Newer diagnostic methods have led to an increased awareness for the role of antibodies in late allograft failure, the commonest cause of transplant kidney loss (1). With improved detection techniques (2, 3), there is now overwhelming evidence that circulating human leukocyte antigen antibodies (HLAab) are associated with graft rejection, delayed graft function, and poor long-term graft survival (1, 2, 4, 5). The presence of HLAab also lengthens waiting times on the deceased donor list which in turn is associated increased mortality (6). HLAab develop in the majority in response to sensitizing events, namely pregnancy, transplantation, and blood transfusion (7, 8). Two large prospective US studies looking at the prevalence of HLAab in the general population showed that pregnancy was the biggest risk for allosensitization and the number of pregnancies correlated with the prevalence of HLAab (9, 10).
The use of blood transfusion in renal patients, especially in potential transplant recipients has long been debated. Donor-specific blood transfusions were undertaken up to the early 1990s because of some evidence suggesting improved graft survival, especially in HLA nonidentical and mixed lymphocyte reactive kidney transplants (11, 12). Some centers routinely administered 3 to 5 units of blood before transplantation and further studies suggested that the transfusion did not need to be donor specific (13). The benefits of donor-specific transfusion were believed to outweigh sensitization rates of 20% to 30% detected by complement-dependent cytotoxicity (CDC) assays. Animal models suggested that the improved graft survival may have been related to the generation of T-regulatory cells and hence donor-specific tolerance (14). Although the mechanism of the donor-specific tolerance was not fully elucidated, the advent of cyclosporine and the use of triple immunosuppression regimens negated the benefits of transfusion; hence, it fell out of favor (15).
Blood transfusions were then avoided in potential recipients to prevent any possibility of sensitization and the subsequent risk of hyperacute or acute rejection. Transfusion avoidance was aided with the discovery of erythropoietin-stimulating agents that led to a marked reduction in the use of red cell transfusions (16). However, with increasing age and cardiovascular comorbidities in renal patients, there are circumstances when a blood transfusion may be necessary. This can occur when there is erythropoiesis-stimulating agents' resistance, a requirement for urgent replacement due to hemodynamic instability or for optimizing cardiovascular function. Where a transfusion was administered, the risk of sensitization was not believed to be as high with leuko-depleted blood (<5×106 white cells).
Whole blood leukodepletion was first explored to avoid some of the transfusion reactions associated with blood transfusion. Complications were a particular problem in hematological patients requiring significant supportive care with blood products such as patients with leukemia receiving chemotherapy, patients with aplastic anemia, or patients undergoing bone marrow transplantation. Early studies with the use of leuko-depleted blood showed a significant reduction in the occurrence of febrile reactions and cytomegalovirus transmission (17). Studies have also now demonstrated a significant reduction in the rates of allosensitization and platelet refractoriness (18–20).
Historically, leukodepletion was generally brought into practice in the 1990s to minimize the complications of “passenger leukocytes” on specific patient populations (some mentioned earlier) and consensus recommendations were made on available evidence at the time. Emerging evidence accompanied by improved and cheaper filtration technology, and better quality assurance and quality control, led to increasing adoption of a leukodepletion. After the demonstration in animal studies that transmissible spongiform encephalopathy infectivity is five to seven times higher in the buffy coat than in plasma (21), the United Kingdom was the first to undertake a universal leukodepletion policy to minimize the potential of a devastating epidemic. It became national policy in 1999, along with stringent standardization of leukodepletion procedures and monitoring according to national protocols and testing schedules. All processing centers in the United Kingdom now undertake in-line filtering and centrifugation to leuko-depleted blood, documentation of all blood transfusion episodes with traceability back to specific unit of blood was also implemented. Currently, 19 countries undertake a universal leukodepletion policy. With the introduction of universal leukodepletion, there was an assumption that HLA allosensitization in renal patients would be minimized. There are little hard data to support this assumption as there are still potential sources of HLA antigens in leuko-depleted blood including contaminating leukocytes, platelets (22), free floating major histocompatibility complex molecules (23), and the small amount of major histocompatibility complex expressed by RBCs (24). There may also be some cross reactivity between red cell antigens (25).
