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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 5  |  Issue : 2  |  Page : 155-158

Evaluation of crossmatch techniques for deceased donor transplant program – Need of the hour


Standalone Blood Bank, Rotary Bangalore TTK Blood Bank, Bangalore Medical Services Trust, Bengaluru, Karnataka, India

Date of Submission29-Feb-2020
Date of Decision30-Mar-2020
Date of Acceptance24-Sep-2020
Date of Web Publication13-Nov-2020

Correspondence Address:
Ankit Mathur
Standalone Blood Bank, Rotary Bangalore TTK Blood Bank, Bangalore Medical Services Trust, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/GJTM.GJTM_20_20

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  Abstract 


Background and Objective: It is important to strengthen deceased donor organ transplant program to help the patients who do not have living kidney donor available. However, there are lots of challenges in deceased donor program. There are challenges to convince families of brain dead patients for organ donation, but transportation of organ as well as allocation is also difficult. One of the main challenges for transplant immunology laboratory is correct crossmatch technique which is sensitive as well as quick and easy to perform. The aim of this study is to evaluate the results of Luminex lysate donor-specific antibody (DSA) crossmatch (LumCross-match) testing for deceased donor renal transplant cases and correlate with graft dysfunction. Methods: We use Luminex lysate DSA crossmatch for human leukocyte antigen testing for deceased donor renal transplants. In 2 years, we had 177 deceased donations and 1184 crossmatches were done. Results: Total 342 patients received kidneys in 2 years. No hyperacute rejection was reported though 28 cases had signs of antibody-mediated rejection posttransplant. Conclusion: We found Luminex lysate crossmatch useful in deceased donor transplant program because of easy procedure, high throughput, more sensitive, and less laborious as well as for posttransplant monitoring.

Keywords: Deceased donor, Luminex lysate crossmatch, renal transplant


How to cite this article:
Mathur A, Thapa S, Jagannathan L. Evaluation of crossmatch techniques for deceased donor transplant program – Need of the hour. Glob J Transfus Med 2020;5:155-8

How to cite this URL:
Mathur A, Thapa S, Jagannathan L. Evaluation of crossmatch techniques for deceased donor transplant program – Need of the hour. Glob J Transfus Med [serial online] 2020 [cited 2020 Nov 25];5:155-8. Available from: https://www.gjtmonline.com/text.asp?2020/5/2/155/300618




  Introduction Top


Renal transplantation has come a long way since Murray and his team first successfully transplanted a human kidney between identical twins in 1954. Impressive advancements have been witnessed in transplant technology, immunological aspects, and understanding of graft outcomes. Nevertheless, despite such advances in immunological monitoring, chronic rejection remains a challenge, and long-term outcomes following renal transplantation still remain disappointing. While multiple factors have been shown to contribute to a progressive decline in allograft function and ultimate graft loss, increasing attention has been focused on the antibody-mediated component of allograft injury.[1],[2],[3]

The most important factor in organ transplantation is the meticulous evaluation of the possible occurrence of anti-donor antibodies before transplantation. Since antibodies cause graft rejection, technologies for their detection must be sufficiently sensitive to predict hyperacute or humoral rejection and adequately specific to determine immunological failure. An optimal combination of the two elements is necessary to determine the predictive value of individual techniques. Currently, anti-human leukocyte antigen (HLA) antibodies can be detected by a number of techniques that include target donor cell-based crossmatch assays, such as complement-dependent cytotoxicity (CDC) and flow cytometry crossmatch, and HLA protein-based (solid phase) assays, such as an enzyme-linked immune-absorbent assay or HLA antigen-coated fluorescence bead assay systems.

