1. health and fitness
2. disease

Use of Leukocyte-Depleted Platelets and Cytomegalovirus-Seronegative Red
Blood Cells for Prevention of Primary Cytomegalovirus Infection After
Marrow Transplant
By Raleigh A. Bowden, Sherrill J. Slichter, Merlin H. Sayers, Motomi Mori, Monica J. Cays, and Joel D. Meyers
Seventy-seven cytomegalovirus (CMV)-seronegative marrow transplant patients were randomized in a prospective
controlled trial comparing the use of leukocyte-depleted
platelets plus CMV-seronegative red blood cells with standard unscreened blood products for the prevention of primary CMV infection during the first 100 days after transplant.
Eligible patients included CMV-seronegative patients undergoing autologous transplant or seronegative patients undergoing allogeneic ‘ransplant for aplastic anemia or nonhematologic malignancy who had seronegative marrow
donors. Patients and marrow donors were serologically
screened for CMV and randomized before conditioning for
transplant and followed for CMV infection with weekly
cultures of throat, urine, and blood and with weekly CMV
serologies until day 100 after transplant. Leukocyte-depleted
platelets were prepared by centrifugation, a procedure that
removed greater than 99% of leukocytes. There were no
CMV infections observed in 35 evaluable treatment patients
compared with seven infections in 30 evaluable control
patients (P = .0013). There was no statistically significant
difference in the mean number of platelet concentrates in the
treatment patients (164 concentrates) compared with the
control patients(126concentrates).Leukocyte-depleted platelets plus CMV-seronegative red blood cells are highly effective in preventing primary CMV infection after marrow
transplant.
0 1991by The American Society of Hematology.
C
ies in CMV-seronegative marrow transplant patients using
filtered red blood cells (RBCs) and CMV-seronegative
platelets6 and in patients with hematologic malignancies
using centrifuged platelets and filtered RBCs’ suggest that
this approach may be effective. We chose to compare the
use of leukocyte-depleted platelets plus CMV-seronegative
RBCs with the use of unscreened blood products in a
randomized trial for prevention of transfusion-associated
primary CMV infection during the first 100 days after
marrow transplant. The primary rationale for this combination was that the platelet needs of marrow transplant
patients at our center place a much higher demand on
blood bank resources than do RBC needs (ie, patients use a
mean of 161 platelets concentrates and 19 RBC units/
transplant).8 Therefore, the ability to avoid serologic screening of platelet transfusions offers the greatest benefit to the
Blood Center. We also chose to perform this study primarily in autologous patients who, although they are at a lower
risk for symptomatic CMV disease than patients undergoing allogeneic transplant, have a similar risk of CMV
infection.’,’ If we could demonstrate protection in autologous patients, a rationale for similar studies in allogeneic
patients who have been shown to be protected by seronegative blood products could be justified. Our ultimate goal
was to examine the efficacy of leukocyte-depleted blood
products for their potential as an alternative to the use of
CMV-seronegative blood for prevention of primary CMV
infection. Such an approach would provide protection for a
greater number of patients, including those in communities
and areas of the world with an insufficient number of
seronegative donors.
YTOMEGALOVIRUS (CMV) pneumonia remains a
leading infectious cause of death after marrow transplant.’ CMV infection develops as a result of reactivation of
latent virus in CMV-seropositive individuals or is acquired
as a primary infection in CMV-seronegative individuals
from either blood products or marrow from CMVseropositive donors. Primary infection can be successfully
prevented by the use of CMV-seronegative blood products
in allogeneic marrow transplant patients with seronegative
marrow d o n ~ r sas~ well
* ~ as in other seronegative patients at
risk for serious CMV infe~tion.~,’
However, CMV-seronegative blood is not universally available and a need for an
alternative means of protection from primary CMV infection occurs in communities in which: (1) insufficient CMVseronegative blood donors are available; (2) a large number
of marrow transplants, including autologous transplants are
performed; (3) renal, cardiac, and liver transplants are
performed; or (4) physicians wish to protect seronegative
patients with malignancies who might be prospective marrow transplant patients from acquiring CMV from blood
products.
