WBC reduction of RBC transfusions is associated with a decreased incidence of RBC alloimmunization

I M M U N O H E M A T O L O G Y

WBC reduction of RBC transfusions is associated with adecreased incidence of RBC alloimmunization

Neil Blumberg, Joanna M. Heal, and Kelly F. Gettings

Volume 43, July 2003 TRANSFUSION 945

ABBREVIATION: AML = acute myeloid leukemia.

From the Department of Pathology and Laboratory Medicine

and the Department of Medicine, University of Rochester

Medical Center and Strong Memorial Hospital, Rochester, New

York.

Address reprint requests to: Neil Blumberg, MD, Box 608,

University of Rochester Medical Center, Rochester, NY 14642;

e-mail: [email protected].

Received for publication November 13, 2002; revision

received March 10, 2003, and accepted March 12, 2003.

TRANSFUSION 2003;43:945–952.

BACKGROUND: Allogeneic transfusion stimulates Th2 (humoral) immunity. A hypothesis was developedthat WBC reduction, by reducing the Th2 stimulus associated with transfusions, might reduce RBC alloimmunization.STUDY DESIGN AND METHODS: The first retrospec-tive cohort study involved determining the prevalence ofnewly detected alloimmunization in transfused patientswith acute myeloid leukemia (AML) in our hospital inthe period before WBC reduction and after its introduc-tion for this particular group of patients. The secondstudy involved determining the incidence of newlydetected RBC alloimmunization in all transfused hospi-tal patients during three annual periods with WBC-reduction prevalences ranging from 0 to 100 percent.RESULTS: The alloimmunization prevalence rate inAML patients was 8.2 percent in those receiving non-WBC-reduced RBCs and platelets (n = 195) and 2.8percent in those receiving only WBC-reduced compo-nents (n = 215) (p = 0.016). In all patients, the alloim-munization incidence rate decreased from 3.47 per1000 antibody screens in 1987 (no WBC reduction) to2.97 per 1000 in 1999 (40% of transfusions WBC-reduced) to 2.38 per 1000 in 2001 (100% of transfu-sions WBC-reduced) (p = 0.0298). A decrease inalloimmunization was observed in both males andfemales, with the decrease more clearly evident inmales.CONCLUSION: These preliminary data support thehypothesis that WBC reduction may be associated witha reduced frequency of RBC alloimmunization. Thesefindings require confirmation and further investigation.

For the better part of a century, it has beenaccepted that alloimmunization to RBC antigensis a common and potentially serious clinicalproblem after allogeneic blood transfusion and

pregnancy.1 It has been assumed that the major factors inalloimmunization (other than exposure to an antigen therecipient lacks) are the intrinsic immunogenicity of theantigen and ill-defined genetic factors governing immuneresponse to alloantigen.1 Neither of these factors are well understood at the molecular level for RBC alloimmunization.

It has been known for many years that the “immuno-logic milieu” in which alloantigen exposure occurs is alsoof importance. Indeed, most antigens, particularly solubleantigens, evoke no immune response whatever withoutthe additional stimulus of an adjuvant.2 Adjuvants oftencause a mild inflammatory response at the vaccinationsite for cutaneous or intramuscular alloimmunization toantigens such as HBsAg or tetanus toxoid. This observa-tion led to the postulate that the presence of “danger”signals, such as inflammation, determines whether analloimmune response occurs, rather than solely the classicconcept of self versus not self.3

RBC antibody formation is a humoral alloimmuneresponse to allogeneic RBCs introduced into the circula-tion by transfusion or pregnancy. Immunologic responsescan become polarized to favor cells and cytokines of Type1 (Th1) or Type 2 (Th2) responses.4 Each type of response

has a characteristic pattern involving helper and cytotoxicT cells, dendritic cells, and soluble mediators. Type 1responses are those involving cytokines such as g-interferon, IL-12, and lymphotoxin and cellular immuneresponses such as delayed-type hypersensitivity. Type 2responses are those typically involving cytokines such asIL-4, IL-5, and IL-10 and humoral immune responses, par-ticularly those involving specific IgG subclasses, as well as IgA and IgE. Certain disease states are characterized by “immune deviation” favoring one or the other type ofresponses. Anti-tumor immunity, antiviral immunity,inflammatory diseases such as rheumatoid arthritis andregional enteritis, and solid organ allograft rejection arethought by some to be primarily Type 1 responses.5 Suc-cessful defense against some parasites and tolerance ofthe fetus as an allograft are thought to be primarily Type 2responses. Type 2 responses tend to down-regulate Type 1immunity and vice versa.

