longitudinal monitoring of wbc subsets in packed rbc units after filtration: implications for...

ROBACK ET AL.

500 TRANSFUSION Volume 40, May 2000

Passenger WBCs in packed RBC and platelet com-ponents can produce significant adverse effectsin transfusion recipients. These include transfu-sion-transmitted infections (TTIs) caused by

WBC-associated infectious agents, alloimmunization, fe-brile nonhemolytic transfusion reactions, and immunosup-pression.1,2 TTIs, particularly from latent herpesvirusessuch as CMV, are of greatest concern with regard to seroneg-ative transfusion recipients who either are immunocom-promised or have immature immune systems. These popu-lations include immunosuppressed bone marrow and solidorgan transplant recipients, patients with AIDS, prematureand newborn infants, and persons with congenital immu-nodeficiency states.1,3

Transfusion of CMV-unscreened blood components toat-risk patients leads to transfusion-transmitted CMV (TT-CMV) rates of 13 to 37 percent,4-9 although rates as high as67 percent have been reported.10 In contrast, transfusion ofblood components from CMV-seronegative donors reducesthe incidence of TT-CMV to 0 to 3 percent.7,9 Clinical trialshave also demonstrated a marked reduction in the inci-dence of TT-CMV with WBC reduction of unscreened com-ponents,8,11-13 which suggests that filtered components are

Longitudinal monitoring of WBC subsetsin packed RBC units after filtration:

implications for transfusion transmission of infections

J.D. Roback, R.A. Bray, and C.D. Hillyer

BACKGROUND: Specific subsets of peripheral bloodWBCs are reservoirs for infectious agents, such as CMVand EBV, and can serve as vectors for transfusion trans-mission of these agents. While filter WBC reduction hasbeen used to prevent transfusion transmission of infec-tions, its effectiveness has not been documented formany infectious agents and in some instances may bedifficult to demonstrate in clinical trials. Because the ef-fectiveness of filtration depends on the number of in-fected WBCs remaining at transfusion, WBC subpopula-tions in packed RBC units were quantitated afterfiltration and storage.STUDY DESIGN AND METHODS: Packed RBC units (n= 14) were filtered and stored at 4oC for 42 days or werestored without filtration. Serial samples were subjectedto flow cytometric immunophenotyping of WBC subsets:neutrophils, monocytes, CD4+ and CD8+ T cells, Bcells, and NK cells.RESULTS: Filtration produced a mean reduction in totalWBCs of 3.2 log. Monocytes, lymphocytes, and neutro-phils were reduced by 4.1, 3.8, and 2.5 log, respectively.Lymphocyte subsets also demonstrated differential re-duction with filtration. All WBC subsets showed ongoingloss during storage.CONCLUSIONS: Monocyte and lymphocyte subsets areremoved most effectively by prestorage filtration.Postfiltration storage leads to further significant reduc-tions in WBC subsets. The implications of these findingsfor the mitigation of transfusion transmission of infectionare discussed.

ABBREVIATIONS: FCS = fetal calf serum; FSC = forward scatter

characteristics; HHV-8 = human herpesvirus 8; nvCJD = new

variant CJD; PerCP = peridinin chlorophyll protein; SSC = side

scatter characteristics; TT = transfusion-transmitted (w/CMV,

EBV, etc.); TTI(s) = transfusion-transmitted infection(s).

From the Transfusion Medicine Program Research Laboratory,

Department of Pathology and Laboratory Medicine, Emory Uni-

versity School of Medicine Winship Cancer Center, Atlanta,

Georgia.

Address reprint requests to: Christopher D. Hillyer, MD,

Blood Bank, Room D655, Emory University Hospital, 1364

Clifton Road NE, Atlanta, GA 30322; e-mail: [email protected].

Supported in part by a grant from Baxter Healthcare Corpo-

ration.

Received for publication June 11, 1999; revision received

October 25, 1999, and accepted October 31, 1999.

TRANSFUSION 2000;40:500-506.

