The cytomegalovirus-“safe” blood product: Is leukoreduction equivalent to antibody screening?

The Cytomegalovirus-"Safe" Blood Product: Is Leukoreduction Equivalent to Antibody Screening? Jutta K. Preiksaitis

ANY OF THE adverse reactions associated with the transfusion of platelet and red blood cell units are caused by the "unwanted passenger" in these products, the donor leukocyte. Over the past decade, the technology for removal of leukocytes from blood products has improved significantly, and reports of the potential benefits of leukoreduction in specific clinical settings have continued to accrue. 1,2 Several issues with respect to the implementation of this technology remain controversial, including its proven benefits, its cost-effectiveness, and the timing of leukoreduction. Nonetheless, many transfusion services have either implemented or are considering implement. ing leukoreduction of all cellular blood products or are providing leukoreduced blood products for a selected subset of transfusion recipients. The prevention of febrile transfusion reactions and alloimmunization are the major issues driving the requests for the provision of leukoreduced blood products. However, other potential benefits associated with the implementation of this technology include prevention of the immunomodulatory effects of blood transfusion, a reduction in the risk of graft-versus-host disease, transfusion-related acute lung injury, and a reduction or perhaps elimination of the risk of selected transfusion transmitted infections. 1-3 Leukoreduction will not affect the risk of transfusion-acquired hepatitis B, hepatitis C, or human immunodeficiency vires (HIV) infection that results from the direct infusion of infectious virus from the donor to the recipient. Leukoreduction may, however, have a significant effect on the risk of transmission of cell-associated viruses such as cytomegalovirus (CMV), human lymphotropic virus (HTLV HI), and Epstein-Ban" virus (EBV) infection as well as the less wellstudied herpesviruses--human herpesvirus 6, 7,

M

From the Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. Address reprint requests to Jutta K. Preiksaitis, MD, Division of Infectious Diseases, Department of Medicine, 2B1.03 WC Mackenzie Health Sciences Centre, University of Alberta, Edmonton, Alberta T6G 2R7. Copyright 9 2000 by W.B. Saunders Company 0887-7963/00/1402-000353.00/0

112

and 8 (HHV-6, HHV-7, HHV-8). 1-3 The relationship between leukocyte load in blood products and the risk of transfusion-acquired infection has been most extensively studied for CMV infection? -6 Historically, CMV-seronegative cellular blood products have been provided for CMV-seronegative patient populations at high risk for significant morbidity as the result of primary CMV infection. 7 However, the increasing number of immunosuppressed patients who might benefit from receipt of these products, the high prevalence of CMV antibody in some blood donor populations, and the logistics of maintaining multiple inventories of blood products have led to the exploration of leukoreduction as a alternative strategy for the provision of CMV-"safe" blood products? -8 Despite the promising results reported in these studies, the issue of whether leukoreduced blood products are equivalent to CMV-seronegative blood products for the prevention of transfusion-acquired CMV infection remains controversial.6,9 In this article, I review recent data pertaining to the site of CMV latency and factors influencing its reactivation that have significant implications for our understanding of pathogenesis of transfusionacquired CMV infection. Using data from clinical trials and theoretical considerations, the advantages and disadvantages of using CMV-seronegative versus leukoreduced blood products as CMV-"safe" products are discussed. Guidelines for the use of CMV-"safe" blood in specific populations are updated. CMV AND THE LEUKOCYTE

Data from a large number of clinical epidemiological studies dating back to the 1960s 7 and more recent molecular epidemiological data reporting the identity of CMV isolates from a blood donorblood recipient pair 1~ have provided direct and indirect evidence, respectively, that blood transfusion can transmit CMV infection. The high incidence of CMV infection associated with the receipt of granulocyte transfusions 11,12and the observation that the removal of leukocytes from cellular blood products significantly reduces or eliminates the risk of transmitting CMV infection3-8 suggest that CMV is transmitted by leukocytes in blood products.

Transfusion Medicine Reviews, Vo114, No 2 (April), 2000: pp 112-136

THE CMV-"SAFE" BLOOD PRODUCT

During acute CMV infection, infectious virus can be isolated from both polymorphonuclear leukocytes and mononuclear cells in the peripheral blood of both immunocompetent 13 and immunocompromised patients. 14Infectivity is highest in the polymorphonuclear cell fraction. Although high and persistent levels of viremia have been documented in immunocompromised patients experiencing acute CMV infection, 14 immunocompetent patients experience relatively low-grade and shortlived viremia. 13 In a study of 52 immunocompetent patients with symptomatic primary CMV infection, 40 of who were pregnant, viremia could be detected only during the first month after onset of symptoms and was present in only 20.8% of patients. In contrast, using techniques that will not detect latent infection, CMV DNA was found in the leukocytes of 100%, 89.5%, 47.3%, and 26.6% of patients tested at 1, 2, 3, and 4 to 6 months after acute infection. None were positive beyond 6 months. 13 In a prospective study of 6 asymptomatic subjects with primary CMV infection, CMV viremia was documented in only 1 of 6 (16.7%) leukocyte samples collected within 9 to 10 weeks of infection. 15 CMV DNA was detected in the leukocytes of 75% to 80% of these asymptomatic patients within 16 weeks of infection, declining to 0 to 25% after 48 weeks. Seroconversion rates in immunocompetent seronegative health care workers have been found to vary from 0.6% to 3.3% per year. 16 Higher rates have been documented in women who have contact with young children. Middle-class women seroconvert at a rate of 2.0% to 4.6% per year during pregnancies and 4.6% to 6.3% per year between pregnancies. 17 The seroconversion rate in seronegative blood donors is expected to be low and is estimated to be approximately 1.0% per year. This low rate when combined with the low incidence and duration of viremia in immunocompetent seroconverters explains why, aside from a single report in which CMV was isolated from the leukocytes of 2 of 35 (6%) 18 healthy blood donors, repetitive attempts by many laboratories to isolate CMV from the fresh blood or leukocytes of several thousands of blood donors have been unsuccessful.V,12 In contrast to acutely infected CMV patients, the CMV genome is found predominantly in monocytes in the peripheral blood of healthy CMVseropositive subjects. 19 The viral genome persists as a circular plasmid in a subset of CD14+ cells in

113

these subjects. 2~Despite contradictory earlier information, 21 viral gene expression is not observed in nondifferentiated monocytes from these donors. 22 CD14+ monocytes in peripheral blood represent a terminally differentiated cell of the myeloid/ granulocyte lineage. On contact with T and B cells during antigen processing, these monocytes further differentiate into macrophages, which act as immune effector cells. Several in vitro techniques have been developed to mimic the development of monocyte derived macrophages in vivo. After in vitro infection with CMV, infectious virus has been isolated from macrophages, when monocytes were induced to differentiate using these techniques (reviewed by MichelsonZ3). However, attempts to use these techniques to reactivate CMV from latently infected CMV-seropositive subjects have been unsuccessful. 22,23 Recently, Soderberg-Naucler et al24 reported the successful reactivation of CMV from all 7 healthy seropositive subjects studied after allogeneic stimulation of their peripheral blood mononuclear cells. The cell in which CMV reactivated expressed both macrophage (CD14 and CD64) and dendritic cell markers (CD83 and CDla). This cell type has been described as a transient phenotype during the differentiation of CD34+ hemopoietic progenitor cells. These investigators estimate that the frequency of peripheral blood mononuclear cells latently infected with CMV and capable of reactivating infectious virus in healthy subjects is 0.01% to 0.12%. Their study suggests that CMV reactivation requires a very specific cellular differentiation pathway that involves an allogeneic stimulus. Human hemopoietic precursors in bone marrow have long been suspected as a site of CMV latency. The CMV genome has been detected in CD34+ hemopoietic progenitors of normal CMV-seropositive subjects in the absence of any evidence of lytic gene expression. 25 However, CD34+ cells support virus replication during acute infection in immunosuppressed patients in v i v o 26 and after in vitro infection. 27 During the course of studies in experimentally infected granulocyte-macrophage progenitors (GM-P), specific CMV latency-associated transcripts encoded within the iel/ie2 region of the genome were identified. 28 These transcripts were found in 0.01% to 0.001% of CD33+ but not C D 3 3 - cells in the bone marrow of naturally infected CMV seropositive subjects. 28,29They were also found in CD33+ cells in peripheral blood of

114

healthy seropositive subjects but not in T cells, B cells, and C D 3 3 - mature granulocytes and macrophages. 3~ This population of myeloid lineagecommitted progenitor cells containing CLTs were defined further as cells coexpressing CD33 and CD15 or CD33 and CD14 along with the dendritic cell markers CDla and CD10. This cell type is similar to that described by Soderberg-Naucler et al. 24 Studies by Mocarski's group28,29 suggest that GM-P latently infected with CMV have a normal growth rate, cell surface phenotype, and differentiation state. Under standard culture conditions, uniform latency is maintained during hemopoiesis without spontaneous or differentiation-dependent production of infectious virus. However, CMV could be reactivated from these myeloid cells when they differentiated in the presence of human fibroblasts, fibroblast-conditioned medium, or medium supplemented with specific cytokines (interferon gamma, tumor necrosis factor, alpha-interleukin-4, or granulocyte-macrophage stimulating factor)? ~ This suggests that cellular differentiation pathways act as determinants of reactivation, and virus reactivation may be triggered by factors associated with the inflammatory response. Based on this recent evidence, the following model of CMV latency and reactivation has been proposed. 3~A primitive hematopoietic cell population, possibly a stem cell, is infected with CMV as a result of the viremia that follows primary infection. The viral genome is maintained in these cells with limited gene expression as they reside quiescently in the bone marrow or self-renew. As these cells differentiate into lineage-committed progenitors, latency-associated transcripts and proteins are expressed in a subset of CD33+ cells that are progenitors of dendritic cells as well as granulocytes and macrophages. If these cells differentiating along specific pathways are exposed to a particular proinflammatory cytokine milieu, reactivation of lyric virus occurs, and CMV replication ensues in fully differentiated tissue dendritic cells or macrophages. Although these data provide important new insights into the pathogenesis of CMV infection, many questions remain unanswered. It is not clear whether CD33 + progenitors of dendritic and myeloid lineage cells are the major or only sites of CMV latency in healthy seropositive subjects. It is also uncertain whether latently infected cells are susceptible to reactivation triggers only at specific