Allogeneic transfusion has been shown in bothanimal model and clinical studies to elicit immune devi-ation favoring Type 2 responses and down regulation ofType 1 responses.6–8 This mechanism could in partaccount for the unfavorable associations of allogeneictransfusion with the development of antibodies to RBCs,WBCs, platelets, and plasma proteins; increased tumorrecurrence; and postoperative bacterial infection, andfavorable associations with reduced spontaneous abor-tions and increased tolerance of solid organ allografts.9

WBC reduction has been convincingly shown to reducethese effects in animal models and some clinical settings,but this broad scope of “transfusion immunomodulation”remains a subject of continued investigation and controversy.10,11

There is evidence from an animal model that donordendritic cells are required for alloimmunization, and thisrepresents another potential mechanism by which WBCreduction might alter RBC alloimmunization rates.12

Finally, unmodified RBCs often contain large numbers ofplatelets, which are rich in both cell surface and solubleCD40L (CD154).13,14 This costimulatory molecule is wellknown to activate B cells and is critical for IgM-to-IgGclass switching during the immune response toantigen.15,16 WBC reduction filters remove most of thedonor platelets from RBC transfusions and thus may abrogate the CD40L stimulation of recipient humoralimmunity, at least for transfusions WBC-reduced beforestorage.

We hypothesized that WBC reduction of allogeneicRBC transfusions might reduce the Type 2 stimulus asso-ciated with such transfusions, and remove donor den-dritic cells and platelet CD40L. These changes mightdecrease the likelihood of RBC alloimmunization throughdirect removal of antigen-presenting cells and reductionof indirect effects. Speculative indirect effects might bemediated by inflammatory and costimulatory molecules

activated and released due to interactions between donorand recipient WBCs in vivo. To assess this possibility, weretrospectively investigated de novo RBC alloimmuniza-tion in patients before, during, and after the implementa-tion of universal WBC reduction of RBC transfusions inour hospital. To control for possible changes in patientmix, we also investigated RBC alloimmunization in asingle disease, acute myeloid leukemia (AML), in patientsreceiving either exclusively WBC-reduced or unmodifiedRBC and platelet transfusions.

MATERIALS AND METHODS

PatientsThe study is a retrospective blood bank and clinical labo-ratory record review. Information on immunohemato-logic testing results over time, transfusion history, gender,age, and admitting diagnosis were retrieved.

Patients with AML. The first cohort of patients wasemployed to study alloimmunization prevalence inpatients with a specific disease (AML) at high risk of RBCalloimmunization. In previous work, the sensitizationprevalence in these patients, despite their relatively shortmedian survival, was about 16 percent.17 All transfusedpatients during the years 1978 to 2001 with the diagnosisof AML were identified from laboratory records, and theirtransfusion histories reviewed. Patients were divided intothose treated after 1978 but before 1989 when bedside(after storage) WBC reduction of both RBC and platelettransfusions was introduced for all patients with AML, andthose treated between 1990 and 2001 who received onlyWBC-reduced blood components. Patients who receivedboth types of transfusions were excluded from the analysis.

Only antibodies reactive at 37°C and/or antiglobulinphase, newly detected at least 10 days after a transfusionwere considered in the analysis. Anti-D was excludedbecause virtually all of these sensitizations are likely dueto pregnancy or receipt of D+ RBCs in platelet concen-trates, rather than alloimmunization due to RBC transfu-sions. Including anti-D in the analysis yielded identicalresults in any case (results not shown but available onrequest from the authors). Because of referral patterns,AML patients receive transfusions only at our centerduring induction and post-remission therapy, as well asduring relapse. We recognized that a small but unknownnumber of transfusions were probably given to somepatients at referring institutions due to severe anemia orthrombocytopenia at the time of initial diagnosis. Weknow of no reason why this would be more or less likelyto occur during the period 1978 to 1989 as compared with1990 to 2001. During the period 1981 to 2001, our anti-body-screening technique at 37°C and antiglobulin phaseincluded two reagent cells tested against donor serumusing a LISS enhancing medium. Before 1981, an albumin

BLUMBERG ET AL.

946 TRANSFUSION Volume 43, July 2003

enhancement technique was employed rather than lowionic strength. Thus, on average, the WBC-reduced cohortunderwent somewhat more-sensitive immunohemato-logic screening, which might be expected to increase therate of antibody detection.

Newly alloimmunized patients in 1987, 1991, and2001. The second set of patients consists of all patientswho had received at least one RBC transfusion at our hos-pital during the year in question and subsequently devel-oped one or more new alloantibodies reactive at 37°Cand/or antiglobulin phase detected upon subsequenttesting during the years 1987, 1999, or 2001. These yearswere chosen as examples of a year before filtration WBCreduction of RBC transfusions becoming available (1987),the year after universal WBC reduction was introduced inJuly, 2000 (2001), and an example of a year with an inter-mediate proportion (40%) of WBC-reduced transfusionsbeing given (1999). During the period 1990 to 2000,approximately 40 percent of transfusions had been WBCreduced, mostly for patients with hematologic diseases,newborns, transplant recipients, and immunocompro-mised patients.