B L O O D C O M P O N E N T S

WBC SUBSET REDUCTION ON FILTERED RBCS

Volume 40, May 2000 TRANSFUSION 501

essentially equivalent to CMV-seronegative componentswith respect to the rate of TT-CMV.2,13 Nonetheless, in thelargest trial to date,13 five of six BMT patients who con-tracted CMV infection after the transfusion of filtered unitsdeveloped fatal CMV pneumonia, while none of four pa-tients infected with CMV from seronegative componentsdeveloped CMV disease (p = 0.005 by Fisher’s exact test).14,15

As discussed by Landaw et al,14 the trial data suggest thatfiltered components are not precisely equivalent to CMV-seronegative components. Thus, fundamental questionsregarding the prevention of TT-CMV by filter WBC reduc-tion remain to be answered.

Transfusion transmission of other herpesviruses, suchas EBV and human herpesvirus 8 (HHV-8), has proven moredifficult to study. Although TT-EBV has been demon-strated,16 approximately 90 percent of the population is EBVseropositive, which leaves few seronegative patients inwhom to study transmission by transfusion. In contrast, lessthan 20 percent of the population is seropositive for HHV-8. However, evidence of HHV-8 transfusion transmission isinconclusive.17,18 Intracellular prokaryotes like Ehrlichiaspecies, which infect cells of the monocytic and granulo-cytic lineages, could also be transmitted via transfusion.19,20

B cells may play a role in the pathogenesis of transmissiblespongiform encephalopathies such as new variant CJD(nvCJD), although the nature of the interactions betweenprions and B cells is unclear.21 For these and other, lesscommon infectious agents, it is logistically difficult to de-sign clinical trials to document transfusion transmission; itis also difficult to justify the use of WBC-reduction filters toprevent these potential TTIs without evidence that the rel-evant target WBCs are removed with sufficient efficacy.

To address these issues, we have quantitated the reduc-tion in phenotypically identified WBCs in packed RBC com-ponents after filtration and storage. An examination ofthese results in the context of the current understanding ofTTIs can explain the clinically documented success of fil-ter WBC reduction in the prevention of TT-CMV. In addition,these studies provide data with which to evaluate the useof WBC reduction for the prevention of other TTIs.

MATERIALS AND METHODS

Component preparation and sampling

According to institutional guidelines and after informedconsent was obtained, whole-blood units were collectedfrom donors (n = 14) and packed RBCs were prepared bystandard methods. The units were randomly divided into 3groups. Five units were to be filtered, 5 units were left un-filtered, and 4 units were to be filtered for the CD13 flowcytometry studies. Non-WBC-reduced packed RBC unitswere stored at 4oC for the duration of the study. Packed RBCunits to be WBC reduced were filtered (Sepacell R-500(II),Asahi Medical Corp., Tokyo, Japan; Baxter/Fenwal, Deerfield,

IL) at room temperature and then stored at 4oC. Non-WBC-reduced (n = 5) as well as WBC-reduced (n = 5) units weresampled regularly during the course of the study (Days 0,7, 14, 21, 28, 35, and 42) through sterile-site couplers afterthorough mixing. Filtered units were sampled at an addi-tional time point, immediately after filtration (designatedas Day 0.5). Each of these samples was used for quantitationof both total WBCs and all examined WBC subsets, as de-scribed below. The additional WBC-reduced units (n=4)were sampled only on Days 0 and 0.5 and analyzed for to-tal WBCs, major WBC subsets, and CD13+ WBCs.

