|Year : 2021 | Volume
| Issue : 1 | Page : 39-44
Use of panel reactive antibodies for immunological risk stratification and its correlation with antecedent events in live donor kidney transplant: A prospective single-center study
Ashwini B Gadde1, Amit Mahapatra1, Assem Tiwari2, Sidharth Kumar Sethi1, Pranaw Kumar Jha1, Manish Jain1, Dinesh Yadav1, Dinesh Bansal1, Shyam Bihari Bansal1
1 Department of Nephrology and Kidney Transplantation, Medanta Kidney and Urology Institute, Medanta - The Medicity, Gurgaon, Haryana, India
2 Department of Laboratory Medicine, Pathology and Blood Bank, Laboratory Medicine, Pathology and Blood Bank, Medanta - The Medicity, Gurgaon, Haryana, India
|Date of Submission||14-Jun-2020|
|Date of Acceptance||05-Sep-2020|
|Date of Web Publication||31-Mar-2021|
Dr. Shyam Bihari Bansal
Department of Nephrology and Kidney Transplantation, Medanta Kidney and Urology Institute, Medanta - The Medicity, Sector 38, Gurgaon - 122 001, Haryana
Source of Support: None, Conflict of Interest: None
Background: Panel reactive antibody (PRA) is a screening test to identify the immunological sensitization of the transplant recipients. The current study focuses on the role of PRA in immunological risk stratification in live donor kidney transplant and its correlation with antecedent events and posttransplant outcomes. Methods: This was a prospective, single-center, observational study. All patients with end-stage renal disease between January 1, 2016, and October 1, 2016, were included. The patient's sensitization history and other immunological workup were recorded. Patients were followed up for 6 months. Results: PRA positivity was seen in 29/185 (15.7%) patients. Female sex (P = 0.004), longer duration renal replacement therapy (P = 0.037), and hepatitis C virus infection positivity (P = 0.047) were significantly associated with PRA positivity. Only 3/87 (3.4%) patients who had no history of sensitizing events had high PRA. Less number of patients in PRA-positive group received transplant as compared to PRA-negative group (12/29; [41.4%] vs. 143/156 [91.7%, P < 0.0001]). In transplanted patients, there was no significant difference between PRA-positive and PRA-negative group in terms of serum creatinine (1.22 ± 0.26 mg/dl vs. 1.30 ± 0.38 mg/dl, P = 0.63) and number of acute rejection (1 vs. 6, P = 0.507). Conclusions: PRA positivity is not uncommon in live donor kidney-transplant patients. Previous transplants, pregnancies, and multiple blood transfusions are important sensitizing events. PRA testing was useful in identifying high-risk patients and evaluating them further. There was no difference in short term outcomes between PRA-positive and PRA-negative groups after exclusion of high-risk patients from transplantation.
Keywords: Kidney transplant, live donor, panel reactive antibody, sensitization
|How to cite this article:|
Gadde AB, Mahapatra A, Tiwari A, Sethi SK, Jha PK, Jain M, Yadav D, Bansal D, Bansal SB. Use of panel reactive antibodies for immunological risk stratification and its correlation with antecedent events in live donor kidney transplant: A prospective single-center study. Indian J Transplant 2021;15:39-44
|How to cite this URL:|
Gadde AB, Mahapatra A, Tiwari A, Sethi SK, Jha PK, Jain M, Yadav D, Bansal D, Bansal SB. Use of panel reactive antibodies for immunological risk stratification and its correlation with antecedent events in live donor kidney transplant: A prospective single-center study. Indian J Transplant [serial online] 2021 [cited 2021 Jul 30];15:39-44. Available from: https://www.ijtonline.in/text.asp?2021/15/1/39/312753
| Introduction|| |
Kidney transplantation is the preferred treatment modality for most patients with end-stage renal disease (ESRD), because of better long-term survival and quality of life as compared to dialysis patients., There are many barriers to kidney transplant, i.e., donor availability, immunological suitability, medical fitness, and financial implications. Immunological barrier is mainly due to the presence of pretransplant anti-human leukocyte antigen (HLA) antibodies, which are formed as a response to sensitizing events, such as blood transfusions, previous pregnancies, or previous transplant. The aim of pretransplant immunological workup is to detect these anti-HLA antibodies, especially donor-specific antibodies (DSAs).
