J Am Soc Nephrol 15: 818–824, 2004 Leukocyte Reduction of Red Cell Transfusions Does not Decrease Allosensitization Rates in Potential Kidney Transplant Candidates

MARTIN KARPINSKI,* DENISE POCHINCO,† IGA DEMBINSKI,† WILLIE LAIDLAW,† JAMES ZACHARIAS,* and PETER NICKERSON*† *Department of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; and †Immunogenetics Laboratory, Winnipeg Blood Center, Winnipeg, Manitoba, Canada

Abstract. A significant proportion of potential kidney trans- as being at high risk of allosensitization (previous pregnancy, plant candidates continue to periodically require blood trans- transplant, or five or more previous transfusions) or at low risk fusions that carry a risk of allosensitization. Leukocyte reduc- (no previous allogeneic exposures) (high risk: non-LR 52% tion (leukoreduction) of blood products has been proved to versus LR 55%; low risk: non-LR 10% versus LR 8%). Mul- reduce transfusion-associated allosensitization in patients with tivariate analysis revealed previous pregnancy to be the only hematologic malignancies; however, the effect in potential significant risk factor associated with transfusion-associated kidney transplant candidates is unknown. A total of 112 kidney allosensitization (relative risk, 8.2; 95% confidence interval, transplant candidates who received transfusions 2.4 to 24.0; P ϭ 0.0001). Leukoreduction, in particular, was while on the transplant waiting list were identified retrospec- not associated with any protective effect. In summary, leukore- tively. Sixty received a transfusion before leukoreduction (non- duction of red blood cell transfusions does not confer any LR), and 52 received a transfusion after the local implemen- protection against transfusion-associated allosensitization for tation of universal leukoreduction of blood products (LR). potential kidney transplant candidates. Physicians who care for There was no difference in transfusion-associated allosensiti- patients with ESRD must continue to practice careful transfu- zation rates in patients who received a transfusion during the sion avoidance while alternative strategies to minimize trans- two eras (non-LR 27% [16 of 60] versus LR 33% [17/52]; NS). fusion associated allosensitization are sought. Likewise, no difference was observed in subgroups identified

Despite the fact that recombinant erythropoietins have substan- Allosensitization is associated with significant barriers to tially decreased the need for transfusions in patients with successful transplantation in patients with ESRD, including ESRD, United Network for Organ Sharing (UNOS) data indi- prolonged waiting times and inferior graft outcomes (6–8). cate that approximately 30% of wait-listed transplant candi- Accordingly, any measure to limit allosensitization would rep- dates continue to receive red blood cell (RBC) transfusions at resent a substantial advance for ESRD patients. Of the three some point before transplantation (1, 2). In the past, some principal causes of allosensitization—pregnancy, transplanta- transplant programs administered deliberate pretransplant tion, and transfusions—only the last is perhaps modifiable. transfusions aimed at optimizing graft outcomes (i.e., the ben- Leukocyte reduction of blood products (leukoreduction) re- eficial transfusion effect); however, more recent data indicate duces the transfused load of allogeneic leukocytes and has that this beneficial effect is no longer apparent, perhaps as a been proved to limit transfusion-associated allosensitization in result of improving graft outcomes overall (2–5). Concerns of patients with hematologic malignancies undergoing chemo- transfusion-associated allosensitization persist for potential therapy (9). transplant candidates, and it is likely that the majority of The impact of RBC leukoreduction on allosensitization in current transfusions are administered for other clinical ESRD patients is unknown. The few studies that have exam- indications. ined this practice either have been uncontrolled or have screened for allosensitization using technically inferior anti- Received October 9, 2003. Accepted December 12, 2003. HLA antibody screening techniques (10, 11). Several recent Correspondence to Dr. Martin Karpinski, University of Manitoba, Room studies have highlighted the superior sensitivity of flow cyto- GE421B, Health Sciences Centre, 820 Sherbrook Street, Winnipeg, MB, metric anti-HLA antibody screening (FlowPRA) (12–14). We Canada R3A 1R9. Phone: 204-787-1524; Fax: 204-787-3326; E-mail:[email protected] thus set out, in this retrospective cohort study, to use sensitive 1046-6673/1503-0818 flow cytometric techniques to determine whether universal Journal of the American Society of Nephrology RBC leukoreduction has reduced the incidence of transfusion- Copyright © 2004 by the American Society of Nephrology associated allosensitization in potential kidney transplant can- DOI: 10.1097/01.ASN.0000115399.80913.B1 didates within our center. J Am Soc Nephrol 15: 818–824, 2004 Leukocyte Reduction and Allosensitization Rates 819

