| Abstract|| |
Presence of antibodies against human leukocyte antigen (HLA) molecules, which may be may be directed against HLA class I or/and class II antigens, is a known risk factor for acute rejections and graft loss. Pre-transplantation panel reactive antibody (PRA) estimation is done to identify sensitized patients prior to solid organ transplantation and also forms the basis of cadaver organ allocation. The aim of this study is to evaluate the PRA in 52 patients awaiting first renal transplant, identify various factors contributing to high PRA, and observe its influence on graft survival. This was a case control study performed in a tertiary care hospital. Eighty-five samples including 63 from 52 patients with end-stage renal disease (ESRD), 10 from healthy volunteers, and 12 from presumed sensitized individuals were tested for class I and/or II PRA by enzyme-linked immunosorbent assay (ELISA) using Quik ID ® GTI kits. PRA for both class I and II was zero in all healthy controls and 19/46 (37%) patient samples; while individually, PRA class I and II were zero in 32/60 (53%) and 39/45 (86.3%) samples, respectively. PRA exceeded 10% in 16 samples from 12 patients with peak class I and II PRA of 100% and 46%, respectively. Posttransplantation, 27/31 patients are doing well, while four died with a functioning graft. Patient reactivity to antigen stimulation is the most important factor determining the PRA levels, and class I PRA is more relevant for detection of sensitization in first-time recipients and adversely affects the graft outcome.
|How to cite this article:|
Mishra MN, Baliga KV. Significance of panel reactive antibodies in patients requiring kidney transplantation. Saudi J Kidney Dis Transpl 2013;24:495-9
|How to cite this URL:|
Mishra MN, Baliga KV. Significance of panel reactive antibodies in patients requiring kidney transplantation. Saudi J Kidney Dis Transpl [serial online] 2013 [cited 2020 Jun 5];24:495-9. Available from: http://www.sjkdt.org/text.asp?2013/24/3/495/111019
| Introduction|| |
Pre-transplantation antibodies against human leukocyte antigen (HLA) molecules are known risk factors for both rejections and graft loss.  These antibodies may be quantified by panel reactive antibody (PRA) estimation. PRA is defined as the percentage of HLA antigens singly or in combination out of a panel reacting with a patient's serum and may reflect the percentage of donors expected to react with the patient's serum. Till date, PRA is the sole established quantitative indicator of pre-transplantation immunologic responsiveness.  The exact incidence of humoral alloimmune responses after kidney transplantation is still uncertain, as routinely post-transplantation monitoring of antibodies is not performed. An allograft is the most important cause for development of antibodies, but transfusions, infections, and pregnancy can also stimulate antibody formation; the degree of sensitization is stronger and more prolonged when different causes act together in the same person.  This study was carried out to observe the role of PRA in kidney transplantation in an Indian setting, as there is no universal consensus among nephrologists on the usefulness of PRA in pre-transplant work-up and many centers do not perform PRA for renal transplantations. Among the centers in India that carry out PRA testing, very few routinely carry out both class I and II PRA estimation. This is largely due to lack of agreement about its usefulness and also partly on account of the economic constraints. Many studies have described simultaneous presence of antibodies against class I and II HLA in patients with end-stage renal disease (ESRD) , and their association with poor graft outcome. These antibodies can be identified by micro-lymphocytotoxicity - complement dependent cytotoxicity (CDC) enzyme-linked immunosorbent assay (ELISA), luminex, and flow cytometry based methods using a known panel of HLA class I and II antigens, beads, or cells. Prospective kidney recipients are classified into different risk groups based on the percentage of PRA positivity with different levels of cutoff, depending on the method used. ,, Presence of anti-HLA class II antibodies has been shown to confer an increased risk of graft loss before decline in renal function and also for chronic allograft nephropathy.  Advantages of luminex include ability to detect all classes of IgG antibodies including complement-fixing and non-complement-fixing antibodies. It is particularly useful for detection of antibodies against single antigens in characterizing the sera of highly sensitized persons.  Now many labs are performing virtual cross-match, especially for sensitized patients requiring solid organ transplantation including kidney, heart, lung, intestine, and pancreas, based on the antibody testing using single antigen flow or luminex beads. ,
| Subjects and Methods|| |