The source of sensitization in most male renal potential recipients awaiting their first transplant will be blood transfusion. Other sources of sensitization may be cross reactivity between artificial surfaces (26), microorganisms (27), ingested proteins, and allergens (28). Female recipients awaiting their first transplant may be sensitized to all of the above in addition to any previous pregnancies including unknown miscarriages. The only article to examine sensitization rates in potential renal transplant recipients in the postleukodepleted era was by Karpinski et al. (29). This study suggested that the risk of sensitization was not different between the pre- and postleukodepletion era. However, this study was compounded by patients with previous historical sensitization history from pregnancy and transplantation. A recent Dutch national study looked to see whether pretransplant blood transfusions improved outcomes in female recipients by “unmasking” historical sensitization events. They showed that in 15 of 103 (14.6%) patients, antibodies that were detected after transfusion resulted from historical sensitization events as they were directed against the partner and children or both. HLAabs against both the pretransplant blood donor and the partner or children were detected in 67 of 103 (65.0%) (30). This suggested that the detection of HLAab in the patients in the study by Karpinski et al. may have been heavily compounded by historical sensitization. We sought to quantify the risk of developing HLAab after blood transfusion in the postleukodepletion era per se, using the most sensitive techniques currently available. Hence, we studied the cumulative incidence of HLAab detected by solid phase and single bead luminex assays in previously unsensitized male patients awaiting renal transplantation. We also obtained transfusion histories for all these patients.
In all, 126 male patients awaiting their first kidney transplant with no previous history of blood transfusion in the preleukodepleted era (pre-1999) were identified. None of these patients had been the recipient of other transplanted organs. Ten patients were excluded as they had not had antibody testing in the previous 12 months and were suspended from the list. Details of the remaining 116 patients are shown in Table 1. Transfusion events in the postleukodepletion era recollected by patients were corroborated by electronic records on all patients. None of the 116 patients reported transfusion elsewhere or before coming under our care.
HLAab were detected in 10 of the 116 patients. These patients had longer waiting times when compared with unsensitized patients; 40.2 months (range, 2.0–74.4 months) vs. median 18.4 months (range, 0.6–108.0 months). There were no differences in the median age, and sera were tested a similar number of times. Twenty-one of the 106 unsensitized patients had previous surgery compared with 1 of the 10 patients with HLAab.
Relative Risk of Sensitization
Forty-two patients had a history of a blood transfusion in the postleukodepleted era and 74 patients did not receive a transfusion (control patients). Seven of the 42 (16.7%) had detectable HLAab compared with three of 74 control patients (4.1%) (P=0.02). Patients who had a history of a blood transfusion had a relative risk of sensitization of four compared with controls. The association between a blood transfusion and HLAab by chi-square analysis was marginally significant, P=0.05.
There was a trend toward sensitized patients having a higher number of transfusion episodes; median 4 (range, 1–20) against 2 (range, 1–9), P=0.09, and a higher number of total units; 8 (range, 4–42) against 3 (range, 1–29), P=0.04 when compared with those who were not sensitized (Table 2). Based on our cutoff values for detection, all HLAabs attributed to transfusion were directed against HLA class I antigens and none were directed against HLA class II.
Characteristics of the Patients With HLAab
Antibody specificities and characteristics of sensitized patients are shown on Table 3. Four of the seven sensitized transfused patients had unacceptable antigen specificities contraindicating transplantation. However, one of these patients had an initial HLAab screen that was positive, suggesting that only three patients “seroconverted” to the extent of generating unacceptable antigens after a blood transfusion while on the waiting list. The three remaining patients from the sensitized transfused group did not have any unacceptable antigens and therefore no absolute contraindication for transplantation.
There were three patients who developed HLAab with no transfusion history, one had previous surgery elsewhere and one had acute medical care at another hospital before coming under our care. Interestingly, their initial HLAab screen was negative.