Materials and Methods flow cytometric technique (FlowPRA; OneLambda). Both screening Universal Leukoreduction in Canada assays were performed in the Immunogenetics Laboratory at the All blood products within Manitoba are distributed by a single Winnipeg Blood Centre using standard techniques previously de- agency, Canadian Blood Services, and since September 1999, all RBC scribed (13). A patient was considered sensitized before a transfusion Ն units distributed within Manitoba have been leukoreduced in compli- when the AHG-CDC PRA was 10% and/or when the FlowPRA ance with a nationwide Health Canada directive (15). This directive assay revealed any detectable anti-HLA antibodies. Transfusion-as- was issued in response to numerous lines of evidence indicating that sociated sensitization was defined as the de novo appearance of a leukoreduction of blood products likely reduces the incidence of positive FlowPRA or as an increment in the FlowPRA value of Ն several adverse transfusion reactions, including allosensitization. The 10%. Winnipeg Blood Centre now performs universal prestorage leukore- duction of RBC units with commercially available in-line filtration Statistical Analyses systems (Leukotrap WB and RC PL; Pall Medical, East Hills, NY), Statistical analysis was performed using Statview 5.0 software and the maximum accepted residual white blood cell (WBC) count is (SAS Institute, Cary, NC). Values are reported as mean Ϯ SEM or, Ͻ ϫ 6 ϫ 9 5 10 /unit (normal WBC content approximately 5 10 /unit). where indicated, as medians and ranges. The ␹2 test was used for Internal quality control testing is applied to at least 1% of all units, and comparison of categorical variables, whereas the t test was applied to ϫ the actual residual WBC content is observed to be approximately 3 comparisons of continuous variables. P Յ 0.05 was considered to be 5 10 /unit (unpublished data, Canadian Blood Services/Pall Corp.). significant, and values Ͼ0.10 are reported as nonsignificant (NS). In the multivariate analysis of risk factors for allosensitization, univariate Study Procedures risk factors associated with the outcome with P Յ 0.10 were allowed This study was approved by the University of Manitoba Biomed- into the final model. These included a ϩve FlowPRA before transfu- ical Research Ethics Board. The study population consisted of patients sion, previous pregnancy, previous transplantation, previous transfu- who were on the Manitoba renal transplant waiting list and had sions, and the number of RBC units transfused in the episode under received RBC transfusions while wait-listed for transplantation. None study. Leukoreduction was considered in the models despite being of the transfusions administered was prescribed as deliberate pretrans- found to be nonsignificant in univariate analysis. Pregnancy and plant transfusions aimed at optimizing graft outcomes. Sera for anti- previous transfusions were considered as both categorical and contin- HLA antibody screening on wait-listed patients were collected bi- uous variables in the models analyzed. There was no demonstrable monthly during the period of study as well as 2 to 4 wk after any relationship between increasing numbers of pregnancies or transfu- transfusion. Serum collection and transfusions are tracked meticu- sions and an increasing incidence of allosensitization, and the overall lously by local transplant coordinators and Immunogenetics Labora- strength of the model was superior when these were considered as tory technologists. Adult transplant candidates who received RBC categorical variables. For these reasons, five or more previous trans- transfusions between January 1996 and June 2003 thus were identified fusions was chosen as the transfusion variable, and this cutoff is also for retrospective study, and of 112 wait-listed ESRD patients identi- supported by previous studies (16). fied, 60 received RBC units before the implementation of universal leukoreduction and 52 thereafter. Individuals who were broadly sen- sitized (FlowPRA Ն80%) before transfusion were excluded (n ϭ 3). Results Patient data and transfusion records were abstracted from Manitoba During the period of study, 112 individuals on the renal Renal Program database. transplant waiting list received RBC transfusions and had appropriate pre- and posttransfusion serum samples collected Anti-HLA Antibody Screening for anti-HLA antibody screening. Sixty patients received a Transfusion-associated allosensitization was determined by anti- transfusion before universal leukoreduction (non-LR) and 52 HLA panel reactive antibody (PRA) screening pre- and posttransfu- thereafter (LR). There were significant baseline demographic sion. Sera were batched and then screened concurrently by both the differences between these two groups (Table 1). Patients who anti-human globulin cytotoxicity technique (AHG-CDC PRA) and a received leukoreduced transfusions were more likely to have

