].
How the level of proviral DNA in lymphocytes is regulated is
unknown but may involve either the activity of the viral transactivator protein Tax, which differs between HTLV-I and some
HTLV-II subtypes [8], or the degree of lymphocyte turnover
in the infected host [6]. High levels of HTLV-I proviral DNA,
HTLV-I Tax mRNA, and anti-HTLV antibody have been associated with the occurrence of HAM [9, 10]. However, the
pathogenic mechanism is unresolved and may be due to either
direct viral neurotoxicity [11], an autoimmune response directed at viral proteins homologous to neuronal proteins [12],
or “bystander” immunologic damage to neural tissue [13]. Data
are less clear with regard to the association between HTLV-I
provirus load (VL) and ATL, with one study showing significantly higher prediagnostic VLs in patients with ATL than in
asymptomatic carriers [14] and another showing an association
between high HTLV-I VLs and the presence of abnormal lymphocytes thought to be indicative of preleukemia, although this
was not proven prospectively [15].
HTLV-II VL has received comparatively less study than HTLVI VL. One study found no difference in HTLV-II VLs between
IDUs and endemic African Pygmies and no correlation between
HTLV-II VLs and CD8+ lymphocyte counts or stage of AIDS,
in the IDUs who were coinfected with HIV [16]. Other studies
have found high HTLV-II VLs in patients with HIV/HTLV-II
coinfection [17, 18] and, notably, in coinfected patients with
sensory neuropathy [19]. HTLV-II VLs also increased after the
initiation of antiretroviral therapy for HIV in coinfected patients [20, 21].
HTLV-II VL has not been correlated with age or sex, and
neither HTLV-I VL nor HTLV-II VL has been studied in relation
to the route of infection. We therefore measured HTLV-I and
HTLV-II VLs in a well-characterized cohort of former blood
donors and correlated these levels with data on demographic
characteristics, risk factors for infection, and HTLV-II subtype.
SUBJECTS AND METHODS
Study design and population. The present study was a crosssectional analysis of blood samples obtained at the baseline visit
of the HTLV Outcomes Study cohort, formerly known as the
Retrovirus Epidemiology Donor Study HTLV cohort. The study
protocol was approved by the Committee on Human Research
at the University of California, San Francisco, and by institutional review boards at other participating institutions. Enrollment criteria and methods have been reported in detail
elsewhere [22–24]. In brief, blood donors found to be HTLV
seropositive during predonation screening at 5 centers across
the United States in 1988–1992 had confirmation of seropositivity performed by use of Western blot, followed by HTLV
typing by use of type-specific antibody or polymerase chain
reaction (PCR) assays [25]. All subjects were seronegative for
HIV at baseline. Demographic, risk factor, and health history
information was obtained by interview with trained study coordinators. HTLV-II subtypes were determined previously by
use of restriction fragment–length polymorphism analysis [26].
Laboratory methods. Peripheral blood mononuclear cell
(PBMC) samples were stored at ⫺70C until used, and PBMCs
were digested in a PCR solution with proteinase K. Quantitation
of proviral DNA of HTLV-I and HTLV-II was performed by
use of real-time PCR. We used a single set of primers for both
HTLV-I and -II, from highly conserved sequences of the viral
tax regions, designated as HTLV-F5 (5-CGG ATA CCC IGT
CTA CGT GTT T-3) and HTLV-R4 (5-CTG AGC IGA IAA
CGC GTC CA-3). To quantitate the cellular input for each
reaction, HLA-DQ-a copy number was measured separately.
The primers used for HLA-DQ-a were GH26 (5-GTG CTC
CAG GTG TAA ACT TGT ACC AG-3) and GH27 (5-CAC
GGA TCC GGT AGC AGC GGT AGA GTT G-3). For each
sample, 25 mL of DNA lysate was added to the PCR mixture
(5 mL for the HLA-DQ-a reactions). Real-time PCR was performed by use of the GeneAmp 5700 machine (Applied Biosystems), with the following cycle conditions: 10 min at 95C,
followed by 45 cycles of 30 s at 95C, 30 s at 64C, and 45 s
at 72C. Reactions were performed in triplicate [27].