PRA estimation was done on the sera obtained from freshly drawn blood samples in sterile vacutainers from 52 patients with ESRD who were being worked up for their first kidney transplant and from ten healthy age-and sex-matched controls. Additional samples from ten presumed to be highly sensitized individuals were tested only for class II PRA, as we found a low prevalence of PRA class II in a previous study.  Two renal transplant recipients with excellent renal function were also included in this study. When it was not possible to test on the same day, the serum was separated and stored at -40°C until testing. Quik ID ® class I and class II kits from GTI Waukesha (USA) were used. These kits contain combinations of class I (A, B, and Cw) or class II antigens (DR, DQB1) in their respective plates and estimate only IgG anti-HLA antibodies. The panel size was 30 for class II and 40 for class I HLA, and included the most common HLA as well as broad specificities Bw4, Bw6, DR51, DR52, and DR53. Immuno-suppression in all transplant recipients included induction by Dacluzimab followed by administration of mycophenolate mofetil (MMF), prednisolone, and tacrolimus or sirolimus. PRA estimation was carried out according to the procedure mentioned in the product insert. The technique is to add the serum of the patient to microwells coated with HLA glycoproteins, allowing the antibodies present, if any, to bind to the HLA molecules against which they are directed. The unbound antibodies are washed away, after which an alkaline-labeled anti-human globulin reagent (anti-IgG) is added to the wells and incubated. The unbound anti-IgG is washed away and the substrate p-nitrophenyl phosphate (PNPP) is added. After a 30-min incubation period, the incubation is stopped by adding sodium hydroxide solution. The optical density of the color that develops is measured in a spectrophotometer at 405 nm.
Patients were categorized as low-risk group (PRA <10%) and high-risk group (PRA >10%). This study included 52 patients (36 males and 16 females) with ESRD. During pre-transplant work-up, PRA was high (>10%) in 12 patients as shown in [Table 1]. PRA was repeated in seven patients of whom only one patient was not transplanted due to persistent high class I PRA. Class I and class II PRA testing was not done for two and 15 patient samples, respectively, due to nonavailability of kits at the given point of time. All patients were followed up at our center for one to 40 months (mean 25 months) after transplantation at the time of submission of the manuscript.
| Results|| |
Fifty-two patients (M 36, F 16) were worked up at our center, of whom 30 patients received a kidney during the study period. All but three donations were live related. The age of patients ranged from 11 to 53 years (mean 34.7 years). The age of donors (F 27, M 17) ranged from 23 to 62 years (mean 44.5 years). Class I PRA was zero in 32/60 samples. In the sensitized category, PRA positivity was higher for class I (10%-100%) than for class II (13.3%- 46%). [Table 1] shows the peak PRA levels in 12 patients in whom it was ≥10%. All healthy controls had PRA levels of zero for class I and class II. In the presumed sensitized category of 12 controls (F=9, M=3) which included ten subjects who were multiparous and/or received multiple transfusions and two allograft recipients with stable function.
Eight patients with PRA >10% (peak class I/II of 90/10%) had undergone successful renal transplantation without acute rejection and have normal renal function. However, only three recipients had a current PRA >10% (two for class I and one for class II). Four patients died with a functional allograft from seven days to two years after transplantation: three of them died due to infections. The cases of graft dysfunction, rejection, and death are shown in [Table 2]. One female patient had persistent high PRA even at the end of 18 months follow-up, with peak PRA of 100/46% (class I/II) which fluctuated between 67.5% and 100% for class I and between 0 and 46.7% for class II and was refractory to treatment. Hence, she could not be transplanted during the follow-up period as her fourth reading was 100/0 (class I/II). There were only four cases of biopsy-proven acute rejections (12.5%).
| Discussion|| |
The role of PRA in renal transplantation has yet to gain universal acceptance in India; many centers do not carry out PRA estimation and, of these, many do only PRA class I. The test is used worldwide to gauge the level of sensitization of prospective solid organ recipients and may be used to prognosticate the outcome. Many centers are performing only virtual cross-match and no final lymphocyte crossmatch, which is particularly invaluable for sensitized patients. , In this study, of the four patients who died, three had a PRA of zero. One patient with class I PRA of 95% had been transplanted but has made satisfactory progress during a month of follow-up after recovering from delayed graft function. The low rejection rate in this study and one-year graft survival rate of 90% was possible because we identified sensitized patients and gave them higher doses of immunosuppression. In fact PRA is the sole immunologic parameter which provides information regarding the responder status of a prospective solid organ recipient.