Table 1. Baseline demographicsa

Pre-leukoreduction Leukoreduction (n ϭ 60) (n ϭ 52) P

Gender (male/female) 39/21 28/24 NS Age when transfused 40 Ϯ 242Ϯ 2NS Pregnancy 13 20 NS No. of pregnancies (median, range) 2 (0–7) 2 (0–12) NS Previous transplant 6 8 NS Previous RBC transfusion 18 (30%) 33 (63%) Ͻ0.001 Ն5 Previous RBC transfusions 8 18 0.008 ϩve FlowPRA pretransfusion 12 (20%) 20 (38%) 0.03 Units transfused 3 Ϯ 0.4 3 Ϯ 0.3 NS

a RBC, red blood cell. 820 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 818–824, 2004 had a previous transfusion, were more likely to have received in only 22 (67%) of the 33 patients who developed transfusion- five or more RBC transfusions in the past, and were more associated allosensitization as determined by FlowPRA. likely to be allosensitized before the transfusion episode under examination (Table 1). Both groups received the same mean Risk Factors for Transfusion-Associated number of RBC units in the transfusion episode under study. Allosensitization In univariate analysis, factors that correlated with an in- creased likelihood of transfusion-associated allosensitization Transfusion-Associated Allosensitization and included a ϩve FlowPRA before transfusion, previous preg- Leukoreduction nancy, and five or more previous RBC transfusions (Table 3). The overall rates of transfusion-associated allosensitization In multivariate regression analysis, only previous pregnancy were 27% (16 of 60) in the population that received standard was associated with an increased risk of transfusion-associated RBC units and 33% (17 of 52) in those who received leukore- allosensitization (relative risk, 8.2; 95% confidence interval, duced RBC (NS; Table 2). Of the 33 individuals who met the 2.8 to 24.0; P ϭ 0.0001). Leukoreduction per se was not found definition of transfusion-associated allosensitization, 16 were to be protective in either univariate or multivariate analyses. previously unsensitized and 17 demonstrated a ϩve FlowPRA Rates of allosensitization were similar for women who had a before transfusion. Fifteen of the 33 individuals developed history of pregnancy and received either standard or leukore- isolated new HLA class I antibodies, six developed isolated duced transfusions (9 [69%] of 13 non-LR versus 11 [55%] of class II antibodies, and 12 developed new class I and II 20 LR; NS). antibodies. In previously unsensitized patients, the mean HLA Ϯ class I and class II FlowPRA posttransfusion became 53 8% Discussion and 34 Ϯ 11%, respectively, whereas for previously sensitized A significant proportion of patients with ESRD are denied patients, the mean increment in the class I and class II Flow- the full potential benefits of transplantation as a result of PRA was 35% and 39%, respectively (class I pre, 44 Ϯ 8%; allosensitization. Allosensitized patients experience longer post, 79 Ϯ 5% [P Ͻ 0.01]; class II pre, 35 Ϯ 7%; post, 74 Ϯ waiting times for finding compatible donors and are at risk of 9% [P Ͻ 0.01]). There was no significant difference in either inferior graft outcomes transplanted (6). The barrier created by the degree (% ⌬PRA) or the nature of allosensitization (HLA allosensitization is exemplified by the fact that approximately class I and/or class II) that developed in patients who received 30% of UNOS wait-listed renal transplant candidates are al- standard versus leukoreduced transfusions (data not shown). losensitized yet only approximately 10% of transplants are Fifty-two of 112 patients were considered to be at high risk performed in sensitized recipients (6). Of the three principal of transfusion-associated allosensitization on the basis of hav- causes of allosensitization—pregnancy, previous transplanta- ing had previous allogeneic exposures (previous pregnancy, tion, and transfusions—only the last is potentially modifiable. previous transplantation, and five or more previous RBC trans- Nephrologists who care for ESRD patients are well aware of fusions), whereas 44 of 112 were considered to be at low risk the risk of deleterious allosensitization and are careful to avoid (no previous allogeneic exposures). No effect of leukoreduc- unnecessary transfusions; however, this patient population re- tion on allosensitization rates was seen in either of these two mains at risk of periodically requiring allogeneic transfusions. subgroups (high risk, 52% non-LR versus 55% LR [NS]; The UNOS database indicates that approximately 30% of wait- low-risk, 10% non-LR versus 8% LR [NS]; Table 2). listed transplant candidates continue to require blood transfu- AHG-CDC PRA was positive in only six (19%) of 32 sions at some point before transplantation (2). patients who displayed a ϩve FlowPRA before transfusion. Recently, several groups reported their experience with Similarly, AHG-CDC PRA detected new anti-HLA antibodies novel immunosuppressive protocols incorporating intravenous immunoglobulin and to enable the successful transplantation of sensitized recipients (17–19). Although en- Table 2. Transfusion-associated allosensitization ratesa couraging, these protocols are available to only a small pro- portion of sensitized potential recipients and will likely do little Transfusion-Associated to address the disparity in access to transplantation. Strategies Allosensitization P to prevent allosensitization are likely to have a greater impact for ESRD patients. Pre- Leukoreduction leukoreduction Leukoreduction of blood products reduces the load of allo- geneic HLA in transfusions and has been proved to diminish All patients (n ϭ 112) 16/60 (27%) 17/52 (33%) NS allosensitization rates in patients who have hematologic ma- High risk (n ϭ 52) 12/23 (52%) 16/29 (55%) NS lignancies undergoing chemotherapy (9). Several randomized (previous pregnancy, trials have reported a benefit in this population (20–27). The Tx, Ն5 tf) TRAP study, most notably, randomized Ͼ200 patients with Low risk (n ϭ 44) (no 3/31 (10%) 1/13 (8%) NS acute leukemia to receive leukoreduced RBC and either un- previous pregnancy, modified platelet preparations or irradiated, filtered, or aphere- Tx, or tf) sed platelet concentrates (27). Patients who received any of the a Tx, transplant; tf, transfusion. modified platelet products were significantly less likely to J Am Soc Nephrol 15: 818–824, 2004 Leukocyte Reduction and Allosensitization Rates 821