Because the leukocyte fraction of blood products is
thought to be the source of CMV, removal of this fraction
from transfusions should reduce or eliminate the risk of
transmission of CMV infection. Recmt uncontrolled studFrom the Fred Hutchinson Cancer Research Center and the
Departments of Pediatrics, Medicine, and Biostatistics, University of
Washington School of Medicine; and the Puget Sound Blood Center,
Seattle, WA.
Submitted October 22, 1990; accepted February 21, 1991.
Supported by HL 36444, HL 33775, CA 18029, and CA 15704
awarded by the National Institutes of Health.
Address reprint requests to Raleigh A . Bowden, MD, Program in
Infectious Diseases, Fred Hutchinson Cancer Research Center, 1124
Columbia St, Seattle, WA.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 sole& to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971I91I7801-0009$3.0010
246
MATERIALS AND METHODS
Patients. All patients undergoing marrow transplant, and their
donors, were tested for CMV antibody by both latex agglutination
(CMVSCAN Card Test; Becton Dickinson and Co,Baltimore,
MD) and enzyme immunoassay (EIA) (CMV STAT; Whittaker
Bioproducts, Inc, Walkersville, MD) before transplant. Because of
limited CMV seronegative blood resources in our center, CMVseronegative blood is routinely provided only for patients undergoing allogeneic transplant for hematologic malignancy who have a
significantly higher risk for CMV pneumonia compared with
Blood, VOI 78, NO1 (July l), 1991: pp 246-250
PREVENTION OF PRIMARY CMV INFECTION
patients undergoing autologous transplant’ or allogeneic transplant for aplastic anemia.” Therefore, patients were eligible for the
present study if they were CMV-seronegative and were undergoing
either autologous marrow transplant or were undergoing allogeneic transplant for non-hematologic malignancy or aplastic anemia
and had seronegative marrow donors confirmed by both serologic
screening tests. Patients or marrow donors with discrepancies
between the two serologic tests were retested by latex agglutination
and two of three negative tests defined CMV-seronegative status.
Patients were not eligible for this study if they had received more
than 6 units of unscreened blood between the time of CMV
antibody testing and the time of randomization.
Eligible patients were randomized before transplant to receive
leukocyte-depleted platelets plus CMV-seronegative RBCs (treatment arm) or unscreened blood products (control arm). The
randomization was stratified for the patient’s underlying disease
(allogeneic transplant patients with non-hematologic malignancy,
autologous transplant patients). Patients remained on study from
the time of randomization until CMV infection developed or day
100 after transplant, whichever occurred first. Approval was
obtained from the Institutional Review Board for participation in
this study. Patients and marrow donors were informed that samples
for CMV cultures were obtained for research purposes only and
that their privacy would be protected.
Preparation of blood products. The leukocyte-poor platelets
were prepared at the Puget Sound Blood Center from units of
unscreened whole blood. Approximately 50% of blood donors in
Seattle are CMV seropositive. The blood was first centrifuged at
1,025g for 9 minutes, followed by a second spin at 3,OOOg for 20
minutes. The platelet pellet was then resuspended in 50 mL of
platelet-poor plasma. For each transfusion, 4 to 6 platelet concentrates were pooled and subjected to a final centrifugation at 400g
for 10 minutes, which removes greater than 99.8% of the leukocytes. A leukocyte and platelet count was performed on every
transfusion of leukocyte-depleted product to insure quality of the
product and so that subsequent development of CMV infection
might be compared with the actual leukocyte counts of the units
each patient received. This method also resulted in recovery of
86% of the original platelet count.
The CMV-seronegative RBCs were prepared as packed RBC
units from blood donors found to be seronegative by latex agglutination.
Assessment of CMV infection. All patients were followed with
weekly CMV cultures and serologies throughout the study period
for evidence of CMV infection. Viral cultures of urine, throat, and
blood buffy coat were inoculated onto complete monolayers of
human foreskin fibroblasts and observed for 6 weeks by standard
methods. Positive cultures were identified by typical cytopathic
effect and confirmed by immunofluorescence after passage of the
culture. When biopsy tissue was obtained, CMV was identified by
typical viral inclusions, indirect immunofluorescent antibody staining for late CMV antigens with murine monoclonal antibodies
(MoAbs; Genetic Systems Corporation, Seattle, WA), or by
positive culture. Bronchoalveolar lavage was used to diagnose
CMV pneumonia in patients with infiltrates on chest radiograph.