Panel reactive antibody (PRA) is a screening immunological test to assess the sensitization of the recipient to anti-HLA antibodies, which is expressed in percentages. Role of PRA in the organ allocation system in deceased donor waiting list is widely used since many years. Patients with high PRA are likely to have antibodies against many donors; hence, they get preferential allocation in the United Nations of organ sharing and Euro transplant program., In India, the kidney-transplant program is mainly live donor; however, there are little data about PRA and its utility in transplant program.
We undertook this study to see the prevalence of PRA positivity, the causes of PRA positivity, and the effect of PRA positivity on transplant feasibility and short-term outcomes in our living donor kidney-transplant program.
| Methods|| |
It was a prospective, observational single-center study in a private tertiary care hospital in India.
The study was conducted between January 1, 2016, and October 1, 2016, in Medanta-Medicity Hospital, a private tertiary care hospital in Northern India. Patients were included in the study after written and informed consent. All the transplanted patients were followed up 6 months after the transplant.
Inclusion and exclusion criteria
All the prospective transplant recipients between the study dates were included. There were no exclusion criteria to do PRA testing. Patients were classified on the basis of level of PRA - no sensitization: ≤10%, moderately sensitized: 10%–50%, and highly sensitized: >50%. Sensitizing events, namely history of blood transfusions, pregnancy, and previous transplant, were recorded. The complement-dependent cytotoxicity cross-match (CDC), HLA typing, and flow-cytometry cross-match (FCXM) were done in all patients. Luminex single antigen bead (LSA) test was done in patients with high PRA or flow cross-match–positive patients to identify DSAs before taking them for transplant.
As per our departmental protocol, complete blood counts, kidney function test, and other clinically indicated investigations were done daily during till postoperative day 8. Subsequently, patients were followed up twice weekly in the 1st month, once weekly for the next 1 month, once in 2 weeks in the 3rd month, and then once a month till 6 months. In each follow-up visit, complete blood counts and renal function were done, and tacrolimus (TAC) levels were done as required. Urine albumin/creatinine ratio was done at 1, 3, and 6 months, and kidney biopsy was performed if there were graft dysfunction and proteinuria.
All patients were started on capsule TAC at 0.1 mg/kg/day in two divided doses and tablet mycophenolate (MMF)/mycophenolate sodium (MMF Na) at a dose of 1000/720 mg twice daily 1 day before surgery. Induction (either basiliximab or antithymocytic globulin [rabbit ATG-Sanofi Pharma]) was used whenever necessary and as opted by the patient after detailed counseling with patient and family. All patients received one dose of intravenous methylprednisolone (IV MPS) 500 mg perioperatively. Prednisolone was tapered to 5–7.5 mg/day by the end of 3 months.
TAC trough levels were monitored regularly and maintained at 8–12 ng/ml during the 1st 3 months and 6–8 ng/ml over the next 3 months. MMF Na/MMF was tapered to 1080/1500 mg/day at 1 month and 720/1000 mg/day after 3–6 months in most patients. During the study period, decision regarding evaluation (immunological workup, decision of graft biopsy) and treatment was taken by treating nephrologists.
Method of panel reactive antibody estimation
Flow-cytometric bead assay was used for PRA detection. 5 ml of the peripheral blood is collected in a red topped container and processed within 24 h. 20 μl test serum and 20 μl negative and positive control are incubated with Flow PRA Class I and Class II beads in a 1.5 ml microcentrifuge tube for 30 min in dark at 20°C–25°C with gentle shaking; 1 ml of wash buffer is added and then centrifuged at 9000 g for 2 min. Supernatant is discarded. 100 μl of FITC conjugated goat anti human IgG (Fcγ)is added to beads and incubated for 30 minutes in dark at 20-250C.500 μL of fixing solution is added to each tube. Cutoff points on FL1 histogram are set using negative and positive control sera. Percentage of positive reaction is represented by the percentage of events shifted to the right of cutoff point (Flow PRA I and II™; One Lambda, Inc., Canoga Park, CA, USA).