Table 3. Risk factors for transfusion-associated allosensitizationa

Risk Factors (RR, 95% CI)

Univariate P Multivariate P

FlowPRA ϩve pretransfusion 4.5 (1.9–11) Ͻ0.001 2.4 (0.8–7.1) 0.10 Pregnancy 7.8 (3.1–19.5) Ͻ0.001 8.2 (2.8–24) 0.0001 Previous transplant 2.8 (0.9–8.7) 0.08 2.4 (0.6–9.9) NS Ն5 Previous transfusions 5.1 (2.0–13.1) Ͻ0.001 2.6 (0.8–8.8) NS Leukoreduction 0.5 (0.3–1.7) NS 2.0 (0.7–6.0) NS RBC units given (per unit) 1.1 (1.0–1.3) 0.10 1.1 (0.9–1.3) 0.10

a RR, relative risk; CI, confidence interval. develop new anti-HLA antibodies and become refractory to (29–31). These anti-HLA antibodies detected solely by flow platelet transfusions (17 to 21% versus 45%; P Ͻ 0.001). It cytometry are clinically relevant and have been increasingly must be noted, however, that studies that have examined leu- associated with adverse outcomes posttransplantation (12–14, koreduction and allosensitization have almost exclusively been 31–34). performed in this patient population. Patients therein have also We observed previous pregnancy to be the strongest risk received large absolute numbers of transfusions with both factor for transfusion-associated allosensitization, a finding in platelet and RBC preparations (e.g.,14Ϯ 11 platelet and 15 Ϯ keeping with previous observational studies (35, 36). Other 7 RBC transfusions in the TRAP study). allogeneic exposures, such as a previous transplant or previous There are comparatively few data on the impact of leukore- transfusions, have also been reported to be risk factors for duction on allosensitization as a result of RBC transfusions in transfusion-associated allosensitization, although did not reach patients with ESRD. In the 1980s, SanFilippo et al. (10) statistical significance in multivariate analysis herein (35, 36). conducted a randomized study transfusing renal transplant This may represent a limitation of the size of our data set or, candidates with either standard or leukoreduced RBC units and alternatively, an accurate representation of risk factors within found no difference in allosensitization. Importantly, there was our patient population. Notably, leukoreduction was not asso- no assessment of the extent and consistency to which leukore- ciated with any protective effect in either univariate or multi- duction was achieved with the techniques applied, and anti- variate analysis. HLA antibody screening was performed with the AHG-CDC This study is retrospective, and ideally a randomized, con- technique, which is less sensitive than current flow cytometric trolled trial would be performed to evaluate the impact of techniques. Christiaans et al. (11) examined potential trans- leukoreduction on allosensitization in potential renal transplant plant candidates who were given leukoreduced RBC transfu- recipients. This, however, is unlikely to occur. Many blood sions and reported that de novo HLA class I antibodies devel- distribution organizations have in recent years adopted a policy oped in only 6% when screened by flow cytometry. This study, of universal leukoreduction of blood products, and although however, was uncontrolled and examined only low-risk, pre- not without controversy, this practice is now widespread (37, viously unsensitized patients. Limited literature also exists in 38). In Canada and most of Europe, universal leukoreduction other, nonrenal patient populations. Recently, Van de Watering has been in place for several years, whereas approximately et al. (28) randomized Ͼ400 patients who were undergoing 70% of U.S. RBC units are currently leukoreduced before cardiac surgery to receive either standard or leukoreduced RBC distribution. Organizations that monitor transfusion standards transfusions and observed no difference in allosensitization are unlikely to permit a change to previous blood-handling rates in either unsensitized or previously sensitized patients. procedures; thus, retrospective studies such as this are neces- In the current study, we found no significant difference in sary to investigate