We have previously shown that this method is 96% sensitive and
100% specific for the diagnosis of CMV pneumonia when compared with open lung biopsy.” CMV antibody titers were determined by EIA. Sera with an optical density reading at a wave length
of 550 nm of greater than 0.99 were considered positive and
significant seroconversion was defined as a value of 1.47or greater,
according to the manufacturer’s guidelines.
The endpoint of this study was the development of CMV
infection. However, patients were also assessed for the occurrence
of CMV disease. CMV infection was defined as development of a
247
positive CMV culture, seroconversion, or identification of CMV in
tissue (see above) greater than 21 days after transplant. Patients
developing CMV infection during the first 21 days after transplant
were considered not evaluable because these patients were presumed to have been infected at the time of transplant. Because
patients in both study arms were expected to acquire passive
antibody from either standard or leukocyte-depleted products,
seroconversion was defined only when the development of a
positive CMV titer persisted 2 6 weeks after discontinuation of
blood support. CMV disease was defined as tissue evidence of
invasive CMV infection with associated clinical symptoms.
Statistics. The incidence of CMV infection was estimated by
the Kaplan and Meier method, with comparison of the incidence
curves by log-rank test.”.13 Analysis for differences in age and
number of blood products between patient groups was performed
by Wilcoxon rank sum test and analysis of other fixed demographic
variables was performed using either Fisher’s exact or xztests.
RESULTS
Patients. Seventy-seven patients were randomized. The
majority were patients undergoing autologous transplant
for hematologic malignancies (see Table 1). Five patients
randomized to the treatment arm and seven patients to the
control arm were either never transplanted or were found
to be ineligible before transplant because of change in
donor CMV serostatus, leaving 35 treatment and 30 control
patients in the final analysis. There were no significant
differences between the demographic characteristics of
patients in the leukocyte-depleted and control groups.
Because most patients received autologous transplants,
they were given no post-grafting immunosuppression.
Twelve patients in the leukocyte-depleted group and eight
patients in the control groups received post-grafting intraTable 1. Demographic Characteristics
LeukocyteDepleted
Blood
Total patients
Patients excluded before transplant
Total evaluable patients
Sex (ma1e:female)
Median age (y) (range)
Underlying disease
ALL
AN L
AA
Lymphoma
Other
Transplant type
Allogeneic
Autologous
Conditioning regimen
TBI + CTX
CTX + TBI + McAb
CTX + TBI/BU + 4 H C marrow
TBI + other chemotherapy
BU + CTX
40
5
35
20:20
27 (4-50)
Standard
Blood
37
7
30
15:22
26 (2-58)
8
4
2
16
4
5
6
2
13
3
3
32
2
28
6
11
5
3
4
2
11
8
6
5
Abbreviations: ALL, acute lymphocytic leukemia: ANL, acute nonlymphocytic leukemia; AA. aplastic anemia; TBI, total body irradiation;
CTX, cytoxan; McAb, anti T-cell MoAb; BU, busulfan; HC, hydroxycyclophosphamide.
248
BOWDEN ET AL
thecal methotrexate for prophylaxis against recurrent malignancy of the central nervous system. The three patients
undergoing allogeneic transplant in the leukocyte-depleted
group and the two allogeneic patients in the control group
received cyclosporine plus short methotrexate as graftversus-host disease prophylaxis. No patient in this study
received either intravenous Ig as infection prophylaxis,
acyclovir prophylaxis to prevent CMV infection, nor other
experimental antiviral agents during the study.
CMV infection. There were no CMV infections in the
leukocyte-depleted group compared with seven infections
in the control group. The probability of developing CMV
infection is shown in Fig 1 and was significantly greater for
patients receiving standard unscreened blood (P= .0013 by
log-rank test). The manifestations of CMV infection, including the site and time after transplant that the culture
became positive, is shown in Table 2. The median onset of
infection was 59 days after transplant. The patient with
CMV pneumonia was diagnosed by bronchoalveolar lavage
100 days after transplant and the patient with enteritis was
diagnosed by endoscopic biopsy of esophagus and duodenum 60 days after transplant. There were no CMV infections in either group in the first 21 days after transplant.