The incidence of new alloantibody formation wasnormalized to the total number of antibody screens (i.e.,indirect Coombs/antiglobulin) as well as to all RBC-containing transfusions given during that calendar year.This approach was chosen to account for the varyingnumber of patients tested and transfused in each of theseyears. The number of opportunities for alloimmunizationby transfusion and detection of alloimmunization varywith the number of transfusions and number of antibodyscreens performed, although not necessarily in a linearfashion. The actual total number of transfusion episodesor unique transfused patients is not available from ourrecords, thus antibody screens and total number of RBCtransfusions were used as the surrogate denominator for calculating alloimmunization incidence. The policy ofantibody screening remained constant throughout thestudy period, with samples required every three daysduring ongoing transfusion of RBCs.

Testing procedures in each year consisted of identicalmethods employing 37°C and antiglobulin (monospecificanti-IgG) phase testing against a two-cell reagent panelusing a LISS enhancement medium. Only patients with apreviously negative antibody screen who received trans-fusion at least 10 days before the appearance of a newalloantibody were considered de novo alloimmunizations.This period was chosen because it is the shortest periodfor primary sensitization we have detected during the lastquarter-century in our hospital. Antibodies that did notreact at 37°C antiglobulin phase but were detected in ABOand Rh grouping at room temperature are not included.These may represent naturally occurring rather thanimmune antibodies. Anti-Ds are not reported in the analy-sis for reasons previously described, and including them

yielded identical results of slightly greater significance inany case. The number of anti-Ds detected de novo intransfused patients was eight in 1987, six in 1999, andseven in 2001.

We recognized that some of the allosensitizationsdetected would represent secondary responses due to pre-vious exposure to RBC antigens through pregnancy ortransfusion; however, we know of no reason why this pro-portion should have changed during the period 1987 to2001. Indeed, because many of the patients transfused in2001 had received non-WBC-reduced transfusions in pre-vious years (35 of 77), an observed difference in alloim-munization or secondary response rates in this groupwould likely understate any potential effects of WBCreduction. In other words, the study design in thesecohorts is inherently biased against detecting any benefi-cial reduction in alloimmunization that might occur withWBC-reduced RBC transfusions. Alternatives, such asrecalling all transfused patients from 2001 for furtherimmunohematologic testing would have been impracti-cal, expensive, and not comparable to the historical dataavailable on recipients of non-WBC-reduced transfusions.Detailed demographic data on all hospital patients or alltransfused patients during these time periods are notavailable, thus changes in clinical case mix, which mightbe of interest, unfortunately could not be assessed as partof this study.

Statistical analysis was by Chi-square test or the Fisherexact test for categorical variables and ratios, and by theMann-Whitney test for continuous variables (Statview 5.0,SAS Institute, Cary, NC). All p values reported are two sidedand uncorrected for multiple comparisons.

RESULTS

Patients with AMLTable 1 shows the prevalence of patients with AML receiv-ing non-WBC-reduced RBCs with newly detected anti-bodies to RBC antigens was 8.2 percent (16/195; 9 malesand 7 females) as compared with a rate of 2.8 percent(6/215; 2 males and 4 females) in patients with AMLreceiving WBC-reduced transfusions (p = 0.016). Multiplenew antibodies were seen in 5 of 195 patients receivingnon-WBC-reduced versus 1 of 215 patients receivingWBC-reduced transfusions (p = 0.1097). The total numberof alloantibodies detected was 22 in 195 patients in thenon-WBC-reduced group versus 7 in 215 patients in theWBC-reduced group (p = 0.0036). The specificities of theseantibodies are shown in Table 2.

Newly detected Rh blood group antibodies declinedfrom 13 antibodies in 195 patients in the non-WBC-reduced cohort to 2 of 215 in the WBC-reduced cohort (p = 0.003 by the Fisher exact test). The antibodies wereevenly distributed amongst male and female patients sug-gesting they were not primarily pregnancy related.

WBC REDUCTION AND RBC ALLOIMMUNIZATION


The patients in the WBC-reduced transfusion cohortreceived slightly but not significantly more RBC transfu-sions and were followed for longer periods of time thanthe patients in the non-WBC-reduced cohort (Table 1).The two cohorts had similar mean follow-up periods and time from first antibody screen to detection of newalloimmunizations.