JUTTA K. PREIKSAITIS

differentiation stages or during very specific differentiation pathways. The mechanism by which specific cytokines or mixtures of cytokines mediate reactivation events requires further elucidation. DONOR FACTORS THAT INFLUENCE THE RISK OF TRANSFUSION-ACQUIRED CMV INFECTION

It is not known whether blood from all seropositive donors can transmit CMV or whether an infectious subset of donors can be identified. It is possible that donors who have acute primary CMV infection or reactivation of CMV may be more infectious because they have low-grade viremia not readily detectable by standard isolation techniques, or a significantly larger subset of their leukocytes are latently infected. CMV also may be more readily reactivated from the leukocytes of patients who have recently experienced primary or reactivation infection. Using viruria as a measure of recent CMV infection, in 1988 Lentz et aP 1 found that 0.6% of blood donors shed CMV, compared with a prevalence of 3% reported by Kane et a132 in t975. The presence of CMV-specific immunoglobulin M (IgM) has also been investigated as a potential marker of a more infectious subset of blood donors. The reported prevalence of CMV-IgM in blood donor populations is highly variable, 13% (indirect immunofluorescence assay--IFA), 31 6% (IFA), 33 4% (IFA), 34 4.4% (IFA), 35 3% (enzyme-labeled antigen assay--ELA), 3l and 0.9% (enzyme-linked immunosorbent assay--ELISA) 36 and dependent on the specific assay used. Lamberson et aP 3 reported a reduction in the incidence of transfusionacquired CMV infection in neonates, from 7 of 222 (3.2%) to 1 of 114 (0.8%) when CMV IgMnegative blood products were used. Although other investigators have also found an increased risk of transfusion-acquired CMV infection associated with the receipt of CMV IgM-positive units, 34,37 the sensitivity and specificity of this marker for predicting infectivity of the blood product is poor. 33,34,37 In a recent study in children with malignant disease, we were unable to detect an association between the receipt of CMV-IgM-positive blood products and transfusion-acquired CMV infection. 36 An alternate approach for identifying "infectious" blood products has used nucleic acid amplification for the detection of CMV DNA in blood donors. However, the results of polymerase chain reaction (PCR)-based detection are highly variable

THE CMV-"SAFE" BLOOD PRODUCT

115

(Table l). 19'38-44 Krajden et at 42 point out the analytical sampling errors that may occur when studying blood donor samples with low copy numbers of CMV DNA. This was confirmed by Larsson et al,43 who found that if tests were repeated over time, CMV DNA could be detected in all seropositive individuals. Investigators have clearly demonstrated the significant effects of sample preparation, amplification conditions, and detection methodology on the results obtained. Although the use of nested PCR is associated with enhanced sensitivity, these assays are technically difficult, prone to contamination, and poorly reproducible between laboratories. Of particular concern are the high rates of CMV DNA detected in CMVseronegative patients by some investigators. 19,38,39,43 It is possible that some of these results represent false-positive reactions related to technical issues in performance of the assays. These observations are inconsistent with clinical experience that would suggest that current commercial serological assays, particularly ELISA-based assays, are extremely sensitive in identifying the patient who is susceptible (seronegative) to primary CMV infection and capable of transmitting CMV infection (seropositive). This has been most extensively studied in the

setting of donor and recipient screening for solid organ transplantation. Nonetheless, serological tests may not identify all subjects previously exposed to CMV. Soderberg-Nancler showed that CMV could be reactivated after allogeneic stimulation of peripherai blood mononuclear cells from 1 of 6 subjects screened by ELISA and found to be seronegative. 24 CMV DNA was detected in this donor. Whether efforts were made to re-verify the serology results in this donor are uncertain. Further studies of this kind using additional seronegative subjects found to CMV DNA positive would resolve the controversy surrounding this issue. The lack of standardization of methodology and the poor reproducibility of PCR-based detection of CMV DNA prohibits its use as a method to detect an infectious subset of CMV-seropositive donors or as an alternative to serology for the provision of CMV-"safe" blood products. Direct transmission of infectious CMV from viremic blood donors experiencing subclinical acute CMV infection may account for some cases of transfusion-acquired CMV infection. However, the very high incidence of CMV infection that has been reported after exchange transfusion in neonates and granulocyte infusions in adults suggest that CMV is

Table 1. Detection of C M V DNA by PCR in Peripheral Blood Samples From Blood Donors

Amplification Technology Author

Serological Test Used

Extraction Method

Nested PCR

Genome Region(s) Amplified

Stanier et at 38

ELISA

Not given

No

MIE

Bevan et a139

LA

No

HXLF6, IE-1, gp 64

Smith et al 4~

ELISA, LA

No

MIE

Bitsch et a141

N/A

Phenol/chloroform Phenol/chloroform Salting out

No

MIE, pp 65

Krajden et a142

LA

No

MIE

Taylor-Wiedeman et a119 Larsson et a143

ELISA LA

Urishibara et a144 ELISA

ISO QIA AMPLICOR ISO Phenol/chloroform Salting out ISO (not modified)

No No Yes Yes

MIE

Yes

MIE pp 150

Yes

MIE

Product Detection

No. of Subjects in Whom CMV DNA Was DetectedNo. Subjects Tested (%) Seropositive

Seronegative

Hybridization (Southern blot) Size

20/20 (100)

Size/slot blot hybridization Hybridization (Southern Blot) DSSA DSSA DSSA Hybridization (Southern blot) Hybridization (Southern blot) Hybridization (Southern blot) Size

7/74 (10)

0/12 (0)

0/100 (0)

0/22 (0)

(25-79)*

8/101 (8) 0/101 (0) 1/101 (1) 17/50 (34) 6/6 (100) 60/145 (41.4) 0/155 (0)

5/10 (50) (19-35)*

0/13 0/13 0/13 1/10

(0) (0) (0) (0)

3/9 (33) 19/140 (13.6) 0/34 (0)

Abbreviations: ELISA, enzyme-linked immunosorbent assay; LA, latex agglutination; ISO, Isoquick nucleic acid extraction kit (modified); QIA, QIA amp blood kit; AMPLICOR, Amplicor whole blood specimen preparation kit; DSSA, Digene SHARP Signal System Assay. *No. patients tested in subset not clearly stated, prevalence of DNA detection varied with sex and age.

116

JU'I-I'A K. PREIKSAITIS

most often transmitted in a latent form in leukocytes contaminating blood products and is reactivated after transfusion in the recipient. If viable leukocytes are required for the transmission of CMV, the extent to which a blood component is contaminated by leukocytes would affect its infectivity. The approximate leukocyte content of various cellular blood components is summarized in Table 2. It is important to note that donor polymorphonuclear cells are predominantly found in red cell concentrates, and leukocytes in platelets are 80% to 85% small lymphocytes. Although the new generation of filters removes in excess of 3 lOgl0 of total leukocytes, in this process 4 log10 of total B, T cells, and monocytes, the likely site of CMV latency are removed. 45 ff the cells identified by Soderberg-Naucler et al24 and Hahn et a126 are confirmed to be the major site of CMV latency and reactivation, then enumeration of these cells in the peripheral blood of donors and in cellular blood products before and after leukoreduction may provide further insight into the relative infectivity of blood donors and components. A significant difference in the relative risk of platelet and red cell concentrates of comparable age with respect to CMV transmission has not been clinically appreciated. How much leukoreduction of a cellular blood product is required to eliminate the risk of CMV transmission? The American Association of Blood Banks (AABB) has suggested that residual leukocyte levels less than 5 • 10 6 make a blood product CMV-"safe. ''6 In its theoretical limit, the infusion of a single latently infected donor leukocyte may be sufficient to infect a susceptible recipient. However, in practical terms, because the pathogenesis of transfusion-acquired CMV infection involves a Table 2. Approximate Leukocyte Content of Various Cellular Blood Components

Whole blood Red cell concentrates Washed red cell concentrates Deglycerolized red cell concentrates Platelet concentrates (50 mL) Apheresis platelet concentrates (device dependent) Adapted from Dzik. 156