Since Patel and Terasaki first reported the relevance of the pretransplant lymphocyte crossmatch to posttransplant outcomes in 1969, the available test options for crossmatching potential transplant recipients have grown dramatically. The anti-human globulin augmented CDC (AHG-CDC) crossmatch improved upon the sensitivity of the standard CDC crossmatch and thus became the preferred crossmatch before kidney transplantation.[4],[5],[6]

Luminex anti-HLA antibody detection assay is more sensitive and specific than CDC or flow cytometric crossmatch. This technique is a solid-phase assay in which purified HLA molecules (single HLA type or from donor cells) are attached to beads. These molecules will bind to anti-HLA antibodies in the patient's serum. Advantages of the Luminex methodology are the speed, high throughput, less subjectivity, and more sensitivity and specificity of results.[6],[7]

A modification of the Luminex assay (Luminex lysate donor-specific antibody [DSA] crossmatch/LumCross-match) involves the binding of specific donor HLA molecules onto the beads which permit the use of recipient serum in a direct recipient/donor crossmatch.[7]

In India, there is no standard testing protocol followed by all transplant centers. We started Luminex lysate DSA tests in 2009. We use this test for living as well as deceased donor transplants.

It is important to strengthen deceased donor organ transplant program to help the patients who do not have living kidney donor available. However, there are lots of challenges in deceased donor program. There are challenges to convince families of brain dead patients for organ donation, but transportation of organ as well as allocation is also difficult. One of the main challenges for transplant immunology laboratory is correct crossmatch technique which is sensitive as well as quick and easy to perform.

CDC crossmatch was used to be done for all transplant cases. We found this test is labor intensive, time-consuming, and high possibility of false results, especially for deceased donation where there is no time to repeating the tests. We started performing Luminex-based DSA crossmatch (LumCross-match) testing in 2009. The transplant physicians were educated about the new technology and the test.

In our state from South India, we have a well-structured deceased donor transplant program and nearly 100 donations take place every year. Organs used are kidney, liver, heart, and sometimes lungs. The State Registry and Transplant Coordination Committee maintains the list of all prospective organ recipients waiting for deceased donor transplant. Their team performs grief counseling for the family of brain dead individuals and facilitates the entire process of organ donation, retrieval, and transplant. They select the patients for crossmatch and allocate organs mainly on the basis of waiting time. All the crossmatches are done in our laboratory, and the method used is Luminex lysate DSA crossmatch.

Aim and objective

The aim of this study is to evaluate the results of Luminex lysate DSA crossmatch (LumCross-match) testing for deceased donor renal transplant cases and correlate with graft dysfunction.


  Materials and Methods Top


This retrospective observational study is planned to analyze to 2-year data of deceased donor transplantation crossmatch testing. All the samples received in 2 years in 2018 and 2019 for HLA crossmatch from potential recipients were included in the study.

HLA laboratory receives information from the State Transplant Committee about deceased donation, and we collect donor Acid Citrate Dextrose (ACD) samples (40 ml) for crossmatch. Donor lysate is prepared using LIFECODES, Immucor kits (USA). The coordination committee mobilizes the potential recipient according to the waiting list as well as ABO blood group, and the patients ready for transplant are sent to HLA laboratory.

We also collect patient serum samples (5 ml each) who are selected for crossmatch according to their blood type. Luminex lysate DSA crossmatch tests are performed for all the patients' samples. Lysate control is used as a control to check the quality of lysate preparation, and results are validated on the basis of lysate control values.

The method involves preparing a lysate from donor lymphocytes which are incubated with capture beads for 30 min to allow the binding of the HLA molecules onto the beads. After washing with buffer to remove excess lysate, recipient serum is incubated with the beads containing bound donor HLA molecules for 30 min followed by further washes with buffer. Anti-human immunoglobulin G (IgG) conjugated with PE is then added and incubated for a further 30 min. After further washing with buffer, the fluorescence is read on the Luminex instrument, as previously described.

The beads are then incubated with the serum, and any HLA antibodies against the donor are detected using the Luminex analyzer after addition of anti-human IgG conjugated with phycoerythrin.

For interpretation, the cutoff is 1000 for both Classes I and II. All the results are sent to the committee, and organs are allocated on the basis of crossmatch results as well as waiting time.