Newly alloimmunized patients in 1987, 1991, and 2001Fig. 1 shows the incidence of patients with new alloim-munizations during the years 1987, 1999, and 2001 in ourhospital, normalized to the number of antibody screensperformed during that year. A 31-percent decrease from3.47 newly alloimmunized patients (total, n = 70) per 1000antibody screens (total, n = 20,179) to 2.38 (total, n = 67alloimmunized patients and 28,191 antibody screens)accompanied a decrease in the use of non-WBC-reduced

RBC transfusions from 100 to 0 percent (p = 0.026 for the comparison of 1987 vs. 2001). Similar results wereobtained when the results were normalized to the numberof RBC-containing transfusions in each year because thenumber of antibody screens and transfusions were quitesimilar (Table 3).

The incidence of new antibodies (as opposed topatients with antibodies) detected was 4.26 per 1000 anti-body screens in 1987 as compared with 3.82 in 1999 and2.91 in 2001 (p = 0.0161 for the comparison of 1987 vs.

BLUMBERG ET AL.


TABLE 1. RBC alloimmunization, demographic. and transfusion data for patients with AML receiving non-WBC-reduced or WBC-reduced transfusions

Non-WBC-reduced recipients WBC-reduced recipients1978–1989 1990–2001 p value

Number 195 215Patients newly alloimmunized (%) 8.2 2.8 0.016New alloantibodies/1000 RBCs transfused 5.37 1.42 0.0018Patients with multiple new antibodies (%) 2.6 0.5 0.1097New alloantibodies (number) 22 7 0.0036Transfusions before antibody detection (mean) 18 ± 14 17 ±13 NS*Weeks from first antibody screen until detection of new

alloantibody (mean) 27 ± 25 20 ± 21 NS*Total RBC transfusions (mean) 21 ± 20 23 ± 16 0.09Follow-up weeks from first transfusion until last

antibody screen (mean) 29 ± 52 50 ± 65 <0.0001Proportion males (%) 54 57 NS*Mean age (years) 50 ± 24 48 ± 22 0.10Alloimmunization rate in males (%) 8.6 1.6 0.0257Alloimmunization rate in females (%) 7.8 4.3 NS*

* p > 0.20.

TABLE 2. Number of newly detected RBC alloanti-bodies in transfused patients with AML during twoperiods with different types of RBC transfusions

Non-WBC-reduced WBC-reducedtransfusion recipients transfusion recipients

Antigen 1978–1989 1990–2001Number 195 215K 6 4E 8 2C 2 0Jka 1 0S 1 0c 3 0Jkb 1 0Kpa 0 1

Total 22 7

PercentN

umbe

r

Fig. 1. The incidence of newly alloimmunized patients per

1000 antibody screens (– –) in our hospital transfusion

service, and the proportion of total transfused RBCs that were

not WBC-reduced (– –) are shown for 3 different years.

2001). Data on the age, gender, and follow-up of all trans-fused patients in our hospital during these years was notavailable. In the patients making new alloantibodies, thepercentage of female patients in each cohort was similar(Table 3). This lack of gender skewing suggests that few pregnancy-related alloimmunizations are included in the data. When the alloimmunization-incidence-per-antibody screen was calculated by gender, the malealloimmunization incidence decreased 35 percent and thefemale alloimmunization incidence decreased 28 percent(Table 3). Because of the smaller numbers, these results

are not significant, but the trends are similar for bothmales and females, suggesting that the alloimmunizationsare predominately transfusion related. Information on thenumber of antibody screens performed by patient genderwas not available.

The percentage of alloimmunized patients receivingadditional blood components, such as platelets, wassimilar in each group (Table 3). When the analysis wasrestricted to patients who had only received RBC transfu-sions, the patient alloimmunization incidence declined 36percent from 1987 to 2001, similar to the 31 percent seenin the whole cohort. The number of patients making multiple new RBC alloantibodies as a proportion of allpatients making new alloantibodies was remarkably con-stant during the 3 years (Table 3), and the individual speci-ficities detected are shown in Table 4. Of note, slightlyfewer new antibodies and newly alloimmunized patientswere observed in 2001 than in 1987. This is unexpectedgiven that the number of antibody screens and RBC unitstransfused grew by 40 and 36 percent, respectively, duringthis interval. We do not have data on the number ofpatients transfused during these years, thus it is not pos-sible to calculate the alloimmunization rate per transfu-sion episode or per patient transfused.