Unmodified

After ThirdGeneration Filter

106 5 x 106

<106-107 <106-107

106

<106

107-108 5 x 107

<106 106 per pool

106-106

<<106-<106

complex interaction between donor and recipient factors, leukoreduction to less than 5 • 10 6 leukocytes in a blood product may make the sequence of events required to reactivate CMV from latency in almost all transfusion recipients so improbable that the blood product could be considered CMV"safe." The term "safe" may be highly recipient dependent. A non-leukoreduced CMV-seropositive red cell concentrate may be "safe" when transfused into an asymptomatic CMV-seronegative recipient in the setting of uncomplicated elective orthopaedic surgery. In contrast, a platelet concentrate leukoreduced using a third-generation filter may transmit CMV after infusion into a bone marrow transplant recipient who is being aggressively treated for active graft-versus-host disease and has ongoing sepsis. CMV DNA is readily detected in the plasma of patients with acute CMV infection. 15 James et a146 detected infectious CMV in the plasma of red cell concentrate units from CMV-seropositive donors but only after 3 to 4 weeks of storage. Despite this, plasma components, particularly fresh frozen plasma, contain few viable leukocytes and do not appear to transmit CMV. 47 In fact, passive antibody administered in plasma from seropositive donors may protect the recipient from transfusion-acquired CMV infection. 34 Dworkin et al48 showed that storage of CMV viremic blood from patients with acquired immune deficiency syndrome (AIDS) under blood bank conditions was associated with a marked decline in culture positivity after 5 days of storage. The viability of the cells harboring latent CMV in stored blood components is unknown. Early investigators suggested that fresh whole blood was more infectious than stored citrated blood, 49 an observation more recently validated by others. 5~ Proinflammatory cytokines are produced in some leukocyte-contaminated red cell units and platelet concentrates and accumulate with time in storage) 3-56 As suggested by Hahn et al, 3~ these cytokines may be important triggers for both cellular differentiation and CMV reactivation events. This may account for the observation that infectious CMV could be detected in the plasma of red cell concentrates collected from CMV-seropositive blood donors after prolonged storage. 46 Prestorage leukoreduction significantly reduces the levels of these cytokines in stored cellular blood products. Prestorage leukoreduction may reduce the risk of

THE CMV-"SAFE" BLOOD PRODUCT

CMV transmission by blood products not only by reducing the number of latenfly infected cells infused but also by reducing the probability of initiating CMV reactivation events driven by cytokine release from donor leukocytes in a blood component before infusion. RECIPIENT FACTORS THAT INFLUENCE THE RISK OF TRANSFUSION ACQUIRED CMV INFECTION

Several observations support the hypothesis that recipient factors are extremely important in determining the risk of transfusion-acquired CMV infection. In our study of CMV-seronegative transfusion recipients, we observed that patients who had received more than 30 cellular blood products had a significantly higher risk per unit transfused than those who had received fewer blood productsS In the setting of solid organ transplantation, we and others 58 found no association between the risk of transfusion-acquired CMV infection and the number of blood products received by seronegative recipients of seronegative donor organs receiving unscreened and unfiltered blood components. According to the model of CMV reactivation, to produce lytic virus, latently infected cells must be induced to differentiate along specific pathways and must be exposed to a proinflammatory cytokine melieu. 31It is not clear whether the cytokine trigger for reactivation can be transient or must persist throughout the reactivation process. Extrapolating from this model, transfusion recipient populations with high systemic levels of these cytokines, such as those patients with prolonged sepsis, burns, major trauma, or active autoinunune disorders, may be particularly vulnerable to triggering CMV reactivation from the blood products they receive. The transfusion of allogeneic leukocytes constitutes a profound immunologic stimulus to the recipient. 59,6~ This in vivo analog of a mixed lymphocyte reaction can have clinical consequences (alloimmunization or graft-versus-host disease) but is most often subclinical. The allogeneic stimulus has long been suspected to play a major role in the pathogenesis of transfusion-acquired CMV disease, but the results of studies in animal models were inconclusive. 6 However, the recent study of Soderberg-Naucler et al24 dramatically illustrates the efficiency by which an allogeneic stimulus both induces appropriate differentiation in mononuclear cells latently infected with CMV and

117

provides a trigger for CMV reactivation. These events are not aborted by irradiation of blood products to prevent graft-versus-host disease, because CMV transmission clearly occurs in this setting. Allogeneic reactions after blood transfusions are dependent on many factors, including the dose of leukocytes received, HLA matching between donor and recipient, and the immune status of the recipient. 6~ Leukoreduction of blood products may be influencing the incidence of CMV transmission indirectly by reducing the probability of an allogeneic reaction. CMV reactivation from latency is a slow process, as evidenced by both in vitro data and clinical experience. In experiments in which CMV was reactivated from latency in human cells, investigators in 2 studies reported detection of infectious CMV only after 16 to 21 days 3~ and 27 to 61 days 24 of co-culture. The first 21 to 28 days after solid organ transplantation is considered a "honeymoon period," during which infectious lyric CMV reactivated from the donor organ or transplant recipient is not detected. Gilbert et a161 observed an incubation period of approximately 22 and 33 days in 2 neonates with transfusion-acquired CMV infection exposed to a single seropositive blood donor. This suggests that for transfusion-acquired CMV to occur, a state of at least transient microchimerism must occur in the transfusion recipient, with donor cells persisting in the recipient long enough to allow production of infectious CMV. Using a sensitive PCR-based technique, Adams et a162 w e r e able to detect circulating donor white cells in 15 to 20 women for a mean of 2.0 days (range, 1 to 6 days) after their last transfusion. Similarly, Lee et al63 observed that in 6 of 8 female surgery patients transfused with relatively fresh male blood components, donor cells peaked at day 3 or 5, followed by clearance by day 14.63 This short survival time of donor leukocytes may help explain the low risk of transfusion-acquired CMV infection observed in immunocompetent populations. Donor leukocytes have been shown to persist for prolonged periods in neonates, 64 particularly in those who have received transfusions in utero, a factor that may contribute to the high incidence of CMV infection observed in this population. Surprisingly, multiply transfused trauma victims have evidence of donor cell chimerism for up to 1.5 years after receipt of relatively fresh blood without evidence of graft-versus-host disease. 63 Several

118

case series of transfusion-acquired CMV disease have been described in this population. 65-67 Adams et a162 also observed that leukocyte survival was associated with the receipt of large quantities of blood products. Although this may have been related to an increased likelihood of receiving blood with shared HLA antigens, it also may be the result of tolerance induced by sequential transfusion. Pretransplantation blood transfusions are known to promote allograft survival. 6~In the patient transfused repetitively over time, similar immune mechanisms may be allowing longer survival of transfused cells, thereby permitting reactivation of CMV. Chou and Kim 68 found evidence for this effect. They observed that the incidence of transfusion-acquired CMV infection was significantly higher among potential renal transplant recipients who received 2 donor-specific CMVseropositive specific leukocyte transfusions, sepa' rated by a period of 2 weeks compared with patients who received a single infusion. This study also illustrated the importance of HLA matching because donor-specific leukocytes (HLA matched) were associated with a significantly higher incidence of CMV transmission than leukocyte transfusions fi'om random donors. The importance of immunosuppression was highlighted by Suassuna et al,69 who documented an increase in the incidence CMV infection when donor-specific transfusions were given to patients awaiting renal transplantation under azathioprine coverage compared with those who did not receive azathioprine. Based on these observations, several hypotheses can be formulated. The risk of transfusion-acquired CMV infection may be greater in patients who are sequentially transfused over a long period compared with those who receive a comparable amount of blood on a single occasion. The risk also may be increased when HLA-matched donors are used, such as in directed donations from parent to child or when single-donor platelets are used. This risk would be further exacerbated when repetitive transfusions from the same donor are administered, particularly when the recipient is immunosuppressed. The potential prolongation of leukocyte survival in HLA-matched donor recipient pairs must be balanced against the reduced probability of an allogeneic reaction in the setting when assessing the impact of HLA matching on the risk of transfusion-acquired CMV infection. The pathogenesis of transfusion-acquired CMV

JUTTA K. PREIKSAITIS

infection is complex and involves multiple donor and recipient factors. This makes it very difficult to assign a specific risk per CMV-seropositive unit transfused or to definitively extrapolate the efficacy of intervention strategies to reduce the risk of transfusion-acquired CMV infection from one recipient population to another. CLINICAL DATA SUPPORTING STRATEGIES USED TO PROVIDE CMV-SAFE BLOOD PRODUCTS

A large number of studies have been performed evaluating strategies for the prevention of transfusion-acquired CMV infection (Tables 3 through 5). However, many studies were not randomized, failed to include a control group, or used historical controls. Changing transfusion practices over time and geographic variability in risk even in welldefined populations, such as transfused neonates, makes interpretation of these results difficult. Historically, CMV-seronegative cellular blood products have been used extensively for the prevention of transfusion-acquired CMV infection in high-risk populations (Table 3). In prospective, randomized controlled trials, the efficacy of this approach has been validated in both neonates 7~and bone marrow transplant recipients. 71,72However, in bone marrow transplant recipients, "breakthrough" CMV infections have been documented in 1.3% to 4.4% of patients receiving CMV-seronegative blood products. 8,71,72Latex agglutination (LA) assays are often used for the detection of CMV antibody in the blood bank setting because of their simplicity and the short time required for testing. However, other commercially available CMV antibody assays such as ELISA assays are significantly more sensitive than LA assays and have the advantage of not being subjective in the interpretation of results. We, 36 like others,73 have ob served that 1.1% to 1.3 % of blood donors who were seronegative when tested by LA were found to be seropositive when tested by ELISA. False-negative laboratory screening results of both blood donors and recipients may account for much of the "breakthrough" CMV infection observed in recipients of CMV-seronegative blood products. 74 It is not clear why the same high standards with respect to the sensitivity of antibody screening tests for viruses such as HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) are not applied to CMV antibody testing in the blood system. However, it is possible that a very small

THE CMV-'SAFE" BLOOD PRODUCT

119

Table 3. Cytomegalovirus (CMV) infection in CMV-Seronegative Transfusion Recipients Receiving CMV-Seronegative Blood Products No. Patients Infected No. Studied (%) Population Studied Neonates (exchange transfused)

Neonates Bone marrow transplant recipients (seronegative marrow donors)

Heart transplant recipients Pediatric oncology patients

Author (Year)

Trial Design

Screening Test Used

CMV Seronegative Blood

Standard Blood

Luthardt et al (1971) 1~7

Retrospective analysis

CF

0/20 (0)

8/15 (53.3)