WBC countsQuantitation of total WBCs in packed RBC units was per-formed by using a Nageotte counting chamber accordingto published methods.22

Flow cytometric immunophenotypingAt each time point, total WBC preparations were made from1 or 10 mL of blood (non-WBC-reduced or WBC-reducedunits, respectively) by RBC lysis (through addition of 10 volof lysis solution: 150 mM NH4Cl, 7 mM K2CO3, 0.1 mMNa2EDTA [pH 7.4]), which was followed by centrifugationand washing. The resulting concentrated WBC preparationswere resuspended in a 100-µL final volume of DMEM con-taining 10-percent fetal calf serum (DMEM/FCS) andstained with combinations of antibodies labeled with eitherFITC, PE, or peridinin chlorophyll protein (PerCP). All an-tibodies were obtained from Becton Dickinson (San Jose,CA), and appropriate dilutions were determined throughpilot studies. Appropriate isotype antibody controls wereused in all studies. The subsets analyzed (and identifyingantibodies) are as follows: neutrophils (CD15), monocytes(CD14), total lymphocytes (CD45), CD4+ T cells (CD3+/CD4+ dual-staining cells), CD8+ T cells (CD3+/CD8+ dual-staining cells), B cells (CD2–/CD19+), and NK cells (CD16+/CD56+/CD3–). In some studies, CD13-specific antibodieswere used in conjunction with CD14- and CD15-specificantibodies for further evaluation of CMV-infectable cellpopulations.

After antibody staining, WBC samples were examinedby flow cytometry (FACScan, Becton Dickinson). For eachnonfiltered sample, 30,000 to 50,000 events were analyzed.For filtered samples, all events were captured. Each subsetwas first quantitated as a percentage of the total WBCs inthe sample and subsequently as an absolute number of cellsin the packed RBC unit. The following formulae were usedfor these calculations: total WBCs (using Nageotte chamber)= neutrophils (CD15+) + monocytes (CD14+) + total lym-phocytes (CD45+); total lymphocytes = total CD2+ cells (Tcells + NK cells) + B cells (CD2–/CD19+/CD45+); total CD2+cells = total T cells + NK cells (CD16+/CD56+/CD3–); andtotal T cells = CD3+/CD4+ T cells + CD3+/CD8+ T cells.These calculations did not take into account nonstaining

ROBACK ET AL.


(“null”) WBCs, which generally accounted for less than 3percent of all WBCs.

Validation of quantitative flow cytometricimmunophenotypingWBC subset concentrations in peripheral blood samplesfrom healthy volunteers were determined by using an au-tomated hematology analyzer, and the samples were thenserially diluted into WBC-free packed RBCs, which hadbeen prepared by triple-filtering of packed RBC units. Theserially diluted samples were then subjected to flow cyto-metric analysis as described above.

StatisticsAll statistical comparisons were performed by using theMann-Whitney U test. The null hypothesis was rejected atthe 0.05 level of significance.

RESULTSFlow cytometric WBC subset quantitation was validated byanalyzing whole-blood samples, containing known countsof WBCs, after serial dilution into triple-filtered WBC-re-duced packed RBCs. Analysis of concentrated WBC-reducedpacked RBC samples found <100 WBCs per unit (<0.5/mL).Because WBC-reduced samples were concentrated from 10mL of whole blood before analysis, the limit of detectionwas set at 5 WBCs per sample. When expected and observedneutrophil, monocyte, and lymphocyte counts were plot-ted for blood samples serially diluted up to 1 in 1000, theyyielded a linear regression coefficient (r2) of 0.984 (Fig. 1).Analysis of parallel aliquots of whole-blood samples diluted1 in 1000 (3 log WBC reduction) and 1 in 10,000 (4 log WBCreduction) yielded CVs ranging from 12 to 29 percent. Thus,this assay is sensitive, accurate, linear, and precise.

At the time of collection, there was no significant dif-ference (p>0.05) between total WBCs in the group desig-nated to undergo WBC reduction and storage (n = 5; me-dian, 2.16 × 109 WBCs) and in the non-WBC-reductiongroup (n = 5; median, 1.53 × 109)(Fig. 2). A mean 3.04 log re-duction in total WBCs was observed immediately after fil-tration. When these 5 filtered units were analyzed togetherwith 4 additional filtered packed RBC units (see below),there was a mean 3.2 log reduction in total WBCs with fil-tration (median after filtration, 1.16 × 106). During postfil-tration storage of WBC-reduced units, there was a contin-ued decline in WBCs to a median count of 2.38 ×104 on Day42 (p<0.05; Day 0.5 vs. Day 42). Non-WBC-reduced unitssampled after 42 days of storage contained a median of 4.06× 108 residual WBCs, representing a 0.61 log mean declineduring storage (p<0.05, Day 0 vs. Day 42).