To assess whether different degrees of antigenicitywere associated with changes in alloimmunization, wecalculated the incidence of alloimmunization to antigensin the Kell, Duffy, and Kidd blood groups. The alloimmu-nization incidence to these antigens declined 33 percent,which, while not significant due to the small number ofobservations, is similar to the overall reduction in alloim-munization incidence in the whole cohort of 31 percent



TABLE 3. RBC alloimmunization rates, demographics, and transfusion data for patients with newly detected alloimmunization during 1987, 1999, and 2001

1987 1999 2001 p value*Patients newly alloimmunized (number) 70 80 67New alloantibodies (number) 86 103 82Total antibody screens this year (number) 20,179 26,953 28,191Total RBC transfusions this year (number) 19,257 25,084 26,163Previous transfusions/alloimmunized patient (mean) 12 10 9 0.16Previous WBC-reduced transfusions (mean) 0 3.4 6.4 <0.0001Weeks from first historical transfusion to date of new alloimmunization (mean) 260 378 340 NSNew alloimmunization rate† (patients/antibody screen performed) 3.47 2.97 2.38 0.026New alloimmunization rate† (antibodies/antibody screen performed) 4.26 3.82 2.91 0.0161New alloimmunization rate† (patients/1000 RBC transfusions) 3.64 3.19 2.56 0.0488New alloimmunization rate† (antibodies/1000 RBC transfusions) 4.47 4.11 3.13 0.0262Alloimmunized patients female (%) 51 60 54 NSMale alloimmunization rate† 1.68 1.19 1.10 0.109Female alloimmunization rate† 1.78 1.78 1.28 0.19Alloimmunized patients receiving other blood components (%) 29 32 34 NSAlloimmunization rate in patients receiving only RBCs† 3.02 2.60 1.92 0.0137Proportion of patients with multiple new alloantibody specificities (%) 15 18 17 NSAlloimmunization rate to Kell, Duffy, Kidd antigens† 1.33 1.11 0.89 0.18Alloimmunization rate to Rh blood group antigens other than D† 1.4 1.37 1.31 NSAlloimmunization rate to non-Rh, Kell, Kidd, Duffy antigens† 0.74 0.48 0.18 0.0025

* p values are for comparison of 2001 and 1987; NS = p > 0.20.† All alloimmunization rates are expressed as per 1000 antibody screens performed that year unless otherwise specified.

TABLE 4. Number of newly detected RBC alloanti-bodies in transfused patients during years with noWBC reduction (1987), 40-percent WBC reduction

(1999), and 100-percent WBC reduction (2001)Antigen(s) 1987 1999 2001E 20 27 32C 5 5 6c 3 3 2Other Rh 2 4 2K 22 27 20Other Kell 1 0 1Kidd 9 14 7Duffy 10 6 7Ss 0 2 1MN 2 5 3Lewis 1 3 1P1 1 0 0Lutheran 3 1 0HTLA 2 3 0Bg 4 0 0Other 1 3 0

Total 86 103 82

(Table 3). In comparison, alloimmunization to antigens inall non-Rh, Kell, Kidd, and Duffy blood groups declined 76percent. This latter group consisted primarily of newlydetected antiglobulin-phase reactive antibodies to M,Lutheran, Lewis, P, HTLA, Bg, and Diego blood groups.

Alloantibodies to non-D antigens in the Rh bloodgroup did not decline significantly. Females accounted fora slightly increasing proportion of non-D Rh blood groupantigen-immunized patients during the time period: 24percent of newly alloimmunized patients in 1987, 25percent in 1999, and 33 percent in 2001, suggesting a pos-sible role for gender or previous pregnancy.

DISCUSSION

The ideal method for studying the question of whetherWBC reduction decreases RBC alloimmunization wouldbe a randomized trial with frequent immunohematologicscreening of all patients. Given the low incidence of RBCalloimmunization in transfused patients (<1/100), thiswould be a difficult, time-consuming, and expensivestudy to organize. As a first attempt to study this issue, weemployed a more practical preliminary approach to inves-tigating this issue—retrospective cohort studies.

One cohort included patients for whom immunohe-matologic follow-up was frequent and thorough. Patientswith AML in our hospital receive their care and transfu-sions almost exclusively here once the diagnosis has beenmade, and thus it is likely we detected most of the alloim-munizations that occurred in this cohort. The more com-plete follow-up, homogeneity of patient diagnosis, and the quantitatively larger alloimmunization rate decreasesseen in the AML cohort make these more robust observa-tions than the second cohort. However, one limitation tothis study design is that it was a before-and-after study ofWBC reduction and cannot fully account for changes intreatment protocols and patient mix. We doubt that suchchanges alone could account for a 74-percent reductionin alloimmunization prevalence because chemotherapeu-tic protocols for induction therapy in AML have notchanged very much during the past 20 years. However,consolidation therapy and marrow transplantation havebecome much more common during this period andcould conceivably have influenced our observations. Onthe other hand, consolidation therapy is probably lessimmunosuppressive than induction therapy, and marrowtransplant usually occurs a time point beyond the first 4to 6 months when most new alloantibodies were detected.Another limitation to this cohort is that AML patientsreceived myeloablative, immunosuppressive chemother-apy, and the observed results may not be typical of alloim-munization patterns in patients with intact immunologicfunction. Although we think it very unlikely, changes inimmunosuppression by anti-leukemic therapy cannot beruled out as the cause of our findings in this group.