Delivery of blood products. Patients in the leukocytedepleted group received an average of 25 units of RBCs
(range, 4 to 97 units) and 165 platelets concentrates (range,
6 to 568 concentrates) compared with 20 units of RBCs
(range, 7 to 55 units) and 130 platelet concentrates (range,
28 to 428 concentrates) in the control group. The differences were not significant. Standard platelet concentrates
in our laboratory have 1.2 x 10' leukocytes/concentrate.
The method of leukocyte-depletion used in this study
resulted in platelet concentrates with a median residual
leukocyte count of 4 x 106/plateletconcentrate (range, 1to
10 x lo6 leukocytes/concentrate). Patients in the control
group who became infected with CMV received an average
of 22.5 RBC units (range, 14 to 37 units) and 123 platelet
concentrates (range, 40 to 244 concentrates), which was not
Table 2. CMV Infection and Disease
LeukocyteDepleted
Blood
Evaiuable patients
Total CMV infections
Type of CMV infections
Asymptomatic excretion
35
Standard
Blood
0
30
7
0
1
1
1
1
1'
CMV disease:
Pneumonia
0
1
Enteritis
0
1
Site of Infection
(day after marrow
transplant)
Throat (59). urine (59).
blood (66). seroconversion (75)
Urine (98)
Throat (69). seroconversion (69)
Urine (49). throat (49)
Seroconversion only
(40)
Urine (75).throat (75),
BALt (100)
Urine (56), seroconversion (58), esophagus/duodenum
(60). blood (78)
*This is the only patient undergoing allogeneic transplant who
developed CMV infection. All other CMV infections occurred in patients
undergoing autologous transplant.
tBAL, bronchoalveolar lavage.
statistically different from the numbers of units or concentrates for uninfected patients in the control or treatment
groups.
DISCUSSION
This study has demonstrated that leukocyte-depleted
platelet concentrates and CMV-seronegative RBCs are
effective in preventing transfusion-acquired CMV infection
in seronegative marrow transplant patients when compared
with standard blood products (P = .0013). It supports the
observations of Verdonck et al, who administered filtered
RBCs and CMV-seronegative platelets in an uncontrolled
trial to CMV-seronegative allogeneic and autologous marrow transplant patients and found no CMV infections: and
"O1
the study of de Graan-Hentzen et al, who retrospectively
0.8.1
found no CMV infections in 59 seronegative cancer patients administered centrifuged platelets and filtered RBCs.'
A randomized controlled trial of filtered RBCs in seronegative infants born to seronegative mothers found no infections in 30 infants in the filtered arm compared with nine
Standard Blood
r
infections in 42 infants receiving unfiltered RBCs.I4A major
difference between the present study and previous studies
021
P=(.OO13)
f-+
was the number of blood products administered. Our
Leukocyte-poor Blood
patients received an average of 25 units of RBCs (range, 4
0.0 . . . . . . . . . . . . . . . .____________________------__---------------.................................
to 97 units) and 164 platelet concentrates (range, 6 to 568
0
20
40
60
80
100
concentrates), compared with an average of 8 RBC units
DAY after TRANSPLANT
(range, 6 to 18 units) and 6 platelet concentrates (mean, 3
Fig 1. Kapian-Meier product-limit estimates of the probability of
to 31 concentrates) in the study by Verdonck et a1,6 a mean
developing CMV infection in patients receiving leukocyte-depleted
of 15 units of RBCs (range, 0 to 38 units) and 54 platelet
blood (-)
compared with patients receiving standard blood prodconcentrates (range, 0 to 207 concentrates) in the de
ucts (-----). The probability of recovering CMV was different at P =
Graan-Hentzen study,' and a median volume of only 32.5
,0013 (log-ranktest).
'
PREVENTION OF PRIMARY CMV INFECTION
mL of RBCs in the study by Gilbert et al in infants.I4 In
addition to being a controlled trial, the present study
represents a substantial increase in the number of units or
total volume of transfusion products over previous studies
and provides strong evidence that leukocyte depletion can
effectively prevent transmission of CMV infection.