The descriptive statistics were presented in terms of frequency and percentages in different categories for qualitative variables and as range (minimum, maximum), means and standard deviations/median and standard error of the mean for quantitative variables. The statistical significance of quantitative variables having paired observation was carried out by paired t-test/nonparametric Wilcoxon signed-rank sum test, in case data do not follow normal distribution. The statistical significance of qualitative variables for pre–post was determined by Chi-square test and McNemar–Bowker test. The level of significance was taken as 0.05 (5%). The data were analyzed using statistical software SPSS 16.0 (Statistical Package for the Social Sciences; SPSS, Inc., Chicago, IL, USA).
The patient consent has been taken for participation in the study and for publication of clinical details and images. Patients understand that the names, initials would not be published, and all standard protocols will be followed to conceal their identity.
The study is approved by our institutional review board (Ref: MICR-554/2015) dated January 5, 2016, and has followed the ethical guidelines of Declaration of Helsinki. Written informed consent was obtained from the study participants before inclusion in the study.
| Results|| |
During the study period, a total of 185 ESRD patients underwent kidney-transplant evaluation; all of them had PRA estimation as a part of their pretransplant workup. As there were no exclusion criteria, so all were included in the final analysis. There were more males (n = 155, 83.8%) as compared to females (n = 30, 16.2%). Mean age was 41.14 ± 12.75 years. [Table 1] compares the baseline characteristics of PRA-positive and -negative patients.
As per the study criteria, PRA >10% was considered as positive. Of the total 185 patients, 29 had PRA >10%, hence, the prevalence of PRA positivity in our ESRD patients was 15.7%. Of these 29, 10 (34.5%) had Class I PRA, 9 (31%) had Class II PRA, and 10 (34.5%) had both Class I + II PRA. 12/29 (41.3%) were highly sensitized with PRA >50% (predominantly Class II, i.e., 8/12), and remaining 17 (58.7%) had PRA between 10% and 50%.
PRA positivity was significantly more common in females (P = 0.004), in patients with prolonged dialysis vintage (P = 0.037), and in hepatitis C virus (HCV)-positive patients (P = 0.047) [Table 1].
Three main sensitizing events were evaluated in a patient on the basis of history, i.e., history of blood transfusions, history of pregnancy, and previous transplant. 26/29 (89.7%) of positive PRA patients had a history of one of these three events as compared to 45.8% (71/155) in PRA-negative group (P = 0.0001), showing that patients with a history of sensitization are more likely to develop PRA [Table 2].
There were only 3/87 (3.4%) patients who had no history of sensitizing events but had high PRA with moderate sensitization (10%–50%).
When sensitizing events were analyzed independently, they were significantly more common in the PRA-positive group. However, of 87 patients with a history of blood transfusion, 25 had concurrent history of previous pregnancy or previous transplant. Only 62 patients had blood transfusion as the single sensitizing events, 7/62 (11.2%) of these were PRA-positive and 55/62 (88.8%) were PRA-negative (P = 0.244), signifying that blood transfusion alone was not a significant contributor to sensitization.
When we analyzed the number of transfusions received in the past, we had data of 78 patients. 21 received only single transfusion; none developed PRA.
Another sensitization event which was proposed in few studies is viral infections and vaccination. HCV infection was significantly more common in PRA-positive group (P = 0.047) [Table 1]. Hepatitis B vaccination status is known in 136 patients. Eight out of 9 PRA-positive patients (88.9%) were vaccinated for hepatitis B whereas 122/127 (96.1%) patients were vaccinated in the PRA-negative group, which was not statistically significant (P = 0.31). Vaccination data for flu were not available in >90% of patients, so its effect could not be analyzed.
During the study period, 155/185 (83.7%) patients received transplant. In PRA-positive group, 12 out of 29 (41.4%) received transplant during the study duration, as opposed to 143/156 (91.7%) in the PRA-negative group which was statistically significant (P = 0.0001). Thus, positive PRA is one of the important factors for not getting a transplant. In 17 patients with PRA positivity who were not transplanted, two had Class I PRA, five had Class II PRA, and 10 had Class I + II PRA. 12 patients with PRA >50% had predominantly Class II antibodies.