Limitations of this study include small sample size, the fact that all samples were not tested for both class I and II PRA, and inability to determine specificity of the antigens against which the antibodies were specifically directed. It is now possible to do so by flow cytometry, luminex, and two-stage ELISA. , These methodologies are used in centers with high patient workload and where many re-transplants are carried out. At present, in our center, renal transplantation workload is only 10-12 per year and all patients were worked up as first-time recipients, so we have not opted for specification of alleles against which the antibodies are directed. After transplantation, if there is a suspicion of donor-specific antibodies developing, this may be confirmed by microlymphocytotoxicity or ELISA. A couple of transfusions or pregnancies alone may not be immunogenic; however, together they have a potential to sensitize female patients. However, transplanted patients are likely to be most sensitized, especially if they suffer acute rejection or chronic rejection. In this study, in two stable patients with 15 and five years of stable graft function, the PRA was 0 and 20%, respectively.
Workers have differed in the cut-off levels of PRA for labeling a patient as sensitized. Barocci et al have considered PRA >4% as the cut-off for detecting a positive reaction against a single HLA specificity and 80% as highly sensitized, although this percentage may reflect the presence of antibody against a public epitope such as Bw4.  This underscores the relevance of specification of the antibodies, more so for sensitized cases. This will also improve donor recipient matching in dialysis recipients waiting for a subsequent renal transplantation. Premasthian et al have used a cutoff of 10% as in this study to discern between sensitized and non-sensitized patients.  PRA is higher in patients in whom the ESRD resulted due to autoimmune diseases such as systemic lupus erythematosus (SLE), but in this study PRA was <10% in both patients with SLE. Vaidya found significantly higher PRA in women as compared to men waitlisted for renal transplantation, and the result of this study is in agreement with their observations. 
To conclude, PRA is an essential test for the work-up of ESRD patients and may also be used for monitoring of sensitization. PRA levels may be determined by the individual's own immunogenicity besides the well-documented causes. Current PRA is more relevant than peak PRA. There is a need to convince nephrologists to use PRA routinely, as high PRA is a major obstacle to kidney and (also other solid organ) transplantation.
| References|| |
|1.||Cho YW, Cecka JM. Crossmatch tests-an analysis of UNOS data from 1991-2000. Clin Transplant 2001;237-6. |
|2.||Süsal C, Opelz G. Kidney graft failure and presensitization against HLA class I and class II antigens. Transplantation 2002;73:1269-73. |
|3.||Vaidya S. Synthesis of new and memory HLA antibodies from acute and chronic rejections versus pregnancies and blood transfusions. Transplant Proc 2005;37:648-9. |
|4.||Terasaki PI. Humoral theory of transplantation. Am J Transplant 2003;3:665-73. |
|5.||Meng HL, Jin XB, Li XT, Wang HW, Lü JJ. Impact of human leukocyte antigen matching and recipients' panel reactive antibodies on two-year outcome in presensitized renal allograft recipients. Chin Med J (Engl) 2009;122: 420-6. |
|6.||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. |
|7.||Tait BD, Hudson F, Cantwell L, et al. Review article: Luminex technology for HLA antibody detection in organ transplantation. Nephrology 2009;14:247-54. |
|8.||Barocci S, Valente U, Nocera A. Detection and analysis of HLA class I and class II specific alloantibodies in the sera of dialysis patients awaiting for a renal retransplantation. Clin Transplant 2007;21:47-6. |
|9.||Deutsch M-A´, Kauke T, Sadoni S, Kofler S, Schmauss D, Bigdeli AK, et al. Luminex-based virtual crossmatching facilitates com ined third-time cardiac and de novo renal transplantation in a sensitized patient with sustained antibody-mediated cardiac allograft rejection. Pediatr Transplantation 2010;14(8): E96-E100. |
|10.||Zangwill SD, Ellis TM, Zlotocha J, et al. The virtual crossmatch-a screening tool for sensitized pediatric heart transplant recipients. Pediatr Transplant 2006;10:38-41. |
|11.||Mishra MN, Baliga KV, Khanduja R, Sandhu AS, Bhardwaj R. Panel reactive antibodies in patients with end stage renal disease. Tissue Antigens 2009;73:415. |
Mahendra Narain Mishra
Department of Pathology, Command Hospital (Southern Command), Pune 411040
[Table 1], [Table 2]