Even though blood transfusions should be generally avoided in potential transplant recipients, there are clinical scenarios where they are necessary. The decision to transfuse requires careful consideration, balancing the risk of sensitization against the clinical need for rapid hemoglobin correction. The risk of sensitization is believed to be lower in the postleukodepletion era, and this study attempts to quantify this risk accurately using solid phase luminex bead HLA assays. We chose to study male patients awaiting their first kidney transplant to avoid confounding sensitization events such as pregnancy and previous transplantation. We also excluded patients who had transfusions in the preleukodepletion era. The only other study to have looked at allosensitization in the leukodepletion era was by Karpinski et al. (29), this study did not exclude patients with potential confounding factors. A Dutch national study which looked at the use of pretransplant transfusions to unmask historical sensitization and improve graft outcomes in women showed a significant rate of historical sensitization. The article by Karpinski et al. failed to show any difference in the sensitization rates after a transfusion episode in groups that received blood in the pre- and postleukodepletion era. This may be explained by not excluding patients who may have had a historical sensitization event, leading to historical HLAabs being unmasked by the transfusion episode as opposed to de novo sensitization.
The article by Karpinski et al. undertook a subanalysis of a low-risk subgroup with no previous transfusion episodes, no previous transplants or pregnancy and found the risk to be 10% (3 of 31) and 8% (1 of 13) in the pre- and postleukodepletion era, respectively. It is unclear how many patients were male and female in this subgroup as female patients may have historical sensitization to their partners through unknown miscarriages. In our study where we tried to exclude all patients with potential historical sensitization, we found the risk of allosensitization to be 17% in patients who had received one or more blood transfusions, giving a fourfold relative risk when compared with controls.
Our study differs in one important aspect; we have tried to eliminate all confounding factors, namely historical sensitization from previous pregnancy, previous transplantation, and previous transfusion by our case selection. Furthermore, by examining a group of previously unsensitized patients, we are able to obtain the cumulative prevalence of HLAab by using results of repeated testing whereas the patient was on the waiting list as opposed to pre- and posttransfusion sera testing. This would increase the chances of detecting antibodies due to the undulating nature of circulating HLAab. Our use of repeated testing may account for the higher sensitization rates observed in our study than in previously described low risk group in the study by Karpinsky et al. Most importantly, however, is that our sample size for calculating the sensitization rate from the previously unsensitized patients is much larger and therefore we believe it gives a more accurate picture.
We acknowledge that this is a retrospective study using a small cohort of patients. Our primary aim was to show the relative risk between sensitization in the transfused group versus the nontransfused group (control), but we also managed to demonstrate a marginal significance by chi-square analysis in the association between blood transfusion and HLAab-positive patients. We have not made a comparison between the pre- and postleukodepleted era and cannot comment on any possible differences. Leukodepletion has not been shown to be the standard of care in reducing allosensitization rates outside of the hematological malignancy arena.
This study depends heavily on patient recall and although we can corroborate information with electronic data for patients transfused in the posttransfusion era, there will be circumstances where this is not possible. This may be when patients started receiving medical care in another center or in another country and not have reported the event due to lack of recall. Indeed, this may explain the small number of sensitized patients who may not recollect having a blood transfusion. Nonetheless, this limitation would bias the study against us, but we have nevertheless shown a significant difference. Sensitization in our patients with no history of a blood transfusion may have occurred through other unconventional routes such as cross reactivity with microbial antigens, ingested proteins, allergens (22, 24), or foreign objects (22). However, none of the antibodies in these patients were known to be due to cross reaction from microbial antigens. Interestingly, all these patients had initial HLAab screens that were negative and were only shown to be positive with serial testing, demonstrating that historical sensitisation from blood transfusion can occur with men.
With increasing numbers of patient on the transplant waiting list and a shortage of the donor pool, maintenance of a functioning graft for as long as possible should always be the goal. Sensitized patients have longer waiting times on the transplant list and shorter graft survival after transplantation. Although hyperacute rejection is rare in CDC crossmatch negative transplantation, there is now increasing recognition of the role of HLAab as detected by Luminex in delayed graft function, rejection, and chronic graft failure (3). Inadvertent sensitization of a potential transplant patient should always be avoided; however, most clinicians recognize that there are circumstances when the clinical need for a blood transfusion outweighs the risk of sensitization. The data provided in this study will help clinical decision making and appropriate counseling of patients with regards to the risk of allosensitization after transfusion of leuko-depleted blood.