Baseline characteristics of PRA-positive and PRA-negative–transplanted groups were similar, except mean HLA mismatch. PRA-positive patients were better HLA matched [Table 3]. There was no difference in graft outcome between two groups. At 6 months of follow-up, mean creatinine was 1.22 ± 0.26 in the PRA-positive group and 1.3 ± 0.38 in the PRA-negative group (P = 0.63) [Table 4].
A total of 18 graft biopsies were done, of which 7 had acute rejection on biopsy, 6 in PRA-negative group, and 1 in PRA-positive group (P = 0.507) [Table 4]. Overall acute rejection rate was 4.5%. One patient in the PRA-negative group developed delayed graft function (DGF), but none had slow graft function in either group. Baseline immunosuppression was same in the PRA-positive and PRA-negative groups including use of induction with basiliximab or ATG.
No patient lost his/her graft during the follow-up period. One patient had death with functioning graft because of sepsis.
Reason for not getting a transplant
Three patients in nontransplanted group lost to follow-up. Of the remaining 27 patients, 14 were PRA-positive and 13 were PRA-negative. 13/14 patients in the PRA-positive group were not transplanted because of immunological reason (i.e., cross-match positivity either CDC or flow cross-match with high DSA titers). Predominant reason for not getting a transplant in PRA-negative group was nonimmunological, i.e., medical fitness issues of either donor or recipient, financial issues, or lost to follow-up (10/13).
Correlation of panel reactive antibody positivity with cytotoxicity cross-match, flow cross-match, and donor-specific antibodies
12/29 PRA-positive patients got transplanted. Out of these, 11 patients were FCXM-negative and one was FCXM-positive. Further workup with SAB revealed anti-HLA antibodies positivity in four patients, but only one had DSA who was also FCXM-positive. He underwent transplant after desensitization and thymoglobulin induction. Out of 12 patients with PRA positivity, eight received thymoglobulin induction, two received basiliximab, and two patients did not receive any induction, but the use of induction was not different between PRA-positive and PRA-negative group [Table 3].
Of 17 PRA-positive patients, who did not undergo kidney transplant, 12/17 patients had CDC positivity and 5 were CDC negative but FCXM positive. The LSA was done in these five FCXM-positive patients and one patient who was CDC positive; in all these patients, the DSA MFI was >5000 and they did not enroll of desensitization. The rest 11 patients did not undergo LSA testing as they were CDC positive and their PRA values were high; hence, they refused for further testing due to minimal chances of transplantation with the same donor.
| Discussion|| |
To the best of our knowledge, this is the largest Indian study to see the prevalence of PRA positivity in living donor kidney transplant. The prevalence of PRA positivity in our study was 15.7%, which is comparable to others, namely 14% in the USA (defined as ≥20% PRA), 11% in China (defined as PRA >10%), and 20% in Portugal.,, Variations can be explained by the methods used for PRA estimations.
In our study, females had higher PRA positivity (60.3% vs. 34.2% in males; P = 0.001) as seen in other studies also, which is expected considering their exposure to nonself HLA via pregnancy.,, PRA-positive patients had longer time on dialysis (8.17 ± 8.11 months vs. 6.08 ± 11.64 months, P = 0.037). Dialysis vintage was an important risk factor for high PRA in our patients as many patients do not receive erythropoietin due to financial constraints and end up getting multiple blood transfusions.
All three sensitizing events (blood transfusions, pregnancy, and previous transplant) were significantly more common in PRA-positive patients. This is proved in many previous studies in kidney-transplant recipients as well as healthy blood donors.
Multiple blood/blood products transfusions increase HLA antigen exposure, leading to more risk of sensitization. In our study, no patient with a history of single transfusion developed PRA. When we analyzed blood transfusion as the only sensitizing event, it was not statistically significant (P = 0.244). This could be because of use of leuko-depleted RBCs and small sample size though the effect of leukocyte depletion in decreasing sensitization is controversial. It is important to note that blood transfusion is practically the only “potentially preventable” sensitizing event. Hence, it is important to avoid blood transfusion unless absolutely necessary.