It is not known which type or how many leukocytes in
blood from seropositive individuals carry CMV. Previous
studies infecting mononuclear blood cells with clinical
strains of CMV have shown that monocytes, T cells, B cells,
and natural killer cells express immediate early proteins of
CMV,I5,l6including as many as 2% to 3% of peripheral
blood mononuclear cells.16Schrier et a1 has also shown that
2.4% of CD4 helper and 0.8% of CD8 suppressorkytotoxic
T cells express immediate early proteins in healthy CMVseropositive blood donors, for a frequency of 1/500 cells.”
The present study suggests that a majority of these cells do
not result in productive infection. Because patients in the
leukocyte-depleted arm presumably received half of their
platelet transfusions from seropositive donors, or they
received approximately 80 platelet concentrates from seropositive donors in the first 100 days after transplant, they
would have been exposed to approximately 3.2 x 10’
potentially infectious cells of which 0.2% should contain
CMV proteins. The fact that we observed no infections in
the leukocyte-depleted group suggests that we have reduced the number of leukocytes below the critical threshold
for transmission of infection.
A variety of techniques are available for leukocyte
depletion of blood products. While frozen deglycerolized
RBCs have been shown to be effective,” they are not
practical on a large ~ca1e.I~
Previously described cotton
filters result in a 1.5 to 2 log reduction in leukocytes and
have been shown to be effective in uncontrolled studies6.’in
cancer or marrow transplant patients and in a controlled
trial in newborns.” However, they require relatively large
volumes of fluid, which may present problems for fluidrestricted patients. Centrifugation for preparation of leukocyte-depleted platelets was used in the present study and
others7 and is a relatively easy technique also resulting in a
1.5 to 2 log reduction without a clinically significant platelet
loss. More recently, and since the design of this study,
newer platelet and RBC filters have been developed that
249
can be used “in line” at the bedside that result in a 3 log or
greater reduction in leukocytes that show promising preliminary results in the prevention of transfusion-associated
CMV infection after marrow transplant.m
Because allogeneic transplant patients are at significantly
increased risk for morbidity and mortality’ compared with
patients undergoing autologous transplant: this approach
could ultimately have an more important impact on the
ability to deliver CMV-safe products to allogeneic patients.
Data suggests that the risk of CMV infection is directly
related to the exposure number to increased numbers of
seropositive donors,” while the severity of symptomatic
CMV disease increases with the degree of immunosuppression,19supporting the inclusion of allogeneic patients in the
present study where the endpoint of the study is CMV
infection. While the results of the present study and one
other study including allogeneic patients’ appear promising
as a means of preventing transfusion-associated CMV
infection, further studies in large numbers of allogeneic
patients are warranted, however, because of their increased
risk of CMV disease compared with patients undergoing
autologous transplant.
In conclusion, we have shown in a randomized controlled
trial that the use of leukocyte-depleted platelets is an
effective means for the prevention of transfusion-associated
CMV infection. Presumably leukocyte depletion will also
be applied to RBCs, which were not tested in this study, in
part, because the RBC filters available at the time of this
study were not technically easy to use in patients receiving
such large volumes of RBC support. With recent technical
advances that include the development of more efficient
platelet and RBC filters that can now be used at the
bedside, leukocyte depletion of both RBCs and platelets is
now possible. This approach may obviate the need for CMV
serologic screening of blood products for patients at risk for
serious disease associated with primary CMV infection
acquired from blood products. However, further studies are
needed in patients undergoing allogeneic transplant who
are at higher risk for CMV infection and disease before this
can be assumed. This approach may also increase the
availability of “CMV-safe” blood products for a variety of
patients at risk for transfusion-associated CMV infection.
