|Year : 2018 | Volume
| Issue : 1 | Page : 34-38
Luminex-Based Donor-Specific antibody crossmatching for renal transplant: A 3-Year experience in South India
Ankit Mathur, Samrat Thapa, Latha Jagannathan
Transplant Immunology Lab, Rotary Bangalore-TTK Blood Bank, Bangalore Medical Services Trust, Bengaluru, Karnataka, India
|Date of Web Publication||5-Apr-2018|
Dr. Ankit Mathur
Transplant Immunology Lab, Rotary Bangalore-TTK Blood Bank, Bangalore Medical Services Trust, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Although CDC has been the gold standard for many years, this assay is certainly not perfect and its use is associated with several problems. The Luminex anti-HLA antibody detection assay is reportedly more sensitive and specific. One of the test is Luminex DSA cross match which is more sensitive than CDC cross match. It is also considered cost effective tool to evaluate pre and post renal transplant cases. Material and Methods: We started Luminex based Donor specific antibody Cross Match tests (DSA Xm) with lysate by the end of 2009 along with CDC after standardization. This study is planned to evaluate the results of DSA Xm of last three years & also to observe antibody medicated rejections after renal transplant. Results: Total 3137 Luminex DSA Cross Match tests were done & 515 tests were positive cross match with negative CDC xm result. Out of 515 positive results 164 were positive for class I, 223 were positive for class II and 128 were positive for both class I & II. In the three years total 1129 renal transplants were done at various centers and total 42 cases of Antibody medicated rejections were reported. Out of 42 patients, 36 patients had pre transplant history of CDC negative & Luminex DSA xm positive. Conclusion: We evaluated our three year results of DSA Xm test and found a cost effective, sensitive and useful supplementary test to evaluate pre and post renal transplant cases.
Keywords: Complement-dependent cytotoxicity, donor-specific antibody, Luminex
|How to cite this article:|
Mathur A, Thapa S, Jagannathan L. Luminex-Based Donor-Specific antibody crossmatching for renal transplant: A 3-Year experience in South India. Glob J Transfus Med 2018;3:34-8
|How to cite this URL:|
Mathur A, Thapa S, Jagannathan L. Luminex-Based Donor-Specific antibody crossmatching for renal transplant: A 3-Year experience in South India. Glob J Transfus Med [serial online] 2018 [cited 2018 Nov 20];3:34-8. Available from: http://www.gjtmonline.com/text.asp?2018/3/1/34/229342
| Introduction|| |
Patel and Terasaki first reported many years ago that the presence of recipient antibodies to antigens expressed on donor white cells was a major risk factor for immediate graft loss. Thereafter, the first tissue crossmatch technique was derived which is called complement-dependent cytotoxicity (CDC) crossmatch. The introduction of a serological crossmatch pretransplantation using this CDC method was meant to prevent the occurrence of immediate graft loss, and a positive crossmatch was always considered a contraindication for transplantation.
Although CDC has been the gold standard for many years, this assay is certainly not perfect and its use is associated with several problems. As the target in the CDC assay is a lymphocyte, not only human leukocyte antigen (HLA) molecules but also other irrelevant cell membrane structures may be targets for antibody reactivity. Indeed, antibodies reactive with the patients' own lymphocytes (autoantibodies), immune complexes, and immunoglobulin allotypes have shown to interfere in this assay. Furthermore, the assay is based on complement activation, which implies that HLA-specific immunoglobulin G (IgG) antibodies, which are not able to fix complements such as IgG2 and IgG4, are not detected. This was the reason why more sensitive assays have been introduced such as the antiglobulin assay (detection of noncomplement-activating HLA antibodies by addition of a complement-fixing antihuman IgG antibody, which does fix complement) or indirect immunofluorescence (flow cytometry). These assays have indeed been proven to be more sensitive but still have the disadvantage that the target cell is a lymphocyte with many different (non-HLA) target molecules on its surface. To circumvent this problem, solid-phase assays using isolated HLA molecules as targets for antibody detection have been developed.,
The Luminex anti-HLA antibody detection assay is reportedly more sensitive and specific than either the CDC or flow cytometric crossmatches. In fact, some consider the Luminex antibody detection technique to be the new gold standard for identifying anti-HLA antibodies. This technique is a solid-phase assay in which purified HLA molecules (either of a single HLA type or a combination of types) are attached to beads. These molecules will bind to anti-HLA antibodies in the patient's serum. Using single-antigen technology, the Luminex technology can predict a patient's sensitization to particular HLA types before transplantation without performing a physical CDC or flow cytometric crossmatch (termed a “virtual crossmatch”).
The two main advantages of the Luminex methodology for antibody detection when compared to other techniques, particularly CDC, are the speed with which tests can be turned around, and the sensitivity and specificity of the results obtained.
