The human leukocyte antigen (HLA) system has an important role in the regulation of the immune system; the HLA recognizes cells as ‘non-self’ or ‘self’ to prevent the body from attacking its own cells.
For transplant rejection, which is when donor cells are not recognized as ‘self’ by the recipient, the HLA system plays a key factor. It is also known to contribute to some autoimmune diseases, including rheumatoid arthritis and multiple sclerosis.
HLA typing is a genetic test commonly used in diagnostic laboratories all over the world. It minimizes the risk of transplant rejection by using molecular or serological biological methods to find recipients and donors with closely matched HLA patterns.
Cell death is caused if an immune response against the donated is triggered because the HLAs of the donor are different from those of the recipient.
This article will discuss a number of different HLA typing methods that are in use and how the ASSIST PLUS pipetting robot can free up time for lab personnel and improve reproducibility and accuracy of results by automating multichannel pipetting.
Image Credit: INTEGRA Biosciences
Serological HLA typing in a Terasaki tray
Antibodies are used to distinguish between the different variants of HLA antigens in HLA serotyping. The donor serum is mixed with a panel of HLA-specific antibodies to identify which HLA patterns are expressed by the cells and to test if there is an immunological reaction.
Serotyping with a final crossmatch allows screening for pre-existing antibodies to compare HLAs of the recipient and the donor prior to transplantation. During this process, serum from the patient is mixed with the lymphocytes of the donor.
A special plate, known as a Terasaki tray, is used in both applications. The tray contains 72 or 60 wells and has a capacity of about 10 µl per well; evaporation of sample dispensed onto the tray is prevented using mineral oil.
Even the most experienced lab technician can find it tricky to pipet these low volumes precisely into oil in the center of each well. Despite the unique dimensions of Terasaki trays, the process can be automated using the ASSIST PLUS with the VOYAGER adjustable tip spacing pipette.
The exact positioning of the pipette tip is ensured every time through automation, enabling accurate and precise dispensing of small volumes with a consistent speed and immersion depth.
INTEGRA offers a specially designed adapter for this application to maximize efficiency, which allows users increased throughput and walkaway time by processing up to two Terasaki plates in a row.
ELISA-based and Luminex® HLA typing
The reactivity between donor HLAs and recipient antibodies can also be determined by performing serotyping as an ELISA. Luminex technology is a bead-based immunoassay system that allows the detection of up to 100 analytes simultaneously as compared to standard ELISAs; this offers higher sensitivity and specificity for HLA testing.
Therefore, it has become the clinical standard worldwide in solid organ transplantation for the identification of donor-specific HLA-specific antibodies.
Luminex and ELISAs assays can be efficiently processed with VIAFLO electronic multichannel pipettes as they are multi-well microplate-based assays that are typically performed in 96 well plates.
Precious time in busy clinical labs can be freed up, and inter-operator variability of the results can be minimized by clicking the pipette into the ASSIST PLUS, thereby automating the ELISA steps.
All tedious pipetting steps of the immunoassay can be easily automated by the ASSIST PLUS pipetting robot, from stopping the reaction, adding the detection body, transferring samples, to coating the plate.
Image Credit: INTEGRA Biosciences
Molecular HLA typing
When it became apparent that there were HLA variants that couldn't be distinguished serologically but could be by looking at the underlying genes, clinical molecular typing strategies were developed.
Next-generation sequencing (NGS), PCR sequence-based typing (PCR-SBT), PCR sequence-specific oligonucleotide probe (PCR-SSOP) and PCR sequence-specific primer (PCR-SSP) are some of the sequence-based HLA typing protocols in use.
All molecular HLA typing methods, including those listed above, first isolate nucleic acids from the sample, then they use PCR to amplify the target region of interest - either certain HLA exons, full-length genes or the complete HLA region.
Multiple PCR reactions are needed to allow typing of one sample because the HLA system is a large gene complex spanning 3,600 kilobases.
Image Credit: INTEGRA Biosciences
An optimal system for nucleic acid extraction of samples using the ASSIST PLUS pipetting robot is offered by INTEGRA, which can also automate PCR set-up for molecular HLA typing, resulting in lowered overall cost per sample and significantly reduced hands-on time.
Even PCR set-up in 384 well plates can be easily automated, achieving efficient typing of high numbers of samples. Samples stored in tubes can be added to plates using multiple channels in parallel by automating the VOYAGER adjustable tip spacing pipette with the ASSIST PLUS, which guarantees precise and accurate handling of low volumes.
The reservoir’s SureFlo™ anti-sealing array offers the lowest dead volume available on the market, meaning that combining this set-up with INTEGRA's multichannel reagent reservoirs minimizes the consumption of costly reagents.
Users can ensure that samples remain cool during assay preparation by using adapters for reservoir and 96 well or 384 well PCR plate cooling.
Quantification and purification of the amplicons must be carried out after the PCR step for SBT and NGS methods – this is typically done by magnetic bead purification. Users can accommodate magnetic plates to automate tedious pipetting steps during the purification by making use of the flexible deck set-up of the ASSIST PLUS.
Optimal pipetting settings maximize sample recovery, including tip angle, pipetting speeds and tip immersion depth. Consistent positioning of the tip in the sample well is ensured, minimizing the risk of disturbing the beads or bead carryover, providing high sample yields and purities for downstream sequence analysis.
Image Credit: INTEGRA Biosciences
Conclusions
Inaccurate liquid handling or human errors during HLA typing have the potential to lead to serious consequences if donors and patients do not match. These risks can be minimized by automating the workflow.
As compared to a manual workflow, using the ASSIST PLUS pipetting robot means that low volumes are pipetted with higher precision and accuracy, which is particularly important in situations where samples can be precious and small.
Users can easily switch between different processes and applications as their workflow demands and are not restricted to only one HLA typing method through the combining the ASSIST PLUS with one of INTEGRA's 25 electronic multichannel pipettes, which offer a variety of volumes and pipetting channels.
Even Terasaki trays or tube racks can be processed by automating the VOYAGER adjustable tip spacing pipette on the ASSIST PLUS, which increases productivity. Diagnostics labs and transfusion and transplant centers wishing to enhance their HLA typing capabilities will find the ASSIST PLUS a versatile tool.
About INTEGRA Biosciences
INTEGRA provides innovative solutions for Liquid Handling and Media Preparation applications which serve the needs of their customers in research, diagnostics and quality control laboratories.
Their instruments and plastic consumables are developed and manufactured in Zizers, Switzerland and Hudson, NH USA. In order to remain close to their customers, they maintain a direct sales and support organization in North America, the UK, France and Germany, as well as a network of over 100 highly trained distribution partners worldwide.
In recent years they have focused on developing a new and technologically advanced range of handheld electronic pipettes which are simple to use and meet the ergonomic needs of their customers. Today they are proud to offer the widest range of electronic pipettes in the market spanning a range from single channel pipettes up to 384 channel bench-top instruments.
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