Kumar et al (1980) ls8 Yeager et al (1981 )70 Bowden et al (1986) 71 Miller et a[ (1991 )72 MacKinnon et al (1988) 118 Bowden et al (1995) 8 Preiksaitis et al (1983) 139 Preiksaitis et al (1997) 36

Retrospective analysis Prospective randomized Prospective randomized Prospective randomized Uncontrolled Prospective randomized

CF

1/7 (14.3)

1/3 (33)

IHA

0/90 (0)

Historical cohort control Prospective randomized

LA

1/32 (3.1)

10/74 (13.5) 8/25 (32)

IHA and LA

2/45 (4.4)

14/44 (31.8)

ACiF LA

0/16 (0) 2/252 (1.3)*

3/250 (2.4)*

IHA

0/8 (0)

1/5 (20)

LA

0/30 (0)

0/32 (0)

Abbreviations: CF, complement fixation; IHA, indirect hemagglutination; LA, latex agglutination; ACIF, anti-complement immunofluorescence. *Control group received filtered blood; actuarial probability is in parentheses.

subset of blood donors previously exposed to CMV will lack CMV antibody and would not be identified by any antibody screening test. Early strategies for attempting to prevent CMV transmission by leukodepletion of blood products employed saline-washed or frozen and thawed (frozen and deglycerolized) red cells, which removed approximately 90% and 94% to 99% of white cells, respectively (Table 4). Although the incidence of CMV infection in the studies using

these strategies is lower than might be expected in patients receiving standard blood products, many of these studies were uncontrolled 37,75-77 or used historical controls. 7s,79 The true efficacy of these approaches is difficult to determine because in most of these studies some CMV-infected infants received random, untreated platelet units. 37,75-77These strategies are time-consuming and therefore costly and can only be applied to red cell components. In 1977, Lang et al 8~was the first to observe that

Table 4. Cytomegalovirus (CMV) Infection in CMV Seronegative Transfusion Recipients Receiving Washed or Frozen Deglycerolized (FDRC) or Washed Red Cell Concentrates No. of Patients Infected No. Patients Studied (%) Population Studied

Author (Year)

Leukoreduction Method

Leukocyte Reduced

Standard Blood

Dialysis patients Neonates

Tolkoff-Rubin et al (1978) 78 Taylor et al (1986) 75 Brady et al (1984) 76 Simon et al (1987) 79 Luban et al (1987) 77 Demmler et al (1986) 37

FDRC FDRC FDRC FDRC Washed Washed

0/21 (0) 2/24 (8.3)10/106 (0)w 1/26 (3.8) 1/100 (1.0)~ 6/54 (11.1)11

3/3 (100)* --2/16 (12.5)* ---

*Historical cohort control group. 1"Seven infants received unscreened whole blood • platelets; 2 of these infants developed CMV infection. SThirty-seven infants received non-leukoreduced platelets or plasma. w infants received non-leukoreduced platelets. ]lSome infected infants received non-leukoreduced platelets.

120

JU'VI'A K. PREIKSAITIS

Table 5. Cytomegalovirus (CMV) Infection in CMV Seronegative Transfusion Recipients Receiving Centrifuged or Filtered Blood Products WBC Reduction Method Population Studied Neonates

Leukemia/lymphoma patients

Bone marrow transplant recipients$

Cardiac surgery patients

No. of Patients Infected No. Studied (%)

RBCs (Log Reduction)

Platelets (Log Reduction)

Gilbert et al (1989) 61 EinsenfNd et al (1992) ~59 Murphy et al (1988)82t DeGraan-Hentzen et al (1989) 88 Verdonck and deGraan-Hentzen (1987 )84 De Witte et al (1990) 85 Bowden et al (1991)sl

Filtered (2) Spin-cool-filtered (1-2) or filtered (2) Filtered (2-3)

N/A Non-teukoreduction*

0/42 (0%) 0/48 (0%)

9/59 (15.3%) --

Centrifuged (1-2)

0/11 (0%)

2/9 (22.2%)

Filtered (3)

Centrifuged (1-2)

0/59 (0%)

--

Filtered (3)

CMV seronegative

0/11 (0%) 0/18 (0%)w

---

Filtered (2-3) CMV seronegative

Centrifuged (I-2) Centrifuged (1-2)

Van Prooijen (1994) 86

Filtered (3)

Filtered (3)

Bowden et al (1995Plll Lang et al (1977) 80

Filtered (3)

Filtered (3)

0/28 (0%) 0/3 (0%) 0/32 (0%)w 0/16 (0%) 0/37 (0%)w 3/250 (2.4%)

2/252 (1.3%)

2/8 (25.0%)

4/6 (66.7%)t

Author

Centrifugation 82(<1)

Leukoreduced Blood

Standard Blood

-1/2 (50%) 6/28 (21.4%)w --

*Two infants received platelet concentrates. l"Nonrandomized trial. tUnless otherwise specified refers to allogeneic transplant recipients; only those receiving seronegative donor marrow included. w transplant recipients. IIIn this study, both allogeneic and autologous transplant recipients were included; the control group received CMV seronegative, not standard blood and the % actuarial probability is in parentheses. 82 whole blood was used.

centrifugation of fresh whole blood removed approximately 58% of teukocytes and resulted in a decrease in the incidence of CMV infection after cardiac surgery (Table 5). Centrifugation has also been used to achieve a 1- to 2-log reduction in leukocytes in platelet units. The use of platelets processed in this way along with CMV-seronegative red cells resulted in the reduction of transfusionacquired CMV from 23.4% to 0% when compared with standard blood products in a randomized controlled trial of bone marrow transplant recipients, most of whom received autologous marrows. sl Microaggregate filters result in a 1- to 2-log reduction in leukocytes in RBC units. Gilbert et al61 reported the elimination of transfusion-acquired CMV infection in a prospective randomized controlled trial of filtered RBCs compared with 15.3% incidence of infection in neonates receiving standard RBC units. Subsequent uncontrolled trials using filtered RBCs in combination with either centrifuged or CMV-seronegative platelet units in relatively small numbers of patients with leukemia/

lymphoma and bone marrow transplants failed to show any evidence of breakthrough CMV infection.g2-86 Currently available filters, designed for use with either RBC or platelet components, are even more efficient, achieving a 3- to 4-log reduction in leukocytes. The question that then arose was whether leukoreduction of RBCs and platelets using these filters was equivalent to the use of CMV-seronegative blood products with respect to the prevention of CMV transmission by blood products. To address this issue, a multicenter prospective randomized controlled trial comparing these 2 strategies was performed in a study involving 502 CMV-seronegative bone marrow transplant recipients. 8 The 2 study groups were comparable, with the exception that the group receiving filtered blood had a higher percentage of single-family donors. Based on this difference, one might have predicted that there would be an increased risk of transfusion-acquired infection in the group receiving filtered blood. However, the incidence of both CMV infection and CMV disease between days 21

THE CMV-"SAFE" BLOOD PRODUCT

and 100 after transplantation observed in patients receiving CMV-seronegative blood products, 1.3% and 0%, was not significantly different from that observed in patients receiving filtered blood products: 2.4% and 1:2%. The authors of the study concluded that the 2 strategies were comparable. However, protocol errors in the study, the methods used to analyze the data, and the conclusions reached have engendered significant controversy.6,9 The authors did not use an intent-to-treat analysis. Instead, they performed primary (days 21 to 100) and secondary (days 0 to 100) analyses, using the assumption that any infection occurring in the first 21 days is the result of CMV exposure before the intervention. Although our understanding of the pathogenesis of transfusion-acquired CMV disease suggests that this assumption is valid, 5 patients (2 CMV-seronegative arm, 3 filtered arm) developed CMV infection between days 0 and 21. Four of these 5 patients had equivalent or discrepant serological results at the time of study entry and may well have been seropositive rather than seronegative. Of particular concern was the observation that between days 0 and 100, none of the patients who received seronegative blood developed CMV disease, compared with 6 in the group receiving filtered blood. Although overall survival was not significantly different, 5 of the 6 CMV-infected patients in the filtered arm of the study developed CMV pneumonia. The concern was raised that the use of filtered blood products may be associated with an increased risk of CMV pneumonitis and its attendant mortality. 9 The investigators subsequently reported that after day 100, 2 patients in the seronegative arm of the study also developed CMV disease. This makes the increased CMV-associated morbidity in the group receiving filtered blood less dramatic, but the overall observation remains unexplained. In both arms of this study, the technology used for the provision of CMV-"safe" blood was not optimized. In the CMV-seronegative arm, the less sensitive latex agglutination assay was used, and beside filtration was used in the filtered arm of the study. Not only would filtration in the laboratory be associated with better quality control with respect to leukoreduction, there is evidence to suggest that slow filtration at room temperature as occurs in a bedside setting results in suboptimal leukocyte removal. 87 Despite the controversy, the author believes that