Fluorescense intensity of Syber green incorporated into the
amplified product was measured at every PCR cycle, and the
threshold cycle number of each sample was recorded. For each
run, a standard curve was generated from 1:10 serial dilutions
of MT2 (HTLV-I; obtained from American Type Culture Collection) or MoT (HTLV-II) cell lines, with a range of 10⫺1–104
copies/reaction. Mean HTLV copy numbers for these cell lines
were 2.1 HTLV-I copies/cell for MT2, as determined elsewhere
[28], and 11.7 HTLV-II copies/cell for MoT, determined experimentally using HTLV-II plasmids of known concentration
(provided by J. Kropp, Gladstone Institute of Virology and Immunology, San Francisco, CA, and E. Wattel, Centre Leon Bernard, Lyon, France). The number of copies in the test sample
was then calculated by interpolation of the experimentally determined threshold cycle number onto the control standard
regression curve. To determine the VL of each sample, the
number of copies of HTLV-I or HTLV-II was divided by the
HLA-DQ-a copy number. The lower limit of detection for the
assay was 1 copy/105 cells. Reproducibility of the assay was
measured by testing 40 specimens in duplicate and comparing
the results, by use of linear regression with R 2 p 0.97.
Statistical analysis. VLs, expressed as HTLV-I or -II copies
per 106 PBMCs, were log10 transformed for all analyses, to
approximate the normal distribution. Undetectable VLs were
assigned the value of 1 copy/106 cells, which is below the lower
limit of detection of the VL assay, before log10 transformation.
Means, SEs, and ranges were calculated for each HTLV type
and in subsets defined by demographic and risk factor variables.
HTLV-I and -II Provirus Load • JID 2004:190 (1 August) • 505
Table 1. Demographic and risk factor profile of the
study population from the baseline visit of the HTLV
Outcomes Study.
Variable
All subjects
Age, years
!20
20–29
30–39
40–49
50–59
⭓60
Sex
Female
Male
Race/ethnicitya
White
Black
Hispanic
Asian
Other/unspecified
Lifetime no. of sex partnersa
!4
5–8
9–20
⭓21
History of IDUa
No
Yes
History of sex with an IDUa
No/unlikely
Yes/likely
History of blood transfusiona
No
Yes
NOTE.
user.
HTLV-I
HTLV-II
127 (100)
328 (100)
2
9
25
55
16
20
(2)
(7)
(20)
(43)
(13)
(16)
2
19
144
110
35
18
(1)
(6)
(44)
(34)
(11)
(5)
93 (73)
34 (27)
243 (74)
85 (26)
46
54
6
15
4
(36)
(43)
(5)
(12)
(3)
112
107
86
6
14
(34)
(33)
(26)
(2)
(4)
42
31
38
14
(33)
(24)
(30)
(11)
73
81
89
78
(22)
(25)
(27)
(24)
125 (98)
1 (1)
243 (74)
84 (26)
104 (82)
10 (8)
136 (42)
182 (55)
85 (67)
42 (33)
251 (77)
69 (21)
Data are no. (%) of subjects. IDU, injection drug
HTLV-II group. A greater proportion of subjects with HTLVI than subjects with HTLV-II had !4 lifetime sex partners,
whereas a greater proportion of subjects with HTLV-II admitted
to having had 120 partners. A history of IDU was reported by
25% of the subjects with HTLV-II (48 men and 36 women),
although only ∼1% said they currently injected drugs. More
than half of the subjects with HTLV-II (37 men and 145 women) reported (yes or likely) sexual intercourse with an IDU. Onethird of the HTLV-I group and one-fifth of the HTLV-II group
had a history of blood transfusion; in comparison, !10% of
HTLV-seronegative blood donors enrolled in the cohort reported such a history.
VLs for both viral types ranged from 0.05 to 5.95 log10 copies/
6
10 PBMCs. HTLV-I VLs (mean [SE], 3.28 [0.12] log10 copies/
106 PBMCs) were significantly higher than HTLV-II VLs (mean
[SE], 2.60 [0.09] log10 copies/106 PBMCs; P ! .0001; figure 1A
and 1B). Among subjects with HTLV-II, HTLV-II VLs were
significantly higher in the 190 subjects with subtype A (mean
[SE], 2.82 [0.11] log10 copies/106 PBMCs), compared with the
138 subjects with subtype B (mean [SE], 2.29 [0.15] log10 copies/106 PBMCs; P p .005; figure 2D). All analyses by demographic and risk factor variables were performed separately for
HTLV-I and for HTLV-II.