In previous studies of filtered components, flowcytometric analyses utilized CD45-specific antibodies and/or a limited panel of other antibodies, in conjunction withside scatter and forward scatter characteristics (SSC andFSC, respectively) of unfiltered samples to set analysisgates.23,24 This method may underestimate some WBC sub-sets, as filtration could alter identifying characteristics ofWBCs. Thus, we employed an immunophenotyping strat-egy in which each subset was identified by a specificantibody(ies), with minimal reliance on SSC, FSC, and pre-set analysis gates. Figure 3 illustrates the quantitation ofneutrophils, monocytes, and total lymphocytes in a typicalnon-WBC-reduced RBC sample. WBCs, stained with CD15-FITC, CD14-PE, and CD45-PerCP, are initially displayed onan SSC-by-FSC dot plot (Fig. 3A) and subsequently plotted

Fig. 1. Comparison of expected and observed WBC counts with

flow cytometric analysis of serially diluted whole-blood

samples. A representative experiment is shown, which was re-

peated with similar results.

Fig. 2. Total WBC reduction in RBC units after filtration and/or

storage. Each data point represents total WBCs (median and

range) in either unfiltered RBC units (-� -) (n = 5) or

prestorage-filtered RBC units (-��-) (n = 5) over 42 days of stor-

age at 4oC. *p<0.05, as compared to Day 0; †p< 0.05, as com-

pared to Day 0.5 (immediately after filtration).

Observed cell count (cells/µL)

Exp

ecte

d ce

ll co

unt

(cel

ls/µ

L)

Days of storage

Tot

al W

BC

s



on an SSC-by-CD15-FITC dot plot (Fig. 3B) for the identifi-cation of neutrophils (green; CD15mod-hi, SSCmod-hi). After theneutrophils are gated and quantitated, they are excludedfrom further analysis, and the remaining cells are displayedon an SSC-by-CD14-PE dot plot (Fig. 3C). The monocytes(red; CD14hi, SSClo-mod) are gated and quantitated and thenexcluded from further analysis. The remaining cells are dis-played on an SSC-by-CD45-PerCP dot plot (Fig. 3D), andtotal lymphocytes are gated and quantitated (yellow;CD45mod-hi, SSClo-mod).

A comparison of neutrophil, monocyte, and total lym-phocyte populations in the non-WBC-reduced (Fig. 4 A andB) and WBC-reduced (Fig. 4 C and D) packed RBC unitsshows the removal of WBC subsets by filtration. While neu-trophils (green) and lymphocytes (yellow) can still be iden-tified after filtration, only rare monocytes (red) are observed(Fig. 4D). Neutrophil CD45 immunoreactivity and SSC areboth lower in filtered samples (Fig. 4D) than in unfilteredsamples (Fig. 4B), showing that filtration alters some iden-tifying characteristics of WBCs and could displace thesecells from preset analysis gates. In contrast, CD45 immu-noreactivity and SSC of lymphocytes (yellow) are un-changed by filtration.

The degree of WBC reduction by filtration was not uni-form across WBC subsets (Fig. 5). When all WBC-reducedunits (n = 9) were analyzed together, filtration producedmean 4.1 log reduction in monocytes, 3.8 log reduction intotal lymphocytes, and 2.5 log reduction in neutrophils.During storage of WBC-reduced units (n = 5), there weresignificant decreases (Day 0.5 vs. Day 42) in cells of themajor subsets, including a 1.19 log reduction in monocytes(p<0.05). In unfiltered units, storage-related WBC reductionfor these subsets ranged from 0.4 to 0.7 log. A quantitative

Fig. 3. Three-color flow cytometric dot plots demonstrating

sequential gating strategy for quantitation of unfiltered total

cell population (A) (all three colors), neutrophils (B) (green),

monocytes (C) (red), and total lymphocytes (yellow) (D) in

RBC samples. See text for details.

