H&I laboratory tests carried out in support of solid organ transplant programmes can be divided into three categories depending on the phase of the transplant programme. These are the pre-transplant tests, the tests carried out at the time of transplantation and the post transplant tests carried out as part of the post transplant monitoring. The tests in these different phases generally include HLA typing by DNA techniques to a resolution sufficient to identify broad antigens, HLA antibody screening and identification sufficient to identify antibodies to HLA-A, B, C, DR, DQ and DP and crossmatching by flowcytometric techniques. The actual detailed tests carried out and the frequencies with which those are carried out vary depending on the organ to be transplanted.
In the UK, prior to listing patients for renal/pancreas and cardiothoracic but not for liver transplants, patients are HLA typed by DNA based techniques. It is recommended that HLA typing is performed twice to confirm the type before listing. At a minimum, typing must be to a resolution sufficient to define the HLA type at the broad antigen level as this is the resolution at which matching is run. In liver transplantation, HLA matching is not generally undertaken. Patients and donors are instead matched for size and blood group.
Common HLA typing techniques in use include the polymerase chain reaction sequence specific primers (PCR-SSP) and polymerase chain reaction sequence specific oligonucleotide (PCR-SSO) techniques. HLA typing is carried out at HLA-A, B, C, DR (including DR51, 52 and 53) and DQ. HLA-DP and DQA typing are not generally undertaken unless the patient has HLA antibodies to DP and DQA.
For renal/pancreas and cardiothoracic transplantation, HLA antibody screening and identification is required prior to listing. For cardiothoracic transplantation it is recommended that antibody investigation be undertaken on at least two samples, taken no less than 24 hours apart clinical urgency permitting. HLA antibody investigation on at least two samples prior to listing for renal/pancreas is not required but may be good practice. HLA antibody investigation prior to listing for liver transplantation is not mandated. Studies have however shown that liver patients with donor specific antibodies prior to transplant have an increased risk of acute rejection. HLA antibody screening and identification can aid later post transplant management of such patients.
HLA antibody screening and identification has historically been by cell based techniques such as the Complement Dependent Cytotoxicity (CDC) assay or by cell based flowcytometric assays. Currently however, most HLA antibody testing in the UK is by the solid phase Luminex bead based assays. These assays are much more sensitive than the traditional CDC techniques and require careful consideration of the clinical significance of Luminex positive CDC negative tests. For renal/pancreas and cardiothoracic transplantation, H&I laboratories must be able to identify antibodies specific for HLA-A, B, C, DR, DQ and DP. In addition, the testing must be able to distinguish IgG allo and IgM allo and auto antibodies.
For cardiothoracic transplantation, all identified HLA specificities must be listed as unacceptable mismatches. For renal/pancreas transplantation, the patients antibody profile must be carefully assessed to identify unacceptable mismatches. Furthermore, HLA antigen mismatches to previous failed renal/pancreas transplant must also be listed as unacceptable unless it can be definitively shown by a full antibody history that no HLA antibodies to these mismatches have ever been formed. For patients with a functioning graft requiring transplantation with a different organ, e.g. cardiothoracic patients requiring a kidney transplant, previous HLA antigen mismatches are not listed as unacceptable. HLA antigens may also be listed as unacceptable mismatches in order to prevent sensitisation to them if they are antigens present in a potential live donor.
Once a patient is listed for transplantation, in renal/pancreas and cardiothoracic but not liver transplantation, HLA antibody testing must be undertaken every three months and 2 – 4 weeks after potentially sensitising events such as blood transfusion as required by the BSHI/BTS guidelines. This helps build up a full antibody profile for the patient, avoiding the risk of an unexpected positive crossmatch. HLA antibody investigation is to the same level of detail as described for pre listing. i.e. Antibodies to HLA-A, B, C, DR, DQ and DP must be identified and the testing must be able to distinguish IgG allo and IgM allo and auto antibodies. The unacceptable mismatches listed with the organ registry (ODT in the UK) must be updated.