121

the conclusion of the investigators involved in this study is correct. Blood products leukoreduced by filtration are comparable to seronegative blood components with respect to the risk of CMV transmission to bone marrow transplant recipients. Future prospective surveillance in this population would further validate this conclusion. Some preliminary data in this regard are provided by Pamphilon et al, 88 who found no evidence of CMV infection in 62 CMV-seronegative recipients of CMV-seronegative marrow allografts receiving CMV-seronegative RBCs and leukoreduced platelets. The Immunocompetent Patient Studies of transfusion-acquired CMV infection performed during the first decade after the initial descriptions of CMV-induced postperfusion syndrome documented a high incidence of infection, with 16% to 67% of patients developing infection.7 However, the interpretation of the results of some of these studies was complicated by the inclusion of allograft recipients and patients who were CMV seropositive before transfusion. As transfusion practice changed from the use of fresh whole blood to the use of component therapy in the 1970s and 1980s, the epidemiology of transfusion-acquired CMV infection appears to have changed. In the 1980s 2 studies examined the incidence of transfusion-acquired CMV infection in a large number of seronegative transfusion recipients and found it to be extremely low.5~ In a study by Wilhelm et al, 5~ only 7 of 595 transfused seronegative patients (1.2%) developed CMV infection. Of note, 6 of these infected patients had received fresh whole blood. We documented CMV infection in only 6 (0.9%) of 637 transfusion recipients not receiving exogenous immunosuppression. 57 In these studies, the highly transfused patient is at greatest risk of transfusion-acquired CMV. Fifty percent of the infections we observed occurred in patients who had sustained major trauma or burns. Patients who are repetitively transfused in the setting of a pro-inflammatory cytokine environment induced by trauma or sepsis may represent a particularly vulnerable subset of patients for the acquisition of CMV infection from blood products. Prolonged donor leukocyte survival has been described in this setting. 63 This may explain the number of cases of CMV infection that have been described in patients with burns or other

122

major trauma. A high incidence of CMV infection has also been observed in the highly transfused splenectomized patient. 65-67 Whether splenectomy prolongs donor leukocyte survival, thereby potentiating the risk of transfusion-acquired CMV infection, remains uncertain. Primary CMV infection in the immunocompetent patient, when symptomatic, is usually mild and self-limited. CMV-"safe" blood products are not required in immunocompetent patients not receiving exogenous immunosuppression because of the low incidence of transfusion-acquired CMV infection and mild disease in this population. Moreover, the diversion of CMV-seropositive blood products to this population as a result of providing CMVseronegative products for higher-risk populations is unlikely to have a significant impact.

The CMV-Seropositive Patient Outside of the pediatric population, most transfusion recipients are CMV seropositive before transfusion. CMV isolates have significant antigenic variability. Infections with second strains of CMV have been documented in immunocompetent individuals, but morbidity in this setting has not been described. 89 In the transplant setting, reinfection of CMV-seropositive patients with second strains of CMV transmitted from the seropositive donor organ occurs frequently, and these patients suffer significantly greater morbidity than those who simply reactivate endogenous virus (recipients of seronegative donor organs). 9~ The ongoing allogeneic stimulus of the donor organ may account for the preferential reactivation of CMV from this site. Although there is a theoretic risk that CMV seropositive patients may be reinfected by a second strain of CMV transmitted by the blood transfusions, the frequency with which this occurs and its clinical consequences remain speculative. The provision of CMV-"safe" blood to prevent CMV reinfection events even in immunosuppressed CMV-seropositive patients cannot be justified. In addition, there is a concern that allogeneic leukocytes in blood products can result in the reactivation of CMV in the seropositive transfusion recipient. Busch and Lee 93 observed that allogeneic donor leukocytes markedly upregulated HIV-1 expression and dissemination in infected peripheral blood mononuclear cells ex vivo. It has been postulated that donor leukocytes may have a similar effect on latent CMV infection. 2,93 Although

JUTTA K, PREIKSAITIS

data from animal studies have provided conflicting results, Adler and McVoy94 found CMV reactivation rates measured by serological responses of 11% to 14% and 19% to 23% in transfused seropositive cardiac surgery and oncology patients, respectively. This appears to represent CMV reactivation rather than reinfection, because rates were not significantly different in patients who received CMV-seropositive blood when compared with those receiving CMV-seronegative blood products. In a recent report, evidence for active CMV infection, diagnosed using sensitive techniques of CMV antigenemia and CMV DNAemia, was found in 32.4% of septic patients in an intensive care unit. 95 Although the transfusion history of these patients was not reported in this study, most of these patients had conditions that would almost certainly have resulted in the receipt of blood products. CMV reactivation appears to occur relatively frequently in the sick seropositive patient. The relative importance of donor leukocytes in promoting reactivation events is uncertain. Although there were no direct clinical sequelae-associated reactivation in the patients studied by Adler and M c g o y , 94 the indirect effects of CMV infection were not measured. CMV has significant effects on the immune system, 96 and it has been postulated that the immunomodulatory effects of blood transfusion may be attributable to the reactivation of latent viruses such as CMV. 2,59 Whether CMV reactivation induced by allogeneic donor leukocytes results in an increased risk of events such as superinfection with bacterial or other pathogens and cancer recurrence remains speculative and deserves further study.

Pregnant Women Forty percent of women experiencing primary CMV infection during pregnancy give birth to infants with congenital CMV infection, who may develop significant handicaps such as deafness and neurological problems. 9v Although the consequences of acquiring a CMV infection during pregnancy may be serious, the risk of acquiring CMV from blood transfusions during pregnancy is unknown. We were unable to document a single case of transfusion-acqnired CMV infection in 162 seronegative pregnant women who received a total of 450 cellular blood products. 57 However, most transfusions were given in the postpartum period. Only 8 women had received antepartum transfusions. The risk of acquiring CMV infection from

THE CMV-"SAFE" BLOOD PRODUCT

123

blood transfusions during pregnancy may be low, as has been observed in other immunocompetent populations. However, until further information is available, it would be prudent to provide screened cellular blood products to the small group of seronegative women requiring blood transfusions antenatally or intrauterine blood transfusions. It also may be prudent to provide CMV-safe components for intrauterine transfusions administered in the second trimester even to women who are CMV-seropositive. IgG levels of preterm infants (and therefore fetuses as well) are directly proportional to gestational age and might be too low to protect the fetus from transfusion-acquired CMV in the second trimester. Neonates

In the mid-1970s, a high incidence of cytomegaloviruria was observed in exchange-transfused infants and transfused preterm infants 98,99in neonatal intensive care units. Full-term neonates born to CMV-seropositive mothers who acquire CMV infection after birth, as a result of exposure to infected genital secretions or breast milk, are usually asymptomatic and do not suffer the long-term consequences of CMV infection that have been observed in infants infected in utero. 97 However, a selflimited syndrome consisting of respiratory deterioration, hepatosplenomegaly, gray pallor, and lymphocytosis was described in association with CMV infection in preterm infants, l~ CMV infection was thought to have contributed to the death of some of these infants. Blood transfusion was implicated as

the source of infection and the cause of morbidity in these patients. This resulted in a number of studies that examined the incidence of transfusion-acquired CMV infection and evaluated programs of intervention. 33'61't01"108 The results of these studies are summarized in Table 6. Several conclusions can be drawn from these reports. In most of these studies, CMV morbidity was greatest in low-birth-weight infants who had been born to CMV-seronegative mothers. Blood transfusion was the most likely source of CMV infection in these infants. The contribution of CMV infection to symptoms in sick premature neonates who often have multisystem dysfunction is difficult to evaluate. In some studies, particularly those documenting a high incidence of transfusion-acquired CMV infection, not only did a significant proportion of these patients experience CMV-associated morbidity, CMV infection was believed to have contributed to the death of some of these infants. 33,7~ Risk factors for acquiring infection included low birth weight, 33 .61 ,70 ,101 a large number of donor exposures, 61,1~176 receipt of a disproportionately large number of CMV-seropositire units, and receipt of at least 50 mL blood. 7~ However, neither Lamberson et aP 3 nor Tegtmeier 1~ found any significant difference in the number of donor exposures between infected and uninfected infants in their studies. In 2 studies, 4 low-birth-weight infants acquired CMV infection after a single exposure to a relatively small volume of blood from a single seropositive donor. 33,61 Exposure to a large volume of seropositive blood

Table 6. Prospective Studies of the Incidence of Transfusion-Acquired Cytomegalovirus Infection in Neonates Born to Seronegative Mothers Receiving Standard Blood Products

Source (yr)

CMV Seroprevalence in Blood Donors (%)

No. of Patients Infected/No. of Patients Studied (%)

No. of Patients Infected/No. Low Birth Weight Neonates Studied (%)

Weight (g)*

Yeager et al [1981] 70 Adler et al [1983] TM Smith et al [Abstract, 1983] l~ Rawls et al [1984] l~ Tegtmeier [abstract, 1985] TM Lamberson et at [1988] 33 Preiksaitis et al [1988] l~ Griffin et al [1988] l~ Gilbert et al [1989] 6~ Galea and Urbariak [1992] l~ Snydman et al ]1995] lo8

45.3 34.0 Not given 73.3 Not given 38 42.5 47,0 46.0 Not given Not given

10/74 (13.5) 7/76 (9.2) -1/23 (4.3) -7/222 (3.2) 1/126 (0.8) 0/33 {0.0} 9/42 (21.4) ---

7/22 (31,8) 7/29 (24,0) 2/29 (6.9) -2/23 (8.7) 4/83 (4.8) 1/41 (2.4) 0/14 (0.0) 9/29 (31.0) 2/46 (4.4) 5/98 (5.1)

<1,200 <1,250 <1,500 -<1,250 <1,500 <1,250 <1,250 <1,500 <1,500 <1,500

NOTE. Serostatus of 1 patient was unknown. * L o w birth weight as defined b y t h e authors.

