The principles of Luminex technology
The Luminex technology is based on the use of 5.6 micron polystyrene microspheres (beads) each internally dyed with a unique combination of red and infrared dye. The combination of different intensities of the two dyes allows for the identification of each bead by its unique signature when excited by a laser beam. This permits multiplexing of up to 100 reactions in a single tube.
The surface chemistry of the beads allows them to be chemically coated with a number of different targets, including HLA DNA sequences and whole HLA antigens. The beads can therefore be used to interrogate samples for the presence or absence of specific analytes. The Luminex platform uses the principles of flowcytometry to stream beads in single file past a pair of lasers. A red laser is used to excite and therefore identify the specific bead and a green laser is used to excite and therefore identify any reporter dyes captured on the beads during the assay. As both the bead identification and reporter dye readings are made on each individual bead, a multiplex system can be developed with, typically, up to 100 beads. The probes used to coat the 100 beads are carefully selected such that individual analytes of interest can be identified by unique reaction patterns of the beads.
The application Luminex to HLA typing
HLA typing using Luminex is a reverse polymerase chain reaction sequence specific oligonucleotide (PCR-SSO) system which involves PCR amplification of targeted regions within the MHC class I or II regions with group specific primers, followed by a process of probing the amplicon with Luminex beads, each coated with sequence specific oligonucleotide probes to identify the presence or absence of specific alleles. The assignment of HLA type is then based on the reaction pattern observed, compared to patterns associated with published sequences.
Primers used for the amplification are biotinilated. Amplification can then be either symmetrical, which therefore requires a denaturation step to create single strands or can be asymmetrical to generate an excess of a single strand. The single stranded product is then hybridised with a multiplex of up to 100 beads, all of which can be uniquely identified by their internal dyes and all of which are selectively coated with specific oligonucleotide sequences. The amplified DNA hybridise to complementary DNA probed on the beads. A washing stage may then be required depending on the Luminex typing kit used. Bound amplicon is detected by labelling with a Streptavidin – Phycoerytherin (SAPE) conjugate, with Streptavidin binding to the biotin used to label the primers and phycoerytherin serving as the reporter dye for the presence of bound amplicon. Again a wash step may be required depending on the kit in use.
The Luminex platform is used to identify any SAPE bound to the beads. The observed reaction patterns are used to assign HLA type. Positive and negative control beads are used to quality control the typing test.
The current Luminex kits on the market tend to yield mostly medium resolution HLA types, though high resolution results are occasionally obtained. Suppliers are experimenting with high definition beads and increased numbers of beads in the multiplex in order to develop systems for high resolution HLA typing.
The application of Luminex to the detection and definition of HLA specific antibodies
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 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 single antigen 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 HLA typing
The Luminex methodology for HLA typing combines some of the speed typically associated with a PCR-SSP technique, with the high sample throughput of an SSO technique. As laboratory workloads increase, this gives the ability to rapidly type a large number of samples with high reproducibility. The combinations of speed and reproducibility, together with the removal of the need to maintain and validate in-house methods, form the main advantages of Luminex as used for HLA typing. The technique is fairly robust and requires very little DNA. The methodology also makes better use of laboratory staff with fewer staff required to test the same number of samples when compared to SSP or other traditional SSO techniques.
The Luminex technique as used by most H&I laboratories is semi-automated but can be fully automated, contributing to the speed with which results can be obtained. There does however remain a need for experienced scientists to check, confirm and if required modify, the software proposed HLA types.
Another advantage of Luminex compared to PCR-SSP is the reduction in use of or even elimination of the use of agarose gel electrophoresis and its associated use of ethidium bromide and the H&S risks associated with that.
A potential disadvantage of the Luminex methodology is that even though it is rapid, it is still not as rapid as PCR-SSP and may therefore be unsuitable for use in an on call situation where a rapid turnaround of results is needed. In addition the system may be better suited to batch testing of samples rather than the single sample testing typical of the on call situation. In this situation the Luminex system may have a disadvantage both in terms of cost and speed.
Another current disadvantage of the Luminex methodology is that results are low to medium resolution and therefore require further testing by SSP or sequence based typing (SBT) to obtain high resolution results. Laboratories that use Luminex as their main typing methodology may therefore have to retain PCR-SSP and/or SBT for high resolution typing. Luminex suppliers are currently developing sets of beads capable of yielding high resolution results. One potential problem is the current limit of 100 beads used in a multiplex and ways of increasing this number are being examined.
A further limitation of the Luminex technology is that is does produce a small number of heterozygous ambiguities, though suppliers claim this is less than traditional SSO techniques. Where heterozygous ambiguities are identified, specific probes can be developed to help resolve these. This however points to another disadvantage of the Luminex methodology for HLA typing. With traditional in house SSO techniques or with SSP techniques, new probes could be rapidly added to help identify new alleles or resolve ambiguities. Use of Luminex does rely on the suppliers rapidly updating their kits.
Given the rapid turnaround of batched samples and the associated high throughput, the advantages of the Luminex methodology do perhaps outweigh the disadvantages once the initial capital costs of set up are absorbed. The Luminex kits are however relatively expensive compared to other techniques though staff time for these other techniques needs to be taken into account.
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 and specificity 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. 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 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 are already influencing clinical practice in many areas, including in the post solid organ transplant setting, where it is now possible to rapidly obtain a result of a HLA antibody investigation when a suspected rejection episode or reduced graft function is reported. Another area where this speed and specificity are influencing clinical practice is in the antibody removal programs, where the rapid turnaround of samples during both the desensitisation and immediate post transplant phases are now possible.
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 at least until it has been in use for a longer period of time. The sensitivity of the Luminex methodology means it is possible to obtain Luminex positive, flow and CDC negative results the clinical significance of which in the various transplant programs, kidney/pancreas, heart, lung, liver etc remains to be clarified.
In addition to the high cost of the kits, another potential disadvantage of the Luminex platform is that its use limits the laboratory to only 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 allows for identification of allele specific antibodies which if present against alleles of the same antigen as the patients HLA types, would have historically been dismissed as possibly non HLA antibodies. An emerging 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 perhaps outweigh the disadvantages once the initial capital costs of set up are absorbed. Due care is required when considering the clinical significance of Luminex positive results however, especially if negative by other techniques.
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