MATERIALS AND METHODS
The Barts and the London NHS Trust is a tertiary referral transplantation center serving a population of approximately 2.2 million people. We identified all male patients on the renal transplant waiting list, excluding those with previous failed transplants. A complete transfusion history was sought. Transfusion episodes and the number of units used were obtained from the hospital blood bank. Patients were also directly questioned whether they had received a blood transfusion while under our care, elsewhere, or before coming under our services. All transfusion episodes that occurred while the patient was under our care were corroborated by the blood bank records. In addition, we sought a surgical history from patients to look for any possible transfusion event that the patient may be unaware of. Those with a known or documented transfusion in the preleukodepletion era before being listed were excluded from further analysis.
Universal leukodepletion is undertaken at a single blood processing center that is responsible for the supply of blood products to the Royal London Hospital and hospital with its affiliated satellite dialysis units. The UK specification for leukodepletion is that a minimum of 99% of leukocyte-depleted components from relevant processes should have less than 5×106 leukocytes with 95% confidence and more than 90% of the components should contain less than 1×106 leukocytes. Our particular blood processing center undertakes leukodepletion by in-line filtration and centrifugation, with quality control measures undertaken to ensure adequate preparation of blood before transport to the hospital. Filtration is the commonest method of leukodepletion used, but any validated method can be used. Not all components are assessed for effectiveness of leukodepletion, but a recognized statistical process monitoring methodology is used for surveillance (31).
Solid Phase Testing
Sera are tested on patients on the waiting list approximately every 6 months. Patients are usually bled on dialysis or when they attend transplant assessment clinics. Blood is withdrawn in a vacutainer with a clot activator. All samples are sent to the clinical transplantation laboratory at the Royal London where samples are process. Blood is the spun at 1600g and the supernatant/serum is collected and analyzed. Remaining serum is aliquoted and stored at −80°C.
The Royal London Hospital uses the services of a single clinical transplantation laboratory. Luminex Labscreen mixed assay (One Lambda Inc., Canoga Park, CA) was introduced in 2006 and manufacturer's suggested guidelines are used for assay cutoff (normalized background ratio of 2.2). In those in whom HLA antibodies were detected, the HLA specificities were defined using Luminex single antigen beads. A positive result was defined according to the manufacturer's criteria, namely a readout 1.5 times higher than background. This was validated using receiver operator characteristic curve analysis where we successfully selected optimum cut off point 4.5 for HLA class I and 3.5 for class II antibodies. In sensitized patients in the United Kingdom, a calculated HLA antibody reaction frequency is obtained by comparing patient's unacceptable HLA specificities with the HLA types of donors of identical blood group from a pool of 10,000 donors on the UK Transplant database. The definition of an unacceptable HLA specificity varies between centers. We were the first center to published data on the relevance of luminex-defined antibodies and our center is one of few in the United Kingdom that still uses CDC to define unacceptable antigens for class I antibodies. Our center defines unacceptable as such: class I antibody specificities have to be CDC defined and Luminex positive with a mean fluorescence intensity (MFI) more than 2000; specificities positive by Luminex only are listed as “acceptable ”; however, for class II antibody specificities with MFI more than 2000 and any other cross-reactive antigen with MFI less than 2000. For all patients, calculated HLA antibody reaction frequency value is worked out using the class I and class II “unacceptable specificities” present in patient sample and is sent to UK Transplant to indicate level of sensitization.
Information was collated and analyzed in Microsoft Excel. Statistical analysis was performed using SPSS. Relative risk was calculated between the groups. Comparisons between groups were by Mann-Whitney analysis.
The authors acknowledge Colin Barber, from the Blood Bank, Department of Hematology, The Royal London Hospital, for his help in obtaining the transfusion histories.
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