124

and multiple seropositive donors does not appear to be necessary for the transmission of CMV infection to neonates. The incidence of transfusion-acquired CMV infection in low-birth-weight (<1,200 to 1,500 g) infants born to seronegative mothers was similar in 3 studies at 31.8%, 70 24.0%, 1~ and 31.0%. 61 However, a number of other recent studies have found significantly lower rates of transfusion-acquired CMV infection in their neonatal population, with the incidence of CMV infection in low-birthweight infants born to CMV-seronegative mothers varying from 0% to 8%. 33'101"108 A recent randomized placebo controlled double blind trial of CMV immunoglobulin for CMV-associated disease in low-birth-weight, premature neonates was terminated prematurely because of the low incidence of CMV infection observed.l~ Only 5.1% of low-birthweight infants born to CMV-seronegative mothers 'developed CMV infection. The reasons for the geographic variability in the risk of transfusion-acquired CMV infections in neonatal populations are uncertain. The disparate results cannot be attributed to differences in donor antibody seroprevalence, the number of donor exposures, or the age of transfused blood. Details regarding the use of plasma and platelet products are not clearly documented in many of the published studies. It is possible that the use of plasma in some nurseries may be reducing the risk of transfusion-acquired CMV infection through the passive administration of CMV antibody, as has been suggested by Beneke et a134 in adult patients. Although the relative risk of platelet units versus red cell units is unknown, differences in policies regarding the use of platelets in neonatal nurseries may be altering risk. Although often not stated, it is assumed that the neonates in the studies reported did not receive granulocyte transfusions that would significantly increase the risk of transfusionacquired CMV infection. Detailed comparison of blood product use in centers with high and low incidences of infection would eliminate some of these possibilities. The method of blood distribution in nurseries also may influence leukocyte survival after blood transfusion in the recipient and contribute to the geographic variability in risk observed. When a syringe-and-aliquot technique is used in the nursery, the infant may be repetitively exposed to blood from the same donor. If the donor is seropositive,

JUTTA K. PREIKSAITIS

not only would the risk of exposure to a cell latently infected with CMV be increased, the survival of white cells from second or subsequent transfusions from that donor may be prolonged. Repetitive transfusions from random donors also may have this effect. Immunotolerance induced by repetitive transfusions may be a more relevant factor in the immunologically more mature full-term neonate. Very-low-birth-weight infants (<1,250 g) usually have a gestational age of less than 29 weeks. These infants are immunologically immature with respect to both humoral and cell-mediated immune responses. Circulating donor lymphocytes persisted for very short periods and were never seen beyond 6 to 8 weeks after transfusion in term and near-term neonates receiving exchange transfusions of banked blood but were observed for over 2 years in some infants who had received fetal maternal transfusions. 64 The very-low-birth-weight infant may closely resemble a fetally transfused infant even though random donor cells may not survive as long as more antigenically similar maternal cells. Prolonged survival of donor cells in these infants may significantly increase their risk of acquiring CMV infection. Transfusion-acquired graft-versus-host disease has been described in the exchange-transfused and premature infant. 1~176 This diagnosis may be difficult to make in the sick premature infant. 111,112 Allogeneic reactions are believed to be important in the reactivation of CMV from blood products. The variable incidence of transfusion-acquired CMV infection observed in nurseries may be an indirect measure of the incidence of unrecognized or subclinical graft-versus-host disease in these transfused infants. Although the incidence of infection is highly variable, the low-birth-weight neonate born to a CMV-seronegative mother is at highest risk of CMV morbidity as a result of transfusion-acquired infection. This population is most likely to benefit from the provision of CMV-"safe" blood products. Should low-birth-weight infants born to CMVseropositive mothers also receive these products? Prospective studies suggest that CMV infection rates are highest in infants born to CMV-seropositive mothers. 33,7~176176176 However, sources other than blood products such as exposure to breast milk and infected genital secretions appear to be the most important sources of CMV infection in this setting. In 370,101,107 of the 5 studies 33,7~176176176in

THE CMV-'SAFE" BLOOD PRODUCT

which infants born to seropositive mothers were also evaluated, no significant morbidity associated with CMV infection was observed in this subgroup of patients. However, clinical deterioration has been documented in some infants born to seropositive mothers concomitant with the onset of CMV shedding by others. 33,1~ Snydman et al, 1~ who observed a low incidence of CMV infection in low-birth-weight infants born to seronegative mothers, found that 67% of the CMV-associated disease in his study occurred in tow-birth-weight infants born to seropositive mothers. Whether the use of CMV-safe blood products would have any impact on this morbidity is not clear. Yaeger et al's study 7~ suggests that use of seronegative red cells will not influence the incidence of CMV infection in these infants, but in their study no CMV morbidity was observed in this subgroup of infants. They suggested that the presence of passive CMV antibody protected these infants against CMV-associated morbidity. The low-birth-weight neonate of low gestational age often has very little maternal antibody, and these antibodies can be rapidly diluted depleted by venipuncture and the dilutional effects of blood transfusion. Yeager et al H5 suggested that the loss of maternal CMV antibody may be hastened by the dilutional effect of administration of seronegative blood products to low-birth-weight neonates born to seropositive mothers. This may result in earlier virus shedding and a subsequent increased risk of symptomatic CMV infection when the infant is exposed to infected genital secretions or breast milk. Given these concerns, the use of leukoreduced blood products from random blood donors may be a preferable strategy when compared with the use of seronegative blood products for the provision of CMV-"safe" blood products in this subgroup of neonates. The former strategy would result in the transfer of passive antibody. If CMV-"safe" blood products are provided for low-birth-weight infants born to seropositive mothers, it is also important to ensure that all maternal and banked breast milk given to these neonates is pasteurized to prevent the transmission of CMV from this source to these vulnerable babies. 116 Would infants of higher birth weight ( > 1,500 g), particularly those born to CMV-seronegative mothers, receive benefit from CMV-"safe" blood products ? Transfusion-acquired CMV infection has been described in mature full-term neonates, but these

125

infants were asymptomatic. 33 Extracorporeal membrane oxygenation (ECMO) has been used in neonatal nurseries for the treatment of some term or near-term infants. These infants sometimes have significant transfusion requirements. A death caused by transfusion-acquired CMV infection in a fullterm infant treated with ECMO has been reported. 117 The birth weight of this child would not have placed him in a high-risk category for symptomatic transfusion-acquired CMV infection. Repetitive blood transfusions and the cytokine milieu induced by the ECMO procedure itself may promote reactivation of latent CMV from blood products in this setting. No serious dr fatal cases of transfusion-acquired CMV infection have been reported in term neonates undergoing exchange transfusion or open heart surgery. Although it is difficult to make recommendations based on a single case, physicians may wish to consider providing CMV-safe components in for term or near-term neonates born to both seronegative and seropositive mothers undergoing massive transfusion (exchange transfusion, ECMO, open heart surgery). Neonates represent a high-risk population for transfusion-acquired CMV infection. Although the low-birth-weight infant born to a seronegative mother is likely to benefit most from the receipt of CMV-"safe" products, arguments can be made that attempts should be made to prevent all cases of transfusion-acquired CMV infection, even if relatively rare in this population. The proven indications for the use of CMV-"safe" blood products must be balanced against the logistics of providing these products to subgroups of infants in the nursery, which would include the serological screening of infants and the maintenance of multiple blood inventories. Priorities for the distribution of these products are outlined in Table 7. In many centers, CMV-"safe" blood is given to all neonates or low-birth-weight neonates without regard to their serostatus. From a practical point of view, this may be the optimal solution. Indications for the use of granulocyte transfusions in the rieonate are infrequent and controversial.118 The incidence of transfusion-acquired CMV infection associated with granulocyte transfusions in the neonate is unknown. Extrapolation of data from bone marrow transplant recipients H,le suggests that the risk of transmission of CMV infection is high, and passive antibody is less likely to protect against CMV-associated morbidity in this setting. It

126

JU-I-I'A K. PREIKSAITIS Table 7. Indications for the Use of CMV-"Safe" Cellular Blood Products

Category A: (Populations in whom the use of CMV-"safe" cellular blood products have been proven to reduce the incidence and morbidity of CMV infection using controlled trials) 9 Low birthweight infants born to seronegative mothers 9 Seronegative recipients of seronegative donor bone marrow (allogeneic) 9 Seronegative recipients of autologous bone marrowtransplants Category B: (Populations at high risk of significant morbidity as the result of transfusion-acquired CMV infection, but the incidence of transfusion-acquired CMV infection in these populations has not been clearly documented or the benefit of using CMV-"safe" cellular blood products has not been proven) 9 Seronegative pregnant women requiring antepartum transfusion or intrauterine blood transfusions and seropositive women requiring intrauterine blood transfusions in the second trimester 9 Low birthweight infants born to seronegative or seropositive mothers or other seronegative immunosuppressed patients requiring granulocyte transfusions 9 Seronegative recipients of seronegative donor lungs and livers and possibly other organs excluding heart and kidney recipients 9 Seronegative HIV-infected and AIDS patients and children born to HIV-infected mothers Category C: (Populations who may be at higher risk of transfusion-acquired CMV infection or its morbidity, but in whom the incidence or morbidity of transfusion-acquired CMV infection is low or poorly documented) 9 Low birthweight infants born to seropositive mothers 9 Infants with birthweights >1,500 g born to seronegative mothers 9 Neonates receiving ECMO (extracorporeal membrane oxygenation) and other neonates requiring extensive transfusion support (ie, exchange transfusion, cardiovascular surgery) 9 Seronegative recipients of seronegative donor kidneys and hearts 9 Seronegative patients with malignant disease receiving chemotherapy 9 Seronegative patients with hematologic or genetic disorders requiring repetitive transfusions in whom bone marrow transplantation may be a future therapeutic option 9 Seronegative patients experiencing major trauma or splenectomy Category D: (Populations in which the incidence and morbidity associated with transfusion-acquired CMV infection is low, the use of CMV-"safe" cellular blood products is not indicated) 9 Infants with birthweights >1,500 g born to seropositive mothers 9 Other seronegative immunocompetent patients 9 Seropositivetransfusion recipients (excluding neonates)

therefore would appear prudent that CMV-seronegative granulocyte transfusions be provided for both seronegative and seropositive sick premature neonates.