A second cohort was selected consisting of all trans-fused patients with newly detected alloantibodies duringeach of three annual periods. The limitation to this cohortis that follow-up is not always complete. Furthermore,there have no doubt been changes in transfusion practicesand types of patients transfused during the period 1987 to2001, such as increases in liver and marrow or stem celltransplantation, that cannot be fully accounted for andmay affect the results we report. The ratio of antibodyscreens to transfusions did increase marginally from 1987(1.048) to 2001 (1.075), but this is a 2.6-percent increase.The number of RBC transfusions increased by 36 percent,yet the alloimmunization rate per antibody screen ortransfusion decreased 31 and 30 percent, respectively.Thus, the increase in antibody screens per RBC transfu-sion of 2.6 percent, even if it was entirely for nontrans-fused patients, could not mathematically account for a 30- to 31-percent drop in alloimmunization rate overall.Both study cohorts exhibited comparable alloimmuniza-tion incidences per 1000 RBCs transfused: non-WBC-reduced transfusion recipients in the leukemia and whole hospital cohorts developed 5.37 and 4.47 newalloantibodies per 1000 RBCs transfused, respectively, and WBC-reduced transfusion recipients from these twocohorts had 1.42 and 3.13 alloantibodies per 1000 RBCstransfused. One difference between the cohorts is that the denominator consists of all transfusions given specif-ically to the patients involved for the AML cohort and allRBC transfusions given to all patients that year in theannual whole hospital cohort. The alloimmunizationprevalence and incidence decreases associated with WBC-reduced transfusions were greater in male patientsin both cohorts, suggesting that the analysis is not con-founded by alloimmunizations due to pregnancy. Thequantitatively similar alloimmunization rates and qualita-tively similar decreases in these rates seen with WBC-reduced transfusions are consistent in both cohorts. It willbe important to see if similar estimates and trends areobserved in additional cohorts studied by prospectivemethods.

These data support the hypothesis that WBC reduc-tion of RBC transfusions alters the effective immuno-genicity of transfusions in stimulating alloantibodies toRBC antigens. The phenomenon appears general becauseit was observed in patients undergoing chemotherapy for AML, as well as in the hospital population as a whole.In a recent study of antigen-matched transfusions topatients with thalassemia, the authors noted that patientsreceiving WBC-reduced transfusions appeared to have alower RBC alloimmunization rate and speculated thatremoval of WBCs reduced the degree of recipient immuneactivation due to allogeneic transfusion.18

Although our data cannot rule out a role for WBCreduction modulating secondary immune responses toRBC antigens, the data are most consistent with a reduc-

BLUMBERG ET AL.


tion in primary immune responses. This is particularlytrue of the AML cohorts, which were followed longitudi-nally and continuously in our hospital after diagnosis.Most antibodies were not detected during the typical 4- to6-week induction therapy admission during which multi-ple antibody screens are performed, but rather weredetected months after the initial transfusion. Secondaryresponses are not usually impaired by immunosuppres-sive therapy and would be expected to be detectablewithin 1 to 2 weeks of restimulation.

The evidence suggests that the immune response to both moderately immunogenic antigens depending on amino acid sequence variation (such as Kidd, Kell, and Duffy antigens) and weakly immunogenic antigensdepending on carbohydrate structural variation (such asLewis and P) are reduced. The generation of new alloanti-bodies to antigens in the Rh system did not seem to beaffected by WBC reduction in one cohort (all patients) butwas observed to decrease by a factor of approximately six in the AML cohort (from 13 alloantibodies to 2). The reason for this difference is uncertain, but perhapsimmunosuppressive therapy in the AML cohort was addi-tive to the effects of WBC reduction in preventing alloim-munization to these relatively immunogenic antigens inthis group of patients.

