Flowcytometric Crossmatching

The Flowcytometric crossmatch (FCXM) is highly relevant to transplantation and for many H&I labs is the only technique used for crossmatching. It is semi-quantitative, giving Mean Channel Shift (MCS) and Relative Mean Fluorescence (RMF) values that can help inform clinical risk assessment. When used in association with Luminex, it can potentially help the transplant team to take a risk-based approach to transplantation rather than a blanket yes or no to the presence or absence of DSA.

The FCXM is much more sensitive than a CDC XM and can pick up reactivity at MFI’s that would typically be negative by CDC. A negative FCXM can therefore give confidence to the renal team to proceed with a transplant, taking all other clinical information into account. A positive FCXM on the other hand can be used as part of an overall risk assessment, taking into account the nature of any DSA present, i.e. class I or II or both, the MFI levels, the CDC crossmatch result and the patient history. BSHI/BTS guidelines state that a positive T and B cell FCXM in the presence of Class I DSA or a positive B cell FCXM in the presence of class II DSA are intermediate risk and transplant should be avoided if reasonably possible. Transplantation in these circumstances may however proceed as part of a high-risk protocol. The decision to proceed or not will also be influenced by the CDC crossmatch results if available and the actual FCXM MCS/RMF value as well as the DSA MFI.

In settings where a prospective crossmatch is undertaken, such as the live donor transplant or transplantation in sensitised patients with potential DSA’s, the FCXM is relevant for assessing risk and determining whether or not to proceed as part of a standard or high-risk protocol.

In the retrospective transplant setting, such as in cardiothoracic transplants or transplantation in minimally sensitised renal patients following a virtual prospective crossmatch, the FCXM is useful for informing the post-transplant clinical response.

In HLAi, rounds of desensitisation are typically continued until the post plasma exchange FCXM is negative or a decision is made that desensitisation is not a viable option for the given patient.

The FCXM needs to be interpreted in light of patient history. Patients with autoimmune diseases for instance often have auto antibodies which result in a positive Allo FCXM. For such patients and Auto FCXM is required. A Pos. Allo FCXM which is mirrored in the Auto and where DSA is Neg is not a contraindication to transplantation.

Patient treatment can also affect the FCXM. A patient treated with Rituximab for instance prior to and ABO incompatible transplant will have a Pos. B cell FCXM. In the live donor transplant setting, such patients must have final crossmatch sample taken pri0r to commencing Rituximab. Even donor treatment can affect the outcome of the FCXM. Where a donor is treated with Cyclophosphamide for example, will have little or no B cells, making a B cell FCXM impossible.

CDC Crossmatching

Many H&I laboratories have significantly reduced or even stopped doing CDC crossmatching but it remains a true predictor of hyperacute rejection. The BSHI/BTS guidelines state that a T and B cell positive CDC crossmatch in the current sample in the presence of class I DSA has a high risk of hyperacute rejection and is a transplant veto. A B cell CDC positive crossmatch (XM) in the current sample in the presence of class II DSA is high risk and should be avoided if possible. A T and B cell positive CDC XM in the historic sample in the presence of class I DSA or a B cell positive CDC XM in the historic sample in the presence of class II DSA both carry a high risk of an anamnestic reaction and should only be carried out as part of a high-risk immunosuppression strategy.

In highly sensitised patients who would otherwise accumulate on the waiting list, use of CDC crossmatching alongside FCXM is highly effective for patients who can tolerate an enhanced immunosuppression regime. In such patients, a FCXM Pos, CDC Neg XM can potentially be undertaken, taking into account all other factors such as antibody priming source, repeat mismatches, MCS/RMF, DSA MFI, potential for the patient to get better deceased donor offer and live donor options including paired/pooled exchange.

In settings where a prospective XM is undertaken, such as the live donor transplant, the CDC XM is useful, alongside the FCXM, for assessing risk and determining where or not to proceed as part of a standard or high-risk protocol.

In some retrospective transplant settings, such as in minimally sensitised renal patients following a virtual prospective crossmatch, the CDC XM is often not undertaken.

In HLAi transplantation, rounds of desensitisation are typically continued until the post plasma exchange FCXM is negative. In some very highly sensitised patients where IvIG is used as the desensitisation protocol, it often proves useful to also perform CDC crossmatching as part of the immunological risk assessment.

Virtual Crossmatching

A Virtual Crossmatch (vXM) is a crossmatch that involves a determination of the presence or absence of donor HLA specific antibodies (DSA) in a patient by a comparison of the patients’ HLA antibody specificity profile with the HLA type of the proposed donor without carrying out a ‘wet’ crossmatch such as a Complement Dependent Cytotoxic (CDC) or flowcytometric crossmatch.

Patel and Terasaki demonstrated in 1969 that hyperacute rejection can result from allograft injury caused by preformed donor specific anti-HLA antibodies (DSA) and since then a pre transplant crossmatch became a mandatory requirement for renal transplantation with a positive cytotoxic HLA crossmatch a contraindication to transplant. This pre transplant crossmatch almost completely eliminated hyperacute antibody mediated rejection. The single most important technological advance which has allowed this wet crossmatch to be replaced with a vXM in carefully selected patients is the development of solid phase assays such as Luminex for HLA antibody screening and identification. Use of Luminex assays has revolutionised HLA antibody investigation. One of the main advantages is the sensitivity and specificity of the results obtained. When using single antigen bead assays, for each antibody detected, a Mean Fluorescence Intensity (MFI) value is obtained which provides a measure of the strength of the antibody. Studies have found good correlation (>85%) between the vXM as predicted based on MFI values obtained in Luminex and flowcytometric crossmatch results, with MFI figures of greater than 5000 shown to correlate with positive flowcytometric crossmatch. There is however some inter laboratory variation which makes it important for each Transplant unit and their laboratory to establish the MFI values that correlate with positive crossmatch in their hands. The correlation indicated in published literature between a vXM and CDC results has not been as strong.

The concordance between a vXM and a FCXM is not 100% with rare cases of vXM Neg, FCXM Pos., as well as cases of vXM Pos., FCXM Neg. The cases of vXM Neg, FCXM Pos. are possible explained by non-HLA antibodies such as antibodies to HNA or by the presence of auto antibodies. The cases of vXM Pos., FCXM Neg. are possibly explained by denatured epitopes in the solid phase giving false positive.

The current British Transplant Society/British Society for Histocompatibility and Immunogenetics (BTS/BSHI) guidelines permit the use of Virtual Crossmatching for renal transplantation in place of the pre-transplant wet crossmatch provided the laboratory has validated procedures in place. When a Virtual Crossmatch is used, an early post-transplant retrospective wet crossmatch is currently required by for EFI accreditation.