Allograft Recipients: Bone Marrow Cytomegalovirus remains a significant cause of morbidity and mortality after allogeneic bone marrow transplantation despite recent improvements in treatment. 8 Among patients who are seropositive for CMV antibody before transplantation, there is a high incidence of CMV infection (69%) regardless of the serostatus of the donor marrow, presumably because of reactivation of endogenous virus. Although there is a theoretical risk of reinfection by second virus strains transmitted by donor marrow or blood transfusions, the incidence and clinical significance of these events remain unknown. There is no indication for the use of screened cellular blood products for seropositive marrow recipients. In a study by Bowden et al, 71 no significant effect

was seen on the incidence of CMV infection when seronegative blood products were used in seronegative patients receiving seropositive donor marrow, although the number of patients studied was small. Both seronegative recipients of autologous and seronegative allogeneic donor marrows have been extensively studied with respect to the incidence of transfusion-acquired CMV infection. The benefits of providing CMV-"safe" blood products to these patients have been clearly demonstrated in prospective randomized controlled trials (Tables 3 and 5). 71'72'81'84-86'119 In the bone marrow transplant setting, hemopoiesis in the patient's bone marrow is highly stimulated in an environment that often includes both graft-versus-host disease and sepsis. This combined with the massive transfusion support these patients often require make it an ideal setting for the reactivation of CMV from blood products and may explain the high incidence of transfusion-acquired CMV infection that has been described in this population.

THE CMV-"SAFE" BLOOD PRODUCT

Solid Organ Transplant Recipients CMV infection is a major source of morbidity and mortality in solid organ transplant recipients. 58J2~ In this setting there are 3 potential sources of infection: endogenous reactivation, the donor organ, and cellular blood products. In patients who are seropositive for CMV antibody before transplantation, reactivation of endogenous virus occurs frequently. 22~ The most important source of CMV infection in solid organ transplant recipients is the donor organ. CMV is efficiently transmitted to both seronegative ~2~and seropositive recipients 9~ from this source. In the seronegative recipient of seronegative donor organs (R-D-), cellular blood products remain the most likely source of infection. Because primary CMV infection in the posttransplantation period is associated with significant morbidity, the use of CMV-"safe" cellular blood products has been recommended for this subgroup of patients. 238 However, these recommendations are based on limited data with respect to the incidence of CMV infection in this population. No randomized controlled studies have been done to determine either the effect of the use of CMV-safe blood products in transplant recipients on the incidence of CMV infection in this patient subgroup or the impact of the provision of these products on patient morbidity and mortality. We studied the incidence of CMV infection over a 13-year period in all transfused seronegative solid organ transplant recipients of seronegative donor organs (R-D-), all of whom received unscreened and unfiltered blood products. The organ-specific incidence of transfusion-acquired CMV infection in these patients was as follows: kidney, 0 of 57 (0%); heart, 0 of 29 (0%); heart-lung/lung, 1 of 6 (16.7%); and liver, 2 of 20 (10%). We, like other investigators studying transfusion-acquired CMV infection in liver transplant recipients, found no significant difference in the transfusion requirements of CMV-infected and uninfected patients. 136 The incidence of transfusion-acquired CMV infection (2.4%) in R-D- solid organ transplant recipients receiving unscreened blood products at our center is lower than that reported in most other studies. In a multicenter trial, Rubin et a112l documented a 20% incidence of CMV infection in R-Drenal patients that was presumably transfusionacquired. Other investigators reported similar inci-

127

dences of 15% to 20% (3 of 20 to 2 of 10). 122-124 However, like ours, some studies have failed to document any transfusion-acquired CMV infection 125,126 or a very low incidence (1 of 49, 2%) 127 of infection in this subgroup of renal transplant recipients. Because transfusion requirements for recipients of heart, heart-lung, lung, and liver transplants are usually significantly higher than renal transplant recipients, these patients may be at higher risk of transfusion-acquired infection. However, studies reporting the incidence of transfusion-acquired CMV infection in patients receiving unscreened blood products often involve small numbers of patients, and the results reported in these studies are highly variable. In heart transplant recipients, the lack of transfusion-acquired CMV infection observed in our study contrasts with the incidence of 20% (1 of 5) 139and 26% (9 of 34) a28 documented in 2 previous studies. In the latter study, fresh blood was used. In liver transplantation, the documented incidences of transfusion-acquired CMV infection in R-D- patients vary from universal infection, 3 of 3 (100%), 129 through an intermediate incidence of infection, 1 of 4 (25%), 13~4 of 14 (28.6%), 131 9 of 44 (20%), 132 to an incidence comparable to the 10% observed in our study, 1 of 12 (8.3%), 133 2 of 28 (7.1%), 234 2 of 15 (13.3%). 58 Although morbidity associated with primary CMV infection in heartlung and tung transplant recipients is extremely high and presumptive transfusion-acquired disease has been described in this setting, 1 of 3 (33%), 135 3 of 15 (20%), 236 1 of 22 (4.5%), 237 the number of patients studied has been small. Regional variability as well as changes over time in the use and preparation of blood components, the age of the transfused blood, and the seroprevalence of CMV antibody in the donor population axe donor factors that may influence risk. In some studies, particularly multicenter trials, a variety of serological assays of variable sensitivity were used to determine serostatus of both organ donor and recipient. This also may influence the reported incidence of transfusion-acquired CMV infection. Regional and organ-specific differences in immunosuppressive regimens may influence the risk of transfusion-acquired CMV disease by almring the probability of leukocyte survival. In live r transplantation, it has been hypothesized that :the establishment of microchimerism by passenger. :leukocytes

128

transmitted with the graft is important for graft tolerance.14~ It is possible that this microchimerism may extend the limits of tolerance or anergy of the recipient, allowing longer survival of blood donor leukocytes as well, thereby increasing the probability of CMV transmission. 6~Although this process is believed to occur with all organs, the high density and quality of migratory cells in liver allograft may potentiate this effect in the setting of liver transplantation. The greater immunogenicity of some organs such as lung and intestinal transplants also may increase the risk of reactivation of CMV from donor leukocytes transfused into this environment. Early postoperative complications such as sepsis, more common in some subgroups of allograft recipients, also may provide a better cytokine milieu for the reactivation of CMV from blood products. Some investigators have suggested that in the seronegative solid organ transplant recipient CMV infection acquired from blood products is associated with less morbidity than when CMV is transmitted by the donor organ.132,136In CMV-seronegafive recipients of CMV-seropositive liver allografts, a setting in which CMV infection is most likely transmitted by the donor organ, patients who developed CMV pneumonitis were found to have received a higher total number of blood units and CMV-seropositive blood units than did other CMVinfected patients. 58 Whether this observation is a reflection of CMV reinfection events, is a result of the immunomodulatory effects of blood transfusion, or whether large transfusion requirements simply represent a surrogate marker for the underlying morbidity of the allograft recipient remains unanswered. As in other populations, geographic variability in risk makes it difficult to provide clear recommendations for the provision of CMV-"safe" blood products for allograft recipients that can be universally applied. The risk also may be allograft and immunosuppression dependent and independent of transfusion volume. The relative priority for the provision of CMV-"safe" products for these patients is summarized in Table 7.

The HIV-Infected Patient In the current era of highly effective antiretroviral therapy, the incidence of active CMV infection in HIV-infected patients has decreased.141 However, CMV infection continues to result in

JUTTA K. PREIKSAITIS

significant morbidity and mortality as the result of chorioretinitis, esophagitis, colitis, pneumonia, encephalitis, and adrenalitis in patients who develop severe immunodeficiency.142 Homosexual and bisexual men are almost universally seropositive for CMV antibody. CMV disease in this patient group with AiDS is believed to be attributable to reactivation of endogenous virus. However, patients with transfusion-acquired HIV infection, including hemophiliacs, have CMV seroprevalence rates of 49% to 53%, comparable to those found in blood donor populations. 143,144 In many parts of North America, the demographic characteristics of HIVinfected patients are changing. The HIV epidemic is moving into risk groups other than homosexual and bisexual men, such as patients who acquire infection heterosexually, through intravenous drug use or as a result of vertical transmission from HIV-infected mothers. As a result, the overall prevalence of CMV antibody in the HIV-infected population may decrease, and a significantly greater proportion of these patients will be at risk of developing primary CMV infection. As well as causing disease directly, CMV has been implicated as a cofactor in the progression of HIV-1 disease and the pathogenesis of AIDS. 145 In the past, the data to support this hypothesis have been inconsistent. However, in a recent study, investigators observed that HIV-infected infants who acquired CMV infection in the first 18 months of life had a significantly higher rate of disease progression and central nervous system disease when compared with infants infected with HIV-1 alone. 146If these conclusions can be extrapolated to older patients, prevention of primary CMV infection in all HIV-positive, CMV-seronegative populations would be extremely important. CMV infection occurs early in HIV-infected infants, with 39.9% infected by 6 months of age and 80% infected by age 5 years. 146Using a serum bank, seroconversion rates in a cohort of HIVinfected hemophiliac patients were studied and found to be 1.2, 2.6, and 3.9 per 100 person-years for patients ages 0 to 17, 18 to 34, and older than 35 years, respectively.144 Because no control group was studied, it is difficult to determine whether these rates differ from those expected in a normal population when controlled for age, gender, race, and socioeconomic status. The relative importance of blood transfusion compared with other means of acquiring CMV infection in these populations, such