There are different kinds of available Luminex tests depending on the source of the molecule coated to the color-coded microspheres. In the screening and panel reactive antibody (PRA) assays, beads are coated with HLA molecules isolated from human cell lines, whereas in the single-antigen (SA) assays, beads are coated with recombinant antigens. In addition, Luminex donor-specific antibody crossmatch (DSA Xm) tests consisting of beads coated with antihuman Class I and Class II antibodies are available to isolate HLA molecules from a specific donor to be used in donor-specific crossmatches. Xm-DSA permits us to perform a real crossmatch using donor lysates which are easier to keep than living cells for posttransplantation studies.
In India, there is no standard testing protocol followed by all transplant centers. Each center has its own testing strategy. Some centers perform renal transplant on the basis of only CDC crossmatch, but some perform other techniques of antibody detection and crossmatch including flow and Luminex. The variation is due to the availability of technique, trained workforce, and the financial resources. Many HLA laboratories perform Luminex DSA Xm by donor lysate along with CDC crossmatch.
We started Luminex-based DSA Xm tests with lysate by the end of 2009 along with CDC after standardization. This study is planned to evaluate the results of DSA Xm of the past 3 years and also to observe antibody-mediated rejections after renal transplant.
The objective of this study is to evaluate the results of Luminex-based DSA testing for renal transplant cases.
| Materials and Methods|| |
Our HLA laboratory started histocompatibility testing in 1996. The tests are performed on request basis for different hospitals. Each hospital follows different testing protocol for renal transplant since there is no nationwide set protocol. CDC crossmatch was done for all prospective transplant cases. Considering this test is labor-intensive, time-consuming, and high possibility of false results, we started performing Luminex-based DSA testing in 2009. The transplant physicians were educated about the new technology and the test. After that, many of them included this test in the Pretransplant testing protocol along with CDC Xm.
From January 2010, Luminex-based DSA test was done for the transplant cases on the basis of the requisition from the transplant center and physician. All the donor samples were collected in ACD and recipient samples in plain tubes. The procedure was done as per manufacturer's instructions. The kits used for tests were Gen-Probe (Gen-Probe Transplantation Diagnostics, Inc., 550 West Avenue, Stamford, CT 06902).
Donor lysate is prepared using beads provided by the manufacturer. The lysate is preserved at −80°C for 5 years.
Donor lymphocytes isolated from peripheral blood used as the source material for HLA. The isolated cells are solubilized with a nonionic detergent.
The kit includes a single blend of Luminex beads. Two of the beads in the blend are conjugated with monoclonal antibodies specific for Class I HLA or Class II HLA. When mixed with a lysate, these two beads will capture the solubilized HLA, making a donor-specific HLA target for antibodies in a serum sample. The bead blend also includes control beads to monitor the amount of background in the assay and to assure that the appropriate conjugate has been used in the assay. The patient serum, diluted in the Specimen Diluent, is then added and incubated with the beads for 30 min. Following another wash, the diluted antihuman IgG-phycoerythrin (PE) conjugate is added to the beads. After a final 30 min incubation, wash buffer is added to the wells, the plate is placed in the Luminex instrument, and data are collected for analysis.
The test includes a control reagent to be run with each lysate. To assure that HLA has been captured, the lysate control reagent is tested in parallel with the sera samples. The lysate control reagent is a mixture of biotinylated monoclonal antibodies that are specific for and bind to Class I HLA and Class II HLA. The biotinylated monoclonal antibodies are detected by the diluted streptavidin-PE conjugate.
DSA also includes a control system to verify that the entire assay is working properly: a dried lymphocyte control, a negative control serum, and a positive control serum matched to the dried lymphocyte control containing antibodies to both Class I HLA and Class II HLA. The control system is run with every assay.
For CDC XM, lymphocytes were separated using lymphocyte separation medium, complement, and other reagents with proper positive and negative controls. For interpretation and analysis of result, software is used which provides mean fluorescence index (MFI) value. MFI value is used to decide the positive and negative results depend on the cutoff value. We considered 1000 as cutoff for both Class I and Class II.
We retrospectively analyzed the data and the results of DSA Xm tests done in 3 years to evaluate the performance of the test. The clinical follow-up and correlation as well as transplant outcome were not studied and this is the limitation of the study. Furthermore, other tests such as PRA and SA bead assay could not be performed to due to lack of resources.
| Results|| |
A total of 1744 prospective transplant recipients were came for pretransplant testing in 2014, 2015, and 2016 from 12 hospitals of Bengaluru and surrounding cities. For all the patients, CDC Xm and Luminex DSA XM were performed. For these patients, 3137 Luminex DSA crossmatch tests were done as shown in [Table 1]. The test was repeated for many patients when they were waiting for transplant.
