CDC Screening

Many H&I laboratories have significantly reduced or even stopped doing CDC antibody screening but it is potentially a very useful technique for helping the lab to understand the clinical relevance of high MFI Luminex antibodies.

CDC screening helps predict donor mismatches which could potentially lead to a positive CDC crossmatch. CDC screening allows IgM to be separated out form IgG antibodies. All IgG CDC Pos. specificities must be listed as unacceptable mismatches with ODT.

In some patients, some Luminex class I positive specificities, especially where the MFI is less than 5,000-10,000, can turn out to be repeatedly negative by CDC screening (unseparated cells). Although CDC Neg, Luminex Pos. specificities can cause acute and chronic rejection, in carefully selected patients who can tolerate enhanced immunosuppression, use of CDC can potentially allow such specificities not to be listed as unacceptable mismatches, widening the pool of potential donors available to such patients and ensuring that highly sensitised patients do not accumulate on the waiting list. However, in the UK, this approach needs to be considered carefully in light of the new kidney and pancreas allocation scheme introduced in 2019.

CDC screening can also prove useful in addition to acid treatment of Luminex beads, in identifying Luminex reactivity which may be due to denatured antigens on beads.

Luminex Antibody Testing

There are three types of Luminex systems for HLA antibody detection and definition. These are a pooled antigen system, a phenotype panel system and a single antigen bead (SAB) system. The pooled antigen system serves as a screen for determining the presence or absence of HLA antibody though limited specificity information may be gleaned in some cases. Specificity testing can then be performed either by the phenotype panel approach or by the more detailed single antigen method.

The pooled antigen and phenotype panel methods use affinity purified HLA antigens to coat Luminex beads whilst the SAB method uses recombinant HLA proteins. The actual test procedures in all three systems follow the same basic steps. Sample serum is incubated with Luminex beads. Up to 100 different beads, each uniquely internally labelled, can be used. Any HLA antibodies present will bind to their complementary antigens on the beads. A series of wash steps remove unbound antibody. Bound antibody is then labelled with an anti-human IgG – Phycoerytherin conjugate. A washing stage may then be required to remove unbound conjugate depending on the Luminex typing kit used. The Luminex platform is then used to identify each bead and any phycoerytherin and therefore HLA antibody bound to each bead. The reaction pattern is then compared to a predefined reactivity pattern for each batch of beads used. Negative and positive control beads and a negative control sample are used to quality control the tests.

The results may be interpreted using assistance from software supplied with the kits. This must however be done with due consideration of the clinical context. The supplier recommended cut off values for positive reaction may be adjusted up or down depending on locally validated procedures.

The advantages and disadvantages of Luminex for Antibody detection

The two main advantages of the Luminex methodology for antibody detection when compared to other techniques, particularly Complement Dependent Cytotoxicity (CDC), are the speed with which tests can be turned around and the sensitivity, specificity and semiquantitative nature of the results obtained.

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 as is the case with CDC. In addition, with Luminex we are able to detect antibodies directed against HLA-DPA and HLA-DQA which are increasingly being shown to be relevant in some solid organ transplantation. In kidney transplantation, the specificity of the single antigen beads means that most Centres can now list a detailed set of unacceptable mismatches without any residual reactivity.

This and the rapid turnaround time has influenced clinical practice in many areas, including in the post solid organ transplant setting. For post-transplant patients, Luminex gives rapid results of a HLA antibody investigation when a suspected rejection or reduced graft function due to donor specific antibodies (DSA) is suspected. Another area where this speed and specificity proves clinically useful is in HLA antibody incompatible transplantation (HLAi), where the rapid turnaround of samples during both the desensitisation and immediate post-transplant phases allow the residual DSA levels to be rapidly determined (though paired/pooled exchange has reduced the need for HLAi transplantation).

The Luminex technique only requires a small amount of serum and yields a significant amount of information, especially when single antigen beads are used, not just on the specificity of the HLA antibodies present but also on their relative strengths in the form of median fluorescence intensity (MFI).  Information on the MFI of antibodies present allows for a risk based approach to the management of highly sensitised patients where it is no longer a case of an antibody being present or absent but if present, using it’s MFI to tailor clinical response such as changes in immunosuppression.

This leads though to a question of the clinical significance of the reported MFI’s and maybe a disadvantage of the Luminex methodologies. The sensitivity of the Luminex methodology means it is possible to obtain Luminex positive, flow and CDC crossmatch negative results. Transplant units need to work with their laboratories to determine the clinically significant MFI cut off for their various transplant programs – kidney/pancreas, heart, lung, liver etc, taking into account any national guidelines.

One potential disadvantage of the Luminex platform is that its use limits the laboratory to being able to identify antibodies only if its cogent antigen has been included on the beads. In CDC, this situation is overcome by adding more cells to the panel. Also, standard Luminex kits give no information of whether or not detected antibodies are complement fixing. On the other hand, the Luminex methodology does allow for identification of some allele specific antibodies. A disadvantage is the role denatured antigens on beads play in complicating the interpretation of results.

Given the speed, sensitivity and specificity of the Luminex methodology, the advantages do therefore outweigh the disadvantages. Due care is required when considering the clinical significance of Luminex positive results however, especially if negative by other techniques. HLA antibody testing now perhaps is in need of a new next generation antibody detection, identification and quantification technology.

Relevance of Luminex antibody testing

Luminex antibody testing remains relevant to the assessment of immunological risk in transplantation today. Luminex is a solid phase technique which uses microspheres coated with antigen. The single antigen bead in particular has made the identification of antibodies to individual specificities much clearer and faster to obtain than was possible with cell-based techniques such as CDC or even with ELISA.

The use of Luminex, which is semi quantitative, allows risk levels to be assigned in the form of MFI’s to any antibodies present. This has enables virtual crossmatching which has revolutionised the way crossmatching is now done, allowing for transplantation without a pre transplant wet crossmatch to be followed later with a retrospective crossmatch in carefully selected patients.

Use of Luminex also allows the crossmatch results, particularly flowcytometric but also CDC crossmatches, to be put in the clinical context of antibodies present. This allows the interpretation of crossmatch results, taking into acclount reactivity that may potentially not be due to HLA.

HLA incompatibly transplantation (HLAi) would probably not be possible or would be very difficult without Luminex antibody testing. Use of Luminex allow us to predict the potential number of plasma exchanges that may be required to bring a DSA down to a level that would result in a negative crossmatch. We can do pre and post plasma exchange Luminex tests for each round of plasma exchange to assess the level of DSA.

Luminex testing is also vital when assessing the patient post-transplant for potential signs of AMR. In these circumstances, a Luminex result can be turned around the same day to detect donor specific antibodies (DSA) to help inform the clinical response of a poorly functioning graft.

However, caution is required with the use of Luminex as the MFI values are only semi quantitative and do not always correlate in a completely linear manner with crossmatch MCS’s/RMF’s. There are also low-level MFI’s which, if they are DSA, are not necessarily a contraindication to transplant. Also, some Luminex positive reactions can be due to denatured antigens on the beads and may not be true DSA’s.

Overall however, Luminex antibody testing remains very relevant to transplantation today.