Fig. 4. Comparison of WBCs in unfiltered (A,B) and filtered

(C,D) packed RBC units before (A,C) and after (B,D) sequential

gating for identification of neutrophils (green), monocytes

(red), and lymphocytes (yellow). See text for details.

Fig. 5. Reduction in the major WBC subsets after filtration

and/or storage of packed RBC units. Each data point repre-

sents total WBCs of each subset (median and range) in either

unfiltered or prestorage WBC-reduced (LR) RBC units over 42

days of storage at 4oC. *p<0.05 versus Day 0 (unfiltered) or Day

0.5 (WBC-reduced).

Lymphocytes

Lymphocytes

Monocytes

Monocytes

Granulocytes

Granulocytes

WB

C s

ubse

ts

Days of storage

ROBACK ET AL.


comparison also found variations in lymphocyte subset re-moval by filtration (Fig. 6). CD19+ B cells were decreasedby 4.0 log, while NK cells were decreased by 3.4 log andCD4+ and CD8+ T cells by 3.4 log and 3.6 log, respectively.Lymphocyte subpopulations in filtered and unfiltered unitsalso showed ongoing loss with storage. These storage-re-lated declines reached significance in some cases (Fig. 6).

CD13 (aminopeptidase N), a metalloprotease ex-pressed primarily on cells of the monocyte and neutrophillineages, is a putative marker of CMV infectability.25,26

Packed RBC units (n = 4), sampled before and after filtra-tion, were subjected to flow cytometric immunophenotyp-ing with antibodies to CD13, CD14, CD15, and CD45 (Fig.7). Filtration produced equivalent reductions in neutrophilsidentified by either CD15 or CD13 immunoreactivity and inmonocytes identified by CD14 or CD13 immunoreactivity.Thus, immunophenotyping with CD13 antibodies did notprovide additional information on the removal of CMV-infectable WBCs by filtration.

DISCUSSIONTo better understand the mitigation of TT-CMV by filtrationand to examine the applicability of WBC reduction to theprevention of other TTIs, we quantitated major WBC sub-sets in packed RBC units subjected to filtration and/or stor-age. A previous study by Wenz and Burns23 also describedWBC subset analysis of filtered RBC units. However, thepresent investigation differs from that study in a numberof ways. First, we used filters and procedures designed forprestorage WBC reduction, which is becoming the de factointernational standard, while the earlier study was designed

to simulate bedside WBC reduction. Second, Wenz andBurns concluded that RBC filters containing polyester me-dium removed all WBC subsets in an approximately equiva-lent, nonselective fashion.23 In contrast, our studies withfilters of similar composition clearly show a selective bias,with monocytes removed to a greater degree (4.1 log) thaneither lymphocytes (3.8 log) or neutrophils (2.5 log) and Bcells removed to a greater degree (4.0 log) than other lym-phocyte subsets. Third, we quantitated all WBC subsets,including neutrophils, through the use of specific antibod-ies or antibody combinations and did not use analysis gatesset from unfiltered samples. In contrast, Wenz and Burnsused SSC and FSC to quantitate neutrophils in filteredsamples. We have shown in this study (Fig. 4) that flowcytometric characteristics of WBCs, including SSC, changeafter filtration and may not be reliable criteria for neutro-phil quantitation in filtered units. In fact, changes in SSCand CD45 immunoreactivity may be a manifestation ofWBC damage during filtration. Further studies are requiredto more thoroughly document this damage, if it exists, andto determine the effects on WBC survival and function af-ter transfusion.