HTLV-I VLs were lower in white (mean [SE], 3.03 [0.22]
log10 copies/106 PBMCs) than in nonwhite (mean [SE], 3.48
[0.14] log10 copies/106 PBMCs) subjects, although this difference was not statistically significant (P p .08 ; figure 2A). HTLVI VLs were higher in those with a history of blood transfusion
(mean [SE], 3.62 [0.14] log10 copies/106 PBMCs) than in those
without a history of blood transfusion (mean [SE], 3.1 [0.16]
log10 copies/106 PBMCs; P p .04; figure 2B). HTLV-I VLs did
not differ significantly by age, sex, number of sex partners,
a
Nos. may not add to totals, because of missing data (!10%
per variable).
Differences in log10 VLs between groups were tested by use of
2-sample t tests. Associations between log10 VLs and each continuous variable were tested by use of separate linear regression
models. Finally, multivariate linear regression models were constructed to assess independent associations between variables
found to be significantly associated in the univariate analyses.
All analyses were performed using SAS statistical software (version 8.2; SAS Institute).
RESULTS
VLs and epidemiologic data were available for 127 subjects with
HTLV-I and 328 subjects with HTLV-II (∼85 % of the HTLV
cohort). Demographic and risk factor characteristics of the subjects are presented in table 1. Most subjects were middle-aged
and women, and there was a substantial proportion of black
persons in both HTLV groups and of Hispanic persons in the
506 • JID 2004:190 (1 August) • Murphy et al.
Figure 1. Difference in human T lymphotropic virus (HTLV) provirus load
(VL) between HTLV-I– and HTLV-II–infected subjects (P ! .0001), from the
baseline visit of the HTLV Outcomes Study (HOST). VL is presented as
median (central line), 25th and 75th percentiles (box boundaries), and 10th
and 90th percentiles (error bars). PBMCs, peripheral blood mononuclear
cells.
Figure 2. Human T lymphotropic virus (HTLV) provirus load (VL) presented as median (central line), 25th and 75th percentiles (box boundaries), and
10th and 90th percentiles (error bars). A, HTLV-II VL by HTLV-II subtype A vs. B (P p .005). B, HTLV-II VL by sex (P p .007). C, HTLV-I VL by nonwhite
vs. white race/ethnicity (P p .08). D, HTLV-I VL by history of blood transfusion (P p .04). PBMCs, peripheral blood mononuclear cells.
history of IDU, or history of sex with an IDU. In the multivariate linear regression analysis, HTLV-I VL retained its association with a history of blood transfusion (b p 0.68; P p
.02) but had only a borderline association with white race (b
p ⫺0.51; P p .06).
HTLV-II VLs were significantly lower among women (mean
[SE], 2.45 [0.10] log10 copies/106 PBMCs) than among men
(mean [SE], 3.02 [0.18] log10 copies/106 PBMCs; P p .007; figure
2C). There was no association between HTLV-II VL and age,
race, number of sex partners, history of IDU, or history of sex
with an IDU. In the multivariate linear regression analysis, HTLVII VL remained independently and inversely associated with female sex (b p ⫺0.68; P p .01) and positively associated with
HTLV-II subtype A (b p 0.54; P p .008). In an analysis restricted to women only, there was an inverse association between HTLV-II VL and increasing number of sex partners (b
p ⫺0.22; P p .03), which did not persist after adjusting for
other covariates (b p ⫺0.18; P p .12), although the association
with HTLV-II subtype A remained (b p 0.65; P p .004).
DISCUSSION
The present study of HTLV VLs at the baseline visit of a large
cohort of generally asymptomatic former blood donors found
that HTLV-I VLs were significantly higher than HTLV-II VLs
and that HTLV-II VLs were significantly higher in subtype A
than in subtype B infections. Analyses by demographic and
behavioral characteristics revealed higher HTLV-I VLs in those
subjects with a history of blood transfusion and borderline
lower HTLV-I VLs in white subjects. HTLV-II VLs were significantly lower in women than in men. There was a borderline
association of lower VLs with increasing numbers of sex partners, but this did not persist after adjusting for covariates.