For all the tests, Luminex 100i was used and software Match IT (Immucor, USA) was used for interpretation of the results.

The patients who were negative for both Classes I and II were allocated kidneys, and total 342 patients received kidney in 2 years.

Ethical clearance

The study was approved by the institutional ethical committee.

Statistical analysis

Microsoft Excel was used for statistical analysis.


  Results Top


In two years 2018 and 2019, total 177 deceased donations were accepted. At HLA laboratory, we received total 1184 potential kidney recipient samples (male: 733, 61.9%, and female: 451, 38.1%) for crossmatch. Samples found negative for both Classes I and II were 850 (71.7%), and the remaining 334 (28.2%) patients were found to have HLA antibodies [Table 1].
Table 1: Total deceased donations and renal transplants in 2018 and 2019

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We found 66 (5.57%) samples positive for both Class I and II, 92 (7.77%) samples had only Class I, and 176 (14.8%) samples had only Class II HLA antibodies. The range of MFI values for Class I was 1221–19784 and for Class II was 1130–18972 [Table 2].
Table 2: Results of Luminex lysate donor-specific antibody crossmatch in 2 years for deceased donation cases

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As per follow-up information collected, no hyperacute transplant rejection was reported, but 28 (8.18%) patients had some signs of graft dysfunction in the first 3 months.


  Discussion Top


With the emergence of small and nuclear family pattern and the rising prevalence of hypertension and diabetes in India, there has been a significant decline in availability of suitable related donors. This pressing scenario has made “deceased donor transplant program” a compelling need of the hour. Deceased donor renal transplantation is still infrequent in India, constituting <5% of the total renal transplants of about 3500 per year.[8]

Deceased donor renal transplant has lots of challenges at the clinical front as well as laboratory side. We have limited time to complete the pretransplant testing, and on the basis of the testing, organs are allocated. Every HLA crossmatch technology has some advantages and some limitations. We started using Luminex lysate crossmatch because of its speed, high throughput, less subjectivity, more sensitivity and specificity of results in living as well as deceased donor transplantation.

As the Luminex technology has shown more sensitivity than CDC for the detection of HLA antibodies, the Luminex direct crossmatch procedure has potential as an adjunct to Luminex screening as a tool for determining recipient/donor compatibility in organ transplantation. It also has potential as a pretransplant method for monitoring the effectiveness of antibody ablation treatment in immunized patients and posttransplant in the monitoring of the effectiveness of anti-rejection treatment. The advantage of this technique in addition to its sensitivity is the fact that cell lysates can be stored and used when required rather than relying on obtaining viable donor lymphocytes for measuring donor specific sensitization during rejection crisis.[9]

Billen et al. retrospectively studied Luminex crossmatching (LumCross-match) in a cohort of ABO compatible CDC crossmatch-negative renal patients transplanted between 1997 and 2001 with clinical follow-up to 2008. All LumCross-matches were performed with serum samples taken at the time of transplant. In this study, it was found that Class II LumCross-match positivity proved no significant risk factor for graft failure, but the value of the Class II LumCross-match is questionable. A positive Class I LumCross-match resulted in worse long-term graft survival compared with a negative one.[10]

The study from India by Chouwdhary et al. found that LumCross-match is a useful and sensitive technique for the detection of anti-HLA antibodies in pretransplant renal patients. However, other measures such as Luminex antibody screen, single-antigen bead (SAB) assay, history of the donor, and the class of antibodies involved should be taken into consideration for pretransplant workup of renal patients.[11] In this study, we could not perform SAB test due to high cost.

Another study Guillaume et al.[12] demonstrated that the LumCross-match can detect Class I antibodies with an MFI as low as 2300 in the SAB technique and 1300 for Class II.