The practical implications of these findings, if repli-cated by others, may be modest. The newly detected anti-bodies we studied accounted for no more than 10 percentof the antibodies detected in our transfusion service giventhat most antibodies detected are those previously knownto our service or secondary to pregnancy rather thantransfusion. A 30-percent reduction in non-Rh antibodiesand a 66-percent reduction in antiglobulin-reactive anti-bodies in the MN, Lewis, Lutheran, P, and HTLA bloodgroup systems would be a welcome and unexpectedbenefit from WBC reduction but of no great economic orclinical consequence in practice. If, however, the resultsseen in the AML patients turn out to be more typical, a 70-to 75-percent reduction in alloimmunization would be ofgreater importance. Although previous studies haveshown that AML patients are alloimmunized at similar fre-quencies to other patients,17 their receipt of myeloablativeand immunosuppressive therapy renders uncertainwhether our results in this small cohort will be seen inlarger, broader populations of transfused patients.Although the additional immunosuppressive therapygiven to the patients in the WBC-reduced AML cohortduring consolidation and transplant therapy might havereduced the likelihood of forming alloantibodies, thesepatients also received a slightly greater number of RBCtransfusions and were followed for much longer periodsof time. There were no differences in the number of RBCstransfused before, or the time interval to allosensitizationin the AML patients. This argues against increased treatment intensity accounting for the observation of

reduced allosensitization in recipients of WBC-reducedtransfusions.

Assuming they can be replicated by others, from a sci-entific standpoint, these results are of interest regardingthe subject of transfusion immunomodulation. There areextensive data demonstrating down regulation of cellularimmunity after allogeneic transfusion in both animalmodels and patients.10 The clinical consequences that arepotentially mediated by transfusion immunomodulationinclude a variety of outcomes, including reductions insolid organ allograft rejection and repetitive spontaneousabortions, and increases in postoperative bacterial infec-tions and solid tumor recurrence. One common thread inthese observations that could be explained by immuno-logic mechanisms is that each of these clinical outcomesis mediated in large part by Type 1 cellular immunity.9

Type 1 cellular immunity encompasses T helper, T cyto-toxic cell, and dendritic cell inflammatory functions thatfacilitate killing of intracellular microbial organisms andtumor cells.19–21 These functions have been demonstratedto be impaired in experimental studies of the immuno-logic effects of allogeneic transfusion.10 Conversely, Type 2immunity, involving humoral immune responses, partic-ularly to parasites, appears to be up-regulated by allo-geneic transfusion.6–8 The up regulation of Type 2immunity by allogeneic transfusion may account for thedown regulation of Type 1 immunity, as this is a well-established property of “immune deviation” toward Type2 immunity.4

Allogeneic WBCs have been implicated in animalmodels22 and randomized clinical trials23–25 as prime mediators of the clinical effects linked to transfusionimmunomodulation. Our data suggest that humoralimmune responses to RBC antigens may be reduced whenWBC reduction of RBC transfusions is employed. One possible mechanism could be reductions in the Type 2immune stimulation provided by allogeneic WBCs.

Alternative or additional mechanisms are possible.WBC reduction removes donor antigen-presenting cells(e.g., dendritic cells, B cells, and monocytes) bearing RBCantigen epitopes in their HLA peptide-binding sites, andthis may decrease the likelihood of alloimmunization.Non-WBC-reduced RBC transfusions contain largenumbers of platelets expressing CD40L and solubleCD40L (CD154) derived from platelets during storage.13,14

CD40L is a potent activator of B cells and is required forthe class switch from IgM to IgG production.15 Donorplatelet-derived soluble CD40L in stored blood can stim-ulate synthesis of PGE2

14 in vitro and conceivably couldactivate recipient B cells and facilitate antibody produc-tion and class switching. WBC reduction, at least in thecase of WBC reduction before storage, removes not onlyWBCs but also platelets from transfused RBC concen-trates. Thus, the postulated beneficial effects of WBCreduction on RBC alloimmunization could be, in part, due



to thrombocyte reduction. Because much of the WBCreduction performed in our AML cohort was bedside WBCreduction after storage, the removal of soluble CD40L isunlikely to explain the reduced RBC alloimmunizationseen in that group, but removal of platelet CD40L mightreduce the potential for alloimmunization. Finally, viabledonor WBCs in transfused blood may be perceived as dan-gerous nonself, as postulated by the danger model of theimmune system, and act as an adjuvant to stimulate recip-ient immunity in general.3

In summary, these observations suggest that WBCreduction of RBC transfusions may lead to reducedallosensitization to RBC antigens. This novel finding willrequire confirmation by others. In a recent preliminaryreport, Kekomäki and colleagues26 found that small dosesof WBC-reduced RBCs were unable to stimulate a primaryimmune response to the D antigen in healthy volunteers.

ACKNOWLEDGMENTS

We thank Dr. Erika Resetkova and Durga Singh for assistance in

data collection.

REFERENCES

1. Mollison PL, Engelfriet CP, Contreras M. Blood transfusion

in clinical medicine, 10th ed. Oxford: Blackwell, 1997.

2. Janeway CA, Travers P, Walport M, Shlomchik M. Immuno-

biology, 5th ed. Hamden, CT: Garland Press, 2001.

3. Matzinger P. Tolerance, danger, and the extended family

(review). Annu Rev Immunol 1994;12:991–1045.