THE CMV-"SAFE" BLOOD PRODUCT

as perinatal exposure, horizontal transmission, and in the case of adults, sexual transmission of CMV is uncertain. The actual risk of acquiring CMV infection from blood transfusion in HIV-infected patients remains unknown. Of note, there have been no reports of either transfusion-acquired CMV infection or graft-versus-host disease in this population. 147 The risk of transfusion-acquired CMV disease may be dependent on the immune status of the HIV-infected patient. It is possible that in the absence of severe immunodeficiency, the risk may be low, as has been observed in immunocompetent adults. In the severely immunocompromised HIVinfected patient, a deficient cytokine response may prevent the reactivation of CMV from transfused leukocytes. Ideally, a prospective randomized study to more precisely quantify the risk of transfusionacquired CMV infection and its relationship to immunodeficiency in this population should be performed. Ethical issues raised by such a study could be addressed by the retrospective examination of CMV seroconversion rates in transfused and nontransfused seronegative HIV-infected patients, using existing serum banks. In the absence of data, the prudent approach would be to provide CMV-safe blood products for CMV-seronegative HIV-infected patients and all infants born to HIV-infected mothers. Current transfusion practice in HIV-infected patients appears to be highly variable. Popovsky et at 148 reported the results of a survey of transfusion practices in HIV-infected patients, with responses from 43% of the centers approached, which included 47 hospitals with HIV clinical trials units and 14 regional hemophilia centers. CMV-seronegafive blood products were provided for CMVseronegative patients by 50% (RBC transfusions) and 53% (platetet transfusions) of centers surveyed. Thirty-five percent and 32% of centers used leukoreduced RBCs and platelet components, respectively (75% bedside/25% prestorage leukoreduced) for CMV-seronegative AIDS patients. Infection with multiple CMV strains has been clearly documented in HIV-infected patients. 149 There is a theoretical risk of reinfection with a second viral strain transmitted by blood transfusions in the CMV-seropositive patient. This event, however, would be difficult to document and has not been reported. Popovsky et al's survey 148 indicated that 16% and 21% of centers used CMV-seronegative RBCs and platelet products,

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respectively, for CMV-seropositive HIV-infected patients, to prevent reinfection events. 148 However, there is insufficient evidence to support this practice. Of greater concern in both CMV-seronegative and seropositive HIV-infected patients is the potential role of the allogeneic stimulus of blood transfusion on the replication and reactivation of both endogenous HIV and CMV in the transfusion recipient. It would be important to clearly demonstrate these effects in vivo. A Viral Activation Transfusion Study sponsored by the National Institutes of Health in the United States is in progress. 6 This study, which compares the use of leukoreduced blood with non-leukocyte-reduced blood from CMV-unscreened donors on the replication of CMV and HIV in HIV-infected patients should provide more definitive data to support transfusion policies in this population. Infection with other herpesviruses, particularly EBV and HHV-8, has been associated with significant morbidity in HIV-infected patients. 147 Although the risks associated with the transmission of these viruses by blood products have not been clearly defined, both EBV and HHV-8 are highly cell-associated. It is possible that leukoreduction could have a significant impact on the transmission of these viruses by cellular blood products. These additional potential benefits associated with the use of leukoreduced blood components make these products the preferred choice when compared with CMV-seronegative blood products as a means of providing CMV-safe blood for HIV-infected patients.

Patients With Malignant Disease Although early autopsy studies of patients with disseminated CMV infection found that a preponderance of cases had neoplastic disease, primarily lymphomas and leukemias, symptomatic CMV infection is not a common infection in the neutropenic patient with malignant disease. 15~Studies have documented CMV infection rates varying from 12% to 57% in pediatric oncology patients. 15~ Prospective studies of CMV infection in adults with malignant disease are limited. Is Arvin et a1152 found no seroconversion to CMV in 39 initially seronegative patients with lymphoma observed for 16 months, although blood transfusions required by this group of patients were not recorded. Although patients with malignant disease may

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be at increased risk of both CMV infection and CMV disease, the contribution of blood transfusion to this risk has not been clearly delineated. Cox and Hughes 153 describe seroconversion in 5 of 37 (13.5%) seronegative leukemic children who had received a mean of 3.8 RBC transfusions before seroconversion. Murphy et al82 documented CMV seroconversion in 2 of 9 (22%) of adults with acute leukemia receiving a mean of 26 RBC and 15 platelet units but observed no seroconversion among 11 patients receiving a comparable number of leukocyte-poor blood components. Whilhelm et al5~ did not observe any seroconversion in 129 seronegative patients with malignant disease receiving a mean of 6 blood units. However, only 59 of these patients had received chemotherapy. Bowden et a173 observed that 7 of 104 seroconverted in the first 24 days respectively after transplantation, presumably as a result of unscreened blood compofients administered before transplantation. Many of the published studies of the incidence of CMV infection in this population were done in the 1960s and 1970s. Both chemotherapeutic regimens and blood banking practices have changed significantly since that time. The more aggressive chemotherapy often used today not only causes greater immunosuppression in the host, it also increases the number of blood products required to support the patient through prolonged period of neutropenia and thrombocytopenia. Autologous and allogeneic bone marrow transplantation is being used increasingly often for the treatment of genetic disorders, aplastic anemia, and a number of hematologic malignancies and solid tumors. There are distinct advantages to being CMV seronegative at the time of bone marrow transplantation. It would be important to prevent transfusion-acquired CMV infection in CMV-seronegative patients with malignant disease, aplastic anemia, and thalassemia in whom bone marrow transplantation may be a future therapeutic option. To determine the impact of the provision of CMV-"safe" blood products, we studied the incidence of transfusion-acquired CMV disease in 76 CMV-seronegative children with malignant disease, prospectively randomized to receive CMV seronegative or standard blood products. 36 No cases of transfusion-acquired CMV infection were documented either group. The control group had received a median of 7 RBC and 11 platelet units.

JUTTA K. PREIKSAITIS

In contrast, during the study period, 2 children developed community-acquired CMV infection, for an incidence of 1.7% per patient-year of follow-up. For reasons other than CMV prevention, a number of children in the control arm of our study received relatively leukoreduced blood components, including those that were treated by washing or filtration for the prevention of febrile transfusion reactions and single-donor platelet units collected by apheresis for the management of alloimmunization. Recipient host immune factors in this population also may place these children at lower risk of transfusion-acquired CMV infection compared with other populations such as bone marrow transplant recipients. Adults with leukemia who have greater transfusion requirements and patients receiving very aggressive chemotherapy regimens may be at greater risk of transfusion-acquired CMV infection than the children we studied. Nonetheless, based on our study, the risk of transfusion-acquired CMV infection in the average patient with malignant disease would appear to be low. Leukoreduced blood products are often used in this patient population for the prevention of alloimmunization. The use of these products should eliminate any residual risk of transfusion-acquired CMV infection in higher-risk subsets of this population and should be considered the preferred method of delivering CMV-"safe" blood products to these patients. Transfusion policy with respect to prevention of CMV infection in seronegative patients with hematologic or genetic disorders requiring long-term blood product support who may be candidates for future bone marrow transplantation is less certain. The repetitive nature of transfusion requirements, often over many years, and its effect on donor leukocyte survival in the recipient may place these patients at higher risk of transfusion-acquired CMV infection when compared with other immunocompetent populations. Associated immunodeficiency in some genetic disorders may further increase risk. Although a higher prevalence and incidence of CMV infection has been observed in thalassemic children, particularly in those who have been splenectomized, this increased risk has not been clearly linked to the receipt of blood products. 154,155 As in patients with malignant disease, these patients may benefit from the use of leukoreduced blood products for the prevention of alloimmuniza-

THE CMV-'SAFE" BLOOD PRODUCT

131

don, with the added benefit of C M V prevention. There are insufficient data to recommend that C M V - " s a f e " blood products be provided for this group of patients. CONCLUSIONS

M a n y of the populations for whom CMVseronegative blood products are recommended would benefit from the other potential advantages of leukoreduced blood products including prevention of alloimmunization, prevention of infection with other herpesviruses, and a possible reduction in the immunomodulatory effects o f blood transfusion. The use of leukoreduced blood products also permits the infusion of passive CMV-specific antibody from seropositive donors, which m a y have beneficial effects in some settings. After reviewing the evidence from clinical trials and combining these data with theoretical considerations based on our understanding of the pathogenesis of transfusion-acquired CMV infection, the author concludes that prestorage filtration of blood products is the preferred method of providing C M V - " s a f e " blood products for these patients. There is insufficient evidence to suggest that the additional screening of these filtered products for CMV antibody will add any significant incremental safety with respect to C M V transmission. However, to validate these assumptions, it would be prudent to conduct active surveillance for "breakthrough" C M V infection

after the receipt of filtered blood products in what would be expected to be the highest-risk populations for transfusion-acquired C M V infection. These populations would include groups such as CMVseronegative allogeneic bone marrow transplant recipients, low-birth-weight premature infants, HIVinfected patients, and organ transplant recipients. This would confirm the expected safety of these products with respect to C M V transmission. In the past, issues related to C M V infection in transfusion recipients have focused on the prevention of primary C M V infection transmitted by blood products to high-risk populations. However, most adult transfusion recipients are CMV seroposifive. Recent studies suggest that C M V reactivation events in the sick CMV-seropositive adult may be significantly more frequent than previously believed. The role of allogeneic blood transfusion as a stimulus for this process, the contribution of immunomodulation associated with CMV reactivation to the overall immunomodulatory effects of blood transfusion, and its impact on patient morbidity and mortality require further study. The availability of leukoreduced blood products should allow these studies to occur and provide data for the development of rational policies with respect to the prevention of transfusion-associated CMV infection and disease in this larger subset of CMVseropositive transfusion recipients.

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THE CMV-"SAFE" BLOOD PRODUCT

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