the effects of leukoreduction in patient the rate of transfusion-associated allosensitization in renal populations other than those with hematologic malignancies transplant candidates who received either standard or leukore- (39). duced RBC transfusions (27% versus 33%, respectively; mean, Several reasons may underlie why leukoreduction fails to 3 Ϯ 0.3 transfusions). Furthermore, similar rates and degrees diminish allosensitization rates in patients with ESRD. Indi- (i.e., ⌬%PRA) of allosensitization were seen in both low-risk viduals with ESRD are likely more immunocompetent than and high-risk patients who received transfusions of leukore- those who have hematologic malignancies and undergo treat- duced blood. The observed rate of allosensitization in high-risk ment with myeloablative chemotherapy. This is supported by patients is slightly higher than previously reported, and this is the similar sensitization rates in the two populations, despite likely attributable to the superior sensitivity of the flow cyto- the considerably greater overall exposure to allogeneic blood metric screening technique that we used. Studies using CDC products in the latter. The mean number of RBC units trans- techniques have reported allosensitization rates of approxi- fused in the current study was 3 Ϯ 0.3, whereas patients who mately 30% in high-risk recipients, in contrast to the approx- received leukoreduced products in the TRAP trial, in which imately 50% rate that we observed with FlowPRA screening approximately 20% became allosensitized, received fivefold 822 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 818–824, 2004 this amount of platelet and RBC transfusions (27). Similarly, duction of erythropoietin. Nephrol Dial Transplant 13: 2027– the degree of leukoreduction achieved with current techniques 2032, 1998 may be inadequate to prevent allosensitization in ESRD pa- 2. Hardy S, Lee SH, Terasaki PI: Sensitization 2001. Clin Transpl tients. Quality control assessments performed for Canadian 271–278, 2001 Blood Services reveal reliable three to four logfold reductions 3. Hiesse C, Busson M, Buisson C, Farahmand H, Bierling P, Benbunan M, Bedrossian J, Aubert P, Glotz D, Loirat C, Ron- in RBC unit leukocyte content. However, it may be that the deau E, Viron B, Bleux H, Lang P: Multicenter trial of one ϫ 5 residual leukocyte content (approximately 3 10 /unit) rep- HLA-DR-matched or mismatched prior to ca- resents a sufficient residual exposure to allogeneic HLA to daveric renal transplantation. Kidney Int 60: 341–349, 2001 induce an alloimmune response. Current leukoreduction tech- 4. Opelz G, Vanrenterghem Y, Kirste G, Gray DW, Horsburgh T, niques are similar in the degree of leukoreduction achieved, Lachance JG, Largiader F, Lange H, Vujaklija-Stipanovic K, and there are no clinical data to favor current cutoff standards Alvarez-Grande J, Schott W, Hoyer J, Schnuelle P, Descoeudres of approximately 1 to 5 ϫ 106/unit for minimizing allosensi- C, Ruder H, Wujciak T, Schwarz V: Prospective evaluation of tization (9, 38, 40–42). It is interesting that rodent models pretransplant blood transfusions in cadaver kidney recipients. suggest that too great a degree of leukoreduction may in fact Transplantation 63: 964–967, 1997 promote allosensitization, although the clinical relevance of 5. Alexander JW, Light JA, Donaldson LA, Delmonico FL, this is unknown (43). Finally, leukocytes are not the sole Diethelm AG, Wilkinson A, Rosenthal JT, Thistlethwaite JR, Hunsicker LG, Matas AJ, First MR, Reinsmoen NL, Rose SM: source of allogeneic HLA in transfusions as soluble HLA and Evaluation of pre- and posttransplant donor-specific transfusion/ even RBC-bound HLA are present as well, and these are not cyclosporine A in non-HLA identical living donor kidney trans- diminished by leukoreduction (44–46). plant recipients. Cooperative Clinical Trials in Transplantation If standard leukoreduction fails to diminish allosensitization Research Group. 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