Peripheral blood WBCs of the monocyte/macrophagelineage carry latent CMV DNA and can support CMV repli-cation.27-29 Given that latent CMV is present in 1 of every1,000 to 10,000 peripheral blood monocytes from healthyCMV-seropositive individuals,28-30 seropositive packed RBCunits with 1 to 5 × 108 total monocytes should contain be-tween 1 × 104 and 5 × 105 latently infected monocytes, and

Fig. 6. Reduction in the lymphocyte subsets after filtration

and/or storage of RBC units. Each data point represents total

lymphocytes of each subset (median and range) in either un-

filtered or prestorage WBC-reduced packed RBC (LR) units

over 42 days of storage at 4oC. *p<0.05 versus Day 0 (unfil-

tered) or Day 0.5 (WBC-reduced).

Fig. 7. WBC reduction of WBCs that were immunoreactive for

CD13, a putative marker for CMV infectability. Prefiltration

(� ) and postfiltration (� ) samples from RBC units (n = 4) were

subject to quantitation of CD15+ or CD13+ neutrophils and

CD14+ or CD13+ monocytes. Each data point represents total

WBCs of each subset (median and range). There were no sig-

nificant differences (p>0.05) before or after filtration in the

numbers of neutrophils that were immunoreactive with anti-

CD13 versus anti-CD15 or in the numbers of monocytes iden-

tified with anti-CD13 versus anti-CD14.

Lym

phoc

yte

subs

ets

Days of storage



WBC-reduced packed RBC units could contain up to 50 in-fected monocytes after 4 log reduction in monocytes. Asshown in this study, however, the length of storage may af-fect the number of infected monocytes remaining at trans-fusion. Although estimates, these calculations indicate that,while the majority of seropositive WBC-reduced packed RBCunits are “CMV-safe,” as determined in clinical trials, theymay contain residual CMV-infected monocytes. The impli-cations of this finding for an understanding of the biologyof TT-CMV require additional investigation to establish.

An interesting finding of this study is the persistenceof WBCs throughout the 42-day storage period. The objec-tive of this study was to assess the presence of these sub-sets with flow cytometric immunophenotyping, which doesnot require that the cells remain functional. Future inves-tigations will determine the extent to which WBCs remainviable and functional after filtration.

CD13 has been proposed as a marker for CMV-infectable WBCs.25,26 In the present study, anti-CD13 immu-noreactivity was restricted to monocytes and neutrophilsin WBC samples from packed RBC units. The efficiency ofWBC reduction in regard to CD13+ monocytes and neutro-phils was not significantly different than that in regard tomonocytes and neutrophils identified by CD14 and CD15immunoreagents, respectively. These findings suggest thatquantitation of CD14+ monocytes and CD15+ neutrophilsis sufficient to evaluate the CMV-infectable populationsremaining after filtration.

It is more difficult to evaluate the potential efficacy ofWBC reduction for the prevention of TTIs other than TT-CMV. For some of these agents, the basic biology, includ-ing the percentage of WBCs infected, is not well understood.For others, transfusion transmission has still not been docu-mented clinically, which suggests that it may be an ineffi-cient process. Nonetheless, the present data suggest thatWBC reduction would be most effective for infectious agentsthat are carried by monocytes and B cells (EBV, HHV-8,Ehrlichia chaffeensis), as these subsets are reduced by 4 logimmediately after filtration. T cells (which carry HTLV-I and-II) are also efficiently removed by WBC reduction (3.5 log).However, neutrophils are reduced only 2.5 log by filtration,which may have implications for transfusion transmissionof the human granulocytic ehrlichiosis agent, as well as CMV.

While WBC reduction is being applied for the mitiga-tion of a wide array of TTIs, the specific filtration values thatare most important in preventing TTIs (including magni-tude of WBC reduction, differential WBC subset reduction,and prestorage versus bedside WBC reduction) are only nowbeing investigated. Further work will be necessary to guidefuture improvements in filter design and filtration methods.

ACKNOWLEDGMENTS

The authors acknowledge Robert Karaffa and Mary Duenzl for

expert technical advice and assistance with flow cytometry and

Valerie Willis for assistance with obtaining RBC units. Baxter

Healthcare provided the WBC-reduction filters.

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