There were a total of 1210 male patients and 534 female patients [Table 2] and [Figure 1].
The distribution of all the patients according to age is as: 154 patients were age below 20 years, 767 were between 20 and 40 years, 673 between 40-60 years and remaining 150 were above 60 years [Table 3] and [Figure 2].
Out of 1744, totally 332 patients had a history of sensitization events such as blood transfusion, pregnancy, or organ transplant. Totally 16 patients had CDC and Luminex XM both positive and not considered for renal transplant. This category not included in the study also.
Totally 3137 Luminex DSA crossmatch tests were done and 515 tests were positive crossmatch with negative CDC Xm result. Out of 515 positive results, 164 were positive for Class I, 223 were positive for Class II, and 128 were positive for both Class I and Class II.
Decision of transplant was taken by transplant centers depending on clinical condition and other factors. Some of the transplant center considered CDC-negative and Luminex DSA Xm-positive cases also for the transplant.
In the 3 years, totally 1129 renal transplants were done at various centers and totally 42 cases of antibody-mediated rejections were reported. Out of 42 patients, 36 patients had pretransplant history of CDC negative and Luminex DSA Xm positive.
| Discussion|| |
Currently, kidney transplantation has become the preferred choice for treating patients with end-stage renal disease since it not only improves the quality of life as measured by added years but also is cost-effective compared to hemodialysis. In renal transplant program, detecting donor-specific antibodies is very important, and there are several methods to detect donor-specific antibodies less sensitive techniques to very sensitive techniques.,,
The assays based on antibody reactivity against HLA molecules attached to Luminex beads have become popular during the past years. They appear to be very sensitive, and the availability of single HLA antigen beads facilitates the determination of the antibody specificity enormously compared to previous panel analyses.
Many factors impact on the clinical relevance of these detectable low-level antibodies, one being the MFI cutoff for positivity used by the reporting center. There is no recommended “cutoff” value for MFI positivity. Most laboratories set their “cutoff” level for positivity based on levels obtained with relevant controls and also on experience gained from clinical results obtained.
The MFI has been suggested to be a determinative factor, but the MFI determining the border between positive and negative reactions differs between centers and among studies. It is clear that there is a need for standardization, which needs the input of tissue typers, clinicians, and the companies, which developed these products. In the current study, we used MFI value 1000 as a cutoff for both Class I and Class II to report the test positive.
DSA Xm permits us to perform a real crossmatch using donor lysates which are easier to keep than living cells for posttransplantation studies. On the other hand, SA bead assays allow us to take a virtual crossmatch by predicting reactions against HLA specificities. Therefore, both SA assay and Luminex DSA Xm could be used to detect the presence of anti-HLA DSA in patient sera. Each technique has particular advantages and disadvantages. Nevertheless, Luminex DSA Xm is a real crossmatch, and therefore, the existence of DSA against any HLA locus could be theoretically detected without additional donor typing. This employs natural human antigen and it is much cheaper than the SA bead assays.
In our study, we also found this test very sensitive to detect HLA antibodies as compared the CDC Xm. It helped the transplant programs where the resources are limited and the patients cannot afford expensive tests such as SA. This was used as supplementary test which gives additional information about low-level HLA donor-specific antibodies. Since the donor lysate can be preserved for longer period, it can be used as posttransplant monitoring, especially deceased donor program.
To be able to use solid-phase assays more efficiently, standardization is essential. This is only possible by a close collaboration between HLA laboratories, clinicians, and the companies providing these assays. Wet workshops using patient sera and relating the results to clinical consequences such as incidence and severity of rejection, graft function, and graft survival should help the transplant community to implement the results of these assays in a more reliable and reproducible way in clinical decision-making.,,
| Conclusion|| |
Detection of anti-HLA antibodies before kidney transplantation is an evolving science. The CDC crossmatch is considered as classical and gold standard test for detection of pretransplant HLA antibodies. However, this test has low sensitivity and it is time and labor-intensive method. Newer test like Luminex-based DSA Xm is started in India in last few years. In the present study, we evaluated our 3-year results of DSA Xm test and found a cost-effective, sensitive, and useful supplementary test to evaluate pre- and postrenal transplant cases. Due to the constraint of availability and cost, the tests were not compared by SA tests and flow cytometric crossmatch.
Luminex technology can be routinely implemented into pretransplantation workup of renal transplant recipients. Luminex DSA Xm is certainly more sensitive to detect donor-specific antibodies than CDC Xm. A large multicentric study is advisable to compare the results with other tests for Indian patients and also to consider feasibility to include this test as pretransplant testing protocol.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]