HTLV-I VLs have been reported in numerous studies, ranging from !1 copy/10,000 lymphocytes to 11 copy/10 lymphocytes [6, 9, 10, 14]. The results of the present study are consistent with those of previous reports, with a mean HTLV-I VL
of ∼1000 copies/106 PBMCs and a range of 1–100,000 copies/
106 PBMCs. By use of an end-point dilution PCR assay, a
previous study revealed VLs of 20–200,000 copies/106 PBMCs
in 49 subjects with HTLV-II, with and without HIV coinfection
[17]. A competitive PCR assay found 50–162,390 copies/106
PBMCs in Italian IDUs [16], and those with the highest VLs
were found to have clonal expansion of HTLV-II–infected lymphocytes [29]. These VL results are also similar to those of the
present study.
In the only other comparative study of HTLV-I and HTLVHTLV-I and -II Provirus Load • JID 2004:190 (1 August) • 507
II VLs that we were able to find, Kaplan et al. reported that
HTLV-I VLs were significantly higher than HTLV-II VLs [27].
Their study included a subset of individuals from the present
study but used a different assay to measure VLs. The present
report is the only published in vivo study of HTLV-II VLs
according to HTLV-II subtype. Differences in VLs between
HTLV-I and HTLV-II, and between HTLV-II subtype A and
subtype B, may be related to differences in tax gene structure
and protein expression among the HTLV types and subtypes.
HTLV-II Tax has been shown to have a lower transforming
activity than HTLV-I Tax [30], which may account for the lack
of leukemogenesis by HTLV-II. Compared with HTLV-II subtype B, HTLV-II subtype A has less ability to inhibit p53 function in T lymphocytes [8, 31] and to induce CREB- and NFkB–mediated transactivation [32]. These in vitro data would
suggest lower VLs in HTLV-II subtype A infection, which is
the opposite of our finding. One explanation may be that tax
genes from wild-type HTLV-II subtype A isolates may have
different biologic activity than the HTLV-II prototypes commonly used for in vitro experiments [31–33]. It is also possible
that differences in VLs between HTLV types and subtypes are
due to differences in modes of transmission or to host factors,
and not to biological characteristics of each virus.
The association of higher HTLV-I VLs with a history of blood
transfusion is the most suggestive evidence we found that the
route of infection affects HTLV VLs. Fully 34% of our HTLVI cohort had received a blood transfusion, compared with 10%
of our HTLV-seronegative comparison group, which is consistent with our previous report of blood transfusion as a risk
factor for infection (adjusted odds ratio, 4.5; 95% confidence
interval, 2.6–7.8) [34]. These same subjects with HTLV-I and
a history of blood transfusion have now been shown to have
higher VLs than subjects with HTLV-I without this risk factor.
Blood transfusion is associated with a higher dose of HTLV-I–
infected lymphocytes, so it is biologically plausible that VLs are
higher. Since VLs have been shown to remain relatively constant
over time [35], one may infer that a higher infecting dose from
blood transfusion translates to a higher persistent VL, such as
those detected in our subjects. Alternatively, altered immune
function from comorbid illness or severe trauma at the time
of blood transfusion could have affected the VL set point. Our
data are consistent with those of a previous report of median
HTLV-I VLs of 100–200 copies/105 PBMCs in the first year
after blood transfusion–transmitted infection in a Jamaican
study [14]. However, in the same study, asymptomatic carriers
had higher HTLV-I VLs (median, ∼1000 copies/105 PBMCs)
than did asymptomatic carriers in the present study, perhaps
because of a higher proportion of mother-to-child infection.
HTLV-II VLs were significantly lower in women than in men.
Two previous studies of HTLV-I VLs found no differences by
sex among asymptomatic carriers [9, 36], but we were unable
508 • JID 2004:190 (1 August) • Murphy et al.
to find other published reports of differences in HTLV-II VLs
by sex. Other reports found that women with HAM have either
higher or lower HTLV-I VLs than do men with HAM [9, 10].
Women are clearly overrepresented among both patients with
HTLV-I HAM and patients with HTLV-II HAM [37]. Previous
epidemiological data suggest that sexual acquisition of HTLVI is associated with development of HAM, whereas mother-tochild acquisition is associated with ATL [38, 39].
We therefore hypothesize that women are more likely to
acquire HTLV-II sexually, with a smaller infectious dose, and,
hence, to have lower VLs than men, most of whom acquire
the virus by parenteral (IDU or blood transfusion) or motherto-child routes. Against this hypothesis are data from the present study, which has showed no association between HTLV-II
VLs and the lifetime number of sex partners or history of sex
with an IDU, for both sexes. However, in an analysis limited
to women, we did find a nonsignificant trend toward lower
HTLV-II VLs with increasing number of lifetime sex partners.