In our study, only 3/87 (3.4%) patients without a history of sensitizing events developed PRA, which was of moderate strength (10%–50%). In a study by Hyun et al., 5.6% of patients had no sensitization history. The prevalence of HLA antibodies in nontransfused male and nulliparous female blood donors was reported to be 1.0% and 1.6%, respectively., The exact reason of the development of anti-HLA antibodies in these patients is unclear; however, it could be due to cross-sensitization following infections or vaccination.,,, In this study, the incidence of HCV infection was significantly higher in the PRA-positive group, though all three patients who developed PRA without any “identifiable” sensitizing events were HCV negative. Thus, patients with no sensitizing event history are unlikely to develop PRA with good negative predictive value. In these patients, PRA estimation may not be required underscoring the importance of good history-taking.
As per the 201 USRDS data, transplant rates decreased with increasing level of sensitization, ranging from 175.5/100 active waitlist years for candidates with a calculated PRA (cPRA) of <1% to only 6.0 for those with a cPRA of ≥98%, showing that high PRA is an important barrier to kidney transplantation. In the current study as well, PRA-positive patients were less likely to get transplanted, predominantly due to immunological reasons, i.e., cross-match positivity either CDC or flow cross-match with high DSA titers in the PRA-positive group.
In transplanted patients, graft function and rejections were same in both PRA-positive and PRA-negative group; this could be because we excluded patients with high immunologic risk including cross-match positive and high levels of DSAs. A study of 59 re-graft recipients by Cicciarelli et al. demonstrated poor graft survival (63% vs. 86% graft survival at 6 months) when PRA was >10%. Similar effect was seen in the studies by Premasathian et al. However, the study by Malheira et. al. showed that donor-specific anti-HLA antibodies are more important than mere presence of anti-HLA antibodies. Patients with HLA+, DSA + had acute rejection rate of 37%, patients with HLA+, DSA– had 14.5% rejections and HLA − patients had rejection rates of 10.6%. In our institute, LSA testing is not routinely done as it a costly and its routine testing is difficult to do in a resource-poor country like India. The decision to do LSA testing was individualized based on the results of CDC, flow cross-match, and PRA testing.
Acute rejection rate in this study was 4.5% which is low compared to the recent data (8%–10%). These low rates could be because of better immunological workup and numerically more use of ATG in the PRA-positive as compared to PRA-negative patients (66.7 vs. 50%, P = 0.27). PRA positivity had no effect on the incidence of acute rejection.
There are few limitations of this study. The follow-up was only 6 months; however, most acute rejections occur within 6 months, and second complete immunological workup with PRA, CDC cross-match, FCXM, and LSA testing was not available for all the patients.
Interpretation and generalizability
To summarize, this study demonstrates that PRA positivity is common (15%) in our living donor kidney-transplant program. With the help of PRA testing, we can identify patients with higher immunological risks and further evaluate these patients with more specific tests such as flow cross-match and LSA and either exclude patients with high risk of rejection or transplant them after desensitization. We found that patients with high PRA but negative flow cross-match could be transplanted without any further increase in risk of rejection as compared to PRA-negative recipients. However, many patients with high PRA who were not transplanted were either CDC or flow cross-match positive and had high levels of DSAs. Another important message from this study is that single blood transfusion alone is not so important risk factor for sensitization in present context, unless combined with other sensitizing event, but it is better to avoid use of blood transfusions as far as possible.
| Conclusions|| |
This study in live donor kidney transplant in India shows that PRA positivity is common in our kidney failure patients. The patients with positive PRA usually have some sensitizing events such as previous transplant, pregnancies, or blood transfusions. Blood transfusion alone was not a significant contributor to sensitization in this study. Patients with high PRA need further evaluation, and many could not be transplanted because of cross-match positivity or high levels of DSAs. Finally, there was no difference in outcomes between PRA-positive patients who were transplanted after complete workup as compared to PRA-negative patients due to the exclusion of high-risk patients.
We acknowledge Dr. Rajesh Ahlawat and Dr. Prasoon Ghosh – transplant surgeons.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Port FK, Wolfe RA, Mauger EA, Berling DP, Jiang K. Comparison of survival probabilities for dialysis patients vs. cadaveric renal transplant recipients. JAMA 1993;270:1339-43.
Russell JD, Beecroft ML, Ludwin D, Churchill DN. The quality of life in renal transplantation – A prospective study. Transplantation 1992;54:656-60.