The Luminex methodology has completely changed the speed with which it is possible to obtain HLA antibody identification on a sample and the detailed specificity of HLA antibodies that can now be identified. With Luminex, it is possible to look at reactivity against single HLA antigens, which means that the interpretation of results is no longer complicated by issues such as linkage disequilibrium. In addition, we are beginning to understand the prevalence of antibodies directed against HLA-DP and HLA-DQA. In kidney transplantation, the specificity of the SABs means that most centers can now list a detailed set of unacceptable mismatches without any residual reactivity.

The limitation of the study was not performing SAB for each case due to high cost. For the future, we highly recommend to have multicentric prospective study to evaluate the suitable HLA crossmatch test in India for deceased donor transplantation.


  Conclusion Top


In the present study, we found lysate crossmatch very useful because of easy procedure, high throughput, more sensitive, and less laborious. We can also preserve the lysate for posttransplant follow-up.

The rate of graft dysfunction in deceased donation is similar to living donor transplant. On the basis of these results and discussion with transplant committee as well as Nephrology Association, we continued to use Luminex lysate crossmatch as the main crossmatch test for deceased donor kidney transplant program in our state.

Further, follow-up of all the transplanted patients and multicentric study will be required to conclude the most suitable technique of HLA crossmatch for renal transplantation for living as well as deceased donations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Mehra N, Siddiqui J, Baranwal A, Goswami S, Kaur G. Clinical relevance of antibody development in renal transplantation. Translational immunology in Asia-Oceania. Ann N Y Acad Sci 2013;1283:30-42.  Back to cited text no. 1
    
2.
Murray JE, Merrill JP, Harrison JH. Renal homotransplantation between identical twins. Surg Forum 1955;6:432.  Back to cited text no. 2
    
3.
Mehra NK, Kaur G, McCluskey J, Christiansen FT, Frans HJ Claas. The HLA Complex in Biology and Medicine: A Resource Book. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd.; 2010.  Back to cited text no. 3
    
4.
Patel R, Terasaki PI. Significance of the positive cross-match test in kidney transplantation. N Engl J Med 1969;280:735-9.  Back to cited text no. 4
    
5.
Gebel HM, Lebeck LK. Cross-match procedures used in organ transplantation. Clin Lab Med 1991;11:603-20.  Back to cited text no. 5
    
6.
Gibney EM, Cagle LR, Freed B, Warnell SE, Chan L, Wiseman AC. Detection of donor-specific antibodies using HLA-coated microspheres: another tool for kidney transplant risk stratification. Nephrol Dial Transplant 2006;21:2625-9.  Back to cited text no. 6
    
7.
Tait BD, Hudson F, Brewin G, Cantwell L, Holdsworth R. Solid phase HLA antibody detection technology– challenges in interpretation. Tissue Antigens 2010;76:87-95.  Back to cited text no. 7
    
8.
Modi GK, Jha V. The incidence of end-stage renal disease in India: A population-based study. Kidney Int 2006;70:2131-3.  Back to cited text no. 8
    
9.
Lefaucheura C, Suberbielle-Boisselb C, Hillc GS, Nochyc D, Andradeb J, Antoinea C, et al. Clinical relevance of preformed HLA donor-specific antibodies in kidney transplantation. Am J Transplant 2008;8:324-31.  Back to cited text no. 9
    
10.
Billen EV, Christiaans MH, van den Berg-Loonen EM. Clinical relevance of luminex donor-specific cross-matches: Data from 165 renal transplants. Tissue Antigens 2009;74:205-12.  Back to cited text no. 10
    
11.
Chowdhry M, Makroo RN, Thakur Y, Sharma V, Singh M, Kumar M. The good, the bad, and the ugly of luminex donor-specific cross-match. HLA 2018;91:501-6.  Back to cited text no. 11
    
12.
Guillaume N, Mazouz H, Piot V, Presne C, Westeel PF. Correlation between luminex donor-specific crossmatches and levels of donor-specific antibodies in pretransplantation screening. Tissue Antigens 2013;82:16-20.  Back to cited text no. 12
    



 
 
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