Retrospective interview data may not be the most accurate in
assigning the actual mode of transmission. In support of the
hypothesis are data showing lower mean HTLV-I VLs in 11
cases of sexually acquired HTLV-I, compared with the 11 transmitting persons [40].
That infectious dose may predict the chronic set point for
VLs is consistent with the observation that expansion of HTLV
within an infected human is predominantly due to clonal expansion of lymphocytes with integrated HTLV provirus [6].
Thus, the number of initially infected lymphocytes would influence the number of lymphocyte clones, although the size of
each clone may depend on factors that cause multiplication of
lymphocytes. Recent data from the Wattel laboratory support
this hypothesis, in that humans with presumed breast-milk
acquisition of HTLV-II as infants had increased clonality and
VLs, compared with those with risk factors suggesting infection
acquired later in life [41]. An alternative explanation is that
infection during infancy or childhood produces higher VLs
because of a weaker initial immune response.
The borderline association we found between lower HTLVI VLs and white race may be explained either by genetic background or by route of infection associated with a particular
racial group. Bangham et al. have previously reported that
HTLV-I VLs may be correlated with specific HLA subtypes [42]
and that these subtypes are also associated with an increased
risk of developing HAM. Since HLA phenotypes differ by racial
origin, it is conceivable that the race/ethnicity association we
observed is due to genetic background. On the other hand,
other host characteristics, such as route of HTLV infection,
lymphocyte activation, and concomitant infections, may also
differ by race. It has been recognized that the incidence of
HTLV-associated diseases, such as ATL, may differ between
endemic Japanese and Jamaican populations, perhaps because
of differences in biologic response to infection [23]. Furthermore, comparative studies across race and ethnicity populations
using similar methodologies to measure VL and controlling for
route of infection and HLA type should help resolve this issue.
Strengths of the present study include the use of a welldefined, generally asymptomatic group of human subjects and
a sensitive, reproducible, and high-throughput assay to measure
VLs. Potential weaknesses include the relatively large variability
of VLs and the fact that the differences in VLs, although statistically significant, were of relatively small magnitude, compared
with the interpersonal range of observed values. Determining
causality for the associations we observed is also problematic in
this cross-sectional study, since different characteristics of the subjects (i.e., race/ethnicity and mode of acquisition) may be confounded. That associations persisted after multivariable analysis
is reassuring but not definitive.
In conclusion, we have demonstrated associations between
higher HTLV-I VLs and history of blood transfusion and, perhaps, nonwhite race and between lower HTLV-II VLs and female sex. Discovering the biological determinants of VL is important because the incidence of HTLV-associated diseases
seems to be related to VL. Although reasons for the race/ethnicity association remain speculative, we hypothesize that associations with sex and history of blood transfusion result from
a biological relationship between infectious dose of HTLV-I and
HTLV-II and the chronic VL measured in an individual. Further
studies in humans with well-defined time and mode of infection
or experimental studies in animals are required to investigate
this hypothesis.
HTLV OUTCOMES STUDY (HOST) MEMBERS
The HOST is presently the responsibility of the following persons.
Study headquarters. University of California, San Francisco (San Francisco, CA): E. L. Murphy (Principal Investigator)
and J. Engstrom.
Blood centers. American Red Cross Blood Services Greater
Chesapeake and Potomac Region (Baltimore, MD): C. C. Nass,
C. Conry-Cantilena, and J. Gibble; American Red Cross Blood
Services Southeastern Michigan Region (Detroit, MI): B. Newman; American Red Cross Blood Services Southern California
Region (Los Angeles, CA): G. Garratty, S. Hutching, and A.
Ziman; Blood Centers of the Pacific (San Francisco, CA): M.
P. Busch; and Oklahoma Blood Institute (Oklahoma City, OK):
J. W. Smith and E. Moore.
Medical coordinating center. Westat (Rockville, MD): G.B.
Schreiber, D. Ameti, and B. Wang.
Central laboratory. Blood Centers of the Pacific (San Francisco, CA): M. P. Busch and L. H. Tobler.
Diagnostic review panel. E. L. Murphy, R. Sacher, and J.
Fridey.
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