Zhou YC, Cecka JM. Sensitization in renal transplantation. Clin Transpl 1991:313-23.
Israni AK, Salkowski N, Gustafson S, Snyder JJ, Friedewald JJ, Formica RN, et al
. New national allocation policy for deceased donor kidneys in the United States and possible effect on patient outcomes. J Am Soc Nephrol 2014;25:1842-8.
Mayer G, Persijn GG. Eurotransplant kidney allocation system (ETKAS): Rationale and implementation. Nephrol Dial Transplant 2006;21:2-3.
Matas AJ, Smith JM, Skeans MA, Thompson B, Gustafson SK, Stewart DE, et al
. OPTN/SRTR 2013 Annual data report: Kidney. Am J Transplant 2015;15 Suppl 2:1-34.
Shang W, Dong L, Feng G, Wang Y, Pang X, Li J, et al
. Panel-reactive antibody levels and renal transplantation rates in sensitized patients after desensitization and human leucocyte antigen amino acid residue matching. J Int Med Res 2013;41:1333-41.
Malheiro J, Tafulo S, Dias L, Martins LS, Fonseca I, Beirão I, et al
. Analysis of preformed donor-specific anti-HLA antibodies characteristics for prediction of antibody-mediated rejection in kidney transplantation. Transpl Immunol 2015;32:66-71.
Hyun J, Park KD, Yoo Y, Lee B, Han BY, Song EY, et al
. Effects of different sensitization events on HLA alloimmunization in solid organ transplantation patients. Transplant Proc 2012;44:222-5.
Karpinski M, Pochinco D, Dembinski I, Laidlaw W, Zacharias J, Nickerson P. Leukocyte reduction of red blood cell transfusions does not decrease allosensitization rates in potential kidney transplant candidates. J Am Soc Nephrol 2004;15:818-24.
Kakaiya RM, Triulzi DJ, Wright DJ, Steele WR, Kleinman SH, Busch MP. National heart, lung, and blood institute (NHLBI) retrovirus epidemiology donor study-II: Prevalence of HLA antibodies in remotely transfused or alloexposed volunteer blood donors. Transfusion 2010;50:1328.
de Santis C, Lopalco L, Robbioni P, Longhi R, Rappocciolo G, Siccardi AG, et al
. Human antibodies to immunodominant C5 region of HIV-1 gp120 cross-react with HLA class I on activated cells. AIDS Res Hum Retroviruses 1994;10:157-62.
Alberú J, Morales-Buenrostro LE, de Leo C, Vargas-Rojas MI, Marino-Vázquez LA, Crispín JC. A non-allogeneic stimulus triggers the production of de novo
HLA antibodies in healthy adults. Transpl Immunol 2007;18:166-71.
Katerinis I, Hadaya K, Duquesnoy R, Ferrari-Lacraz S, Meier S, van Delden C, et al
. De novo
anti-HLA antibody after pandemic H1N1 and seasonal influenza immunization in kidney transplant recipients. Am J Transplant 2011;11:1727-33.
El Aggan HA, Sidkey F, El Gezery DA, Ghoneim E. Circulating anti-HLA antibodies in patients with chronic hepatitis C: Relation to disease activity. Egypt J Immunol 2004;11:71-9.
Cicciarelli J, Helstab K, Mendez R. Flow cytometry PRA, a new test that is highly correlated with graft survival. Clin Transplant 1992;6:159-64.
Premasathian N, Panorchan K, Vongwiwatana A, Pornpong C, Agadmeck S, Vejbaesya S. The effect of peak and current serum panel-reactive antibody on graft survival. Transplant Proc 2008;40:2200-1.
Campos A, Malheiro J, Tafulo S, Santos S, Dias L, Martins S, et al
. Increase of allosensitization after a kidney graft failure: Predictors and effect on retransplantation outcomes. Nefrologia 2017;37:397-405.
Hart A, Smith JM, Skeans MA, Gustafson SK, Stewart DE, Cherikh WS, et al
. OPTN/SRTR annual data report 2014: Kidney. Am J Transplant 2016;16 (Suppl 2):11-46.
[Table 1], [Table 2], [Table 3], [Table 4]