4. Mosmann TR, Sad S. The expanding universe of T-cell

subsets. Th1, Th2, and more. Immunol Today

1996;17:138–46.

5. Romagnani S. Th1 and Th2 in human diseases. Clin

Immunol Immunopathol 1996;80:225–35.

6. Kirkley SA, Cowles J, Pellegrini VD Jr, et al. Cytokine secre-

tion after allogeneic or autologous blood transfusion.

Lancet 1995;345:527.

7. Babcock GF, Alexander JW. The effects of blood transfusion

on cytokine production by Th1 and Th2 lymphocytes in

the mouse. Transplantation 1996;61:465–8.

8. Kirkley SA, Cowles J, Pellegrini VD Jr, et al. Blood transfu-

sion and total joint replacement surgery: T helper 2 (Th2)

cytokine secretion and clinical outcome. Transfusion Med

1998;8:195–204.

9. Blumberg N, Heal JM. The transfusion immunomodulation

theory: the Th1/Th2 paradigm and an analogy with preg-

nancy as a unifying mechanism. Seminars Hematol

1996;33:329–40.

10. Blumberg N, Heal JM. Transfusion immunomodulation. In:

Anderson KC, Ness PM, eds. Scientific basis of transfusion

medicine, 2nd ed. Philadelpha PA: W.B. Saunders,

2000:427–43.

11. Vamvakas EC, Blajchman MA. Deleterious clinical effects

of transfusion-associated immunomodulation: fact or

fiction? Blood 2001;97:1180–95.

12. Deeg HJ, Aprile J, Storb R, et al. Functional dendritic cells

are required for transfusion-induced sensitization in

canine marrow graft recipients. Blood 1988;71:1138–40.

13. Henn V, Slupsky JR, Grafe M, et al. CD40 ligand on acti-

vated platelets triggers an inflammatory reaction of

endothelial cells. Nature 1998;391:591–4.

14. Phipps RP, Kaufman J, Blumberg N. Platelet derived CD154

(CD40 ligand) and febrile responses to transfusion. Lancet

2001;357:2023–4.

15. Grewal IS, Flavell RA. A central role of CD40 ligand in the

regulation of CD4+ T-cell responses. Immunol Today

1996;17:410–4.

16. Zhang Y, Cao HJ, Graf B, et al. CD40 engagement up-

regulates cyclooxygenase-2 expression and prostaglandin

E2 production in human lung fibroblasts. J Immunol

1998;160:1053–7.

17. Blumberg N, Peck K, Ross K, Avila E. Immune response to

chronic red blood cell transfusion. Vox Sang 1983;44:

212–7.

18. Singer ST, Wu V, Mignacca R, et al. Alloimmunization and

erythrocyte autoimmunization in transfusion-dependent

thalassemia patients of predominantly Asian descent.

Blood 2000;96:3369–73.

19. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2

macrophages and the Th1/Th2 paradigm. J Immunol

2000;164:6166–73.

20. Dong C, Flavell RA. Th1 and Th2 cells. Curr Opin Hematol

2001;8:47–51.

21. Romagnani S. T-cell subsets (Th1 versus Th2). Ann Allergy

Asthma Immunol 2000;85:9–18.

22. Blajchman MA, Bardossy L, Carmen R, et al. Allogeneic

blood transfusion-induced enhancement of tumor growth:

two animal models showing amelioration by leukodeple-

tion and passive transfer using spleen cells. Blood

1993;81:1880–2.

23. Jensen LS, Kissmeyer-Nielsen P, Wolff B, Qvist N. Ran-

domised comparison of leucocyte-depleted versus buffy-

coat-poor blood transfusion and complications after

colorectal surgery. Lancet 1996;348:841–5.

24. Tartter PI, Mohandas K, Azar P, et al. Randomized trial

comparing packed red cell blood transfusion with and

without leukocyte depletion for gastrointestinal surgery.

Am J Surg 1998;176:462–6.

25. van de Watering LM, Hermans J, Houbiers JG, et al. Benefi-

cial effects of leukocyte depletion of transfused blood on

postoperative complications in patients undergoing

cardiac surgery: a randomized clinical trial. Circulation

1998;97:562–8.

26. Salonvaara M, Eronen J, Helminen P, et al. Small doses of

cryopreserved O RhD positive leukocyte-reduced red

cells are not effective in primary immunization of RhD

negative voluntary men (abstract). Vox Sang 2002;83

(Suppl 2):65.

BLUMBERG ET AL.


WBC reduction of RBC transfusions is associated with a decreased incidence of RBC alloimmunization

Documents

Transcript of WBC reduction of RBC transfusions is associated with a decreased incidence of RBC alloimmunization