REFERENCES
1. Meyers JD: Infection in recipients of marrow transplants, in
due to herpesviruses in cardiac transplant recipients: Role of the
Remington JS, Swartz MN (eds): Current Clinical Topics in
donor heart and immunosuppressive therapy. J Infect Dis 147:974,
Infectious Diseases. New York, NY, McGraw-Hill, 1985, p 261
1983
2. Bowden RA, Sayers M, Flournoy N, Newton B, Banaji M,
5. Yeager AS, Grummet FC, Hafleigh EB, Arvin AM, Bradley
Thomas ED, Meyers JD: Cytomegalovirus immune globulin and
JS, Prober CG: Prevention of transfusion-acquiredcytomegaloviseronegative blood products to prevent primary cytomegalovirus
rus infections in newborn infants. J Pediatr 98:281,1981
infection after marrow transplantation. N Engl J Med 314:1006,
6. Verdonck LF, de Graan-Hentzen YCE, Dekker AW, Mudde
1986
GC, de Gast GC: Cytomegalovirus seronegative platelets and
3. Miller WJ, McCullough J, Balfour HH Jr, Haake RJ, Ramsay
leukocyte-poor red blood cells from random donors can prevent
NKC, Goldman A, Bowman R, Kersey J: Prevention of CMV
primary cytomegalovirus infection after bone marrow transplantainfection following bone marrow transplantation: A randomized
tion. Bone Marrow Transplant 273,1987
trial of blood product screening. Bone Marrow Transplant 7:227,
7. de Graan-Hentzen YCE, Gratama JW, Mudde GC, Ver1991
donck LF, Houbiers JGA, Brand A, Sebens FW, van Loon AM,
4. Preiksaitis JK, Rosno S, Grumet C, Merigan T C Infections
The TH, Willemze R, de Gast GC: Prevention of primary cytomeg-
250
alovirus infection in patients with hematologic malignancies by
intensive white cell depletion of blood products. Transfusion
29:757, 1989
8. Bowden RA, Sayers M, Gleaves CA, Banaji M, Newton B,
Meyers JD: Cytomegalovirus-seronegative blood components for
the prevention of primary cytomegalovirus infection after marrow
transplantation: Considerations for blood banks. Transfusion 27:
478,1987
9. Reusser P, Fisher LD, Buckner CD, Thomas ED, Meyers JD:
Cytomegalovirusinfection after autologous bone marrow transplantation: Occurrence of cytomegalovirus disease and effect on
engraftment. Blood 75:1888,1990
IO. Ljungman P, Niedenvieser D, Pepe MS, Longton G, Storb
R, Meyers JD: Cytomegalovirus infection after marrow transplantation for aplastic anemia. Bone Marrow Transplant 6:295,1990
11. Crawford SW, Bowden RA, Hackman RC, Gleaves CA,
Meyers JD, Clark JG: Rapid detection of cytomegalovirus pulmonary infection by bronchoalveolar lavage and centrifugation culture. Ann Intern Med 108:180,1988
12. Kaplan EL, Meier P: Nonparametric estimation from incomplete observations. J Am Stat Assoc 53:457, 1958
13. Mantel N: Evaluation of survival data and two new rank
order statistics arising in its consideration. Cancer Chemother Rep
50:163, 1966
14. Gilbert GL, Hayes K, Hudson IL, James J, the Neonatal
BOWDEN ET AL
Cytomegalovirus Infection Study Group: Prevention of transfusionacquired cytomegalovirus infection in infants by blood filtration to
remove leukocytes. Lancet 1:1228,1989
15. Einhorn L, Ost A: Cytomegalovirus infection of human
blood cells. J Infect Dis 149:207, 1984
16. Rice GPA, Schrier RD, Oldstone MBA: Cytomegalovirus
infects human lymphocytes and monocytes: Virus expression is
restricted to immediate-early gene products. Proc Natl Acad Sci
USA 81:6134,1984
17. Schrier RD, Nelson JA, Oldstone MBA Detection of
human cytomegalovirus in peripheral blood lymphocytes in a
natural infection. Science 230:1048, 1985
18. Tolkoff-Rubin NE, Rubin RH, Keller EE, Baker GP,
Stewart JA, Hirsch MS: Cytomegalovirus infection in dialysis
patients and personnel. Ann Intern Med 89:625, 1978
19. Bowden RA, Meyers JD: Infectious complications following
marrow transplantation. Plasma Ther Transfusion Tech 6:285,
1985
20. Bowden RA, Sayers MH, Cays M, Slichter SJ: The role of
blood product filtration in the prevention of transfusion associated
cytomegalovirus (CMV) infection after marrow transplant. Transfusion 29:5205, 1989
21. Prince AM, Szmuness W, Millian SJ, David DS: Serologic
study of cytomegalovirus infections associated with blood transfusions. N Engl J Med 284:1125, 1971