In the live donor transplant setting for renal transplantation, initial crossmatches may be undertaken well in advance of the actual transplant as part of the donor selection process, with a final crossmatch at the time of transplantation. The purpose of any crossmatch test is to determine whether the patient has any preformed HLA antibodies which will react with antigens expressed by the donor and therefore helps to inform the immunological risk. In the UK, crossmatching is generally undertaken by CDC and/or flowcytometric assays. Cell separation techniques are used to ensure that reactivity to T and B cells can be distinguished. Tests used are also able to distinguish between IgG and IgM antibodies. In some centres, a virtual crossmatch is undertaken as the initial crossmatch in certain carefully selected cases.
Other tests carried out by non H&I laboratories prior to transplantation include ABO typing and various microbiology tests. It is obviously essential that patient and donor ABO types are confirmed prior to transplantation for all solid organ transplants.
At the time of transplant, HLA typing of the donor is undertaken, typing for HLA-A, B, C, DR and DQ but not yet routinely for HLA-DP. Following a matching run, a prospective crossmatch is required for renal/pancreas transplantation but not for cardiothoracic and liver transplantation. As in the live related situation, crossmatching is by CDC and/or flowcytometry. Crossmatching by solid phase bead based techniques have been tried but are not in routine use. The crossmatch test needs to distinguish reactivity to T and B cell populations as well as be able to distinguish between IgG and IgM antibodies.
For cardiothoracic transplantation, the time to transplant is of critical importance and may limit the ability to carry out a prospective crossmatch. For non sensitised patients and for sensitised patients with fully defined specificities a prospective virtual crossmatch is undertaken instead of a full prospective crossmatch, with a retrospective crossmatch being undertaken within 24 – 48 hours of the transplant. For highly sensitised patients with residual unexplained reactivity, a prospective crossmatch may be indicated. The greater degree of sensitivity of the Luminex bead based HLA antibody identification assays does increase the ability of the H&I laboratory to fully define the HLA specificities present and make the use of virtual crossmatching possible in the majority of cases.
No prospective or retrospective crossmatch is indicated in the case of liver transplantation, though some centres do carry out a retrospective crossmatch. T cell positive CDC crossmatching has been shown to be associated with an increased rate of early rejection but graft loss may be prevented by effective management.
The H&I laboratory has a requirement when reporting crossmatch results to provide appropriate advice to the clinical relevance of the results. Such advice will depend on a number of factors including: The actual result, positive or negative; the timing of any positivity seen, current versus historical sample; the cell type the any positivity is seen in, T cells or B cells or both; the presence, strength, specificity and isotype of any donor specific antibodies i.e. IgG HLA class I DSA or IgM autoreactive; and the technique by which the positive reaction is seen, CDC and flow or flow only for instance.
Additional tests and processes are applied in the case of HLA antibody incompatible transplantation. Highly sensitised patients can be transplanted following a desensitisation program that leads to a negative CDC crossmatch at the time of transplantation. The pre desensitisation antibody levels identified by the H&I laboratory, either in the form of Luminex median fluorescence intensity (MFI) or titration levels may help inform the desensitisation methods that are applied. The H&I laboratory will be actively involved in rapid, often daily, antibody testing during the desensitisation phase using the same methods used for routine antibody testing, followed by a prospective crossmatch once donor specific antibody levels have dropped to a target level predicted to lead to a negative crossmatch.
Renal/pancreas and cardiothoracic patients should be screened at regular intervals post transplant for the potential development of DSA and specifically at the time of declining graft function or if otherwise clinically indicated and for renal/pancreas transplant, at the time of biopsy. Routine screening should be every three months and follows the same level of testing as required prior to transplantation. i.e. antibodies to HLA-A, B, C, DR, DQ and DP must be identified and the testing must be able to distinguish IgG allo and IgM allo and auto antibodies. In the case of cardiothoracic transplantation, other non HLA antibodies which have been shown to be involved in the development of vasculopathy should be monitored. Examples include anti vimentin antibodies.
The laboratory tests and procedures undertaken in support of solid organ transplantation therefore are the same basic tests but vary in frequency and timing depending on the organ in question. HLA typing, antibody screening and crossmatching tend to be required for renal/pancreas and cardiothoracic though the timing of crossmatches, prospective vs. retrospective, differs. Cardiothoracic transplantation requires very detailed and careful listing of unacceptable mismatches as prospective crossmatching is not routinely undertaken. Liver transplantation is based mainly on matching the patient and donor for size and ABO, though some antibody investigation prior to transplant may be informative in post transplant patient management.
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