Histocompatibility and immunogenetics

Anthony Nolan’s laboratories, led by Dr Katy Latham, BSc PhD, FRCPath, Laboratory Director and Senior Research Scientist, provide a Histocompatibility and Immunogenetics service, testing the HLA type of donors joining the Anthony Nolan register and potential recipients.

We also work for haematopoietic stem cell transplant centres throughout the world.

Why do donors and recipients need to have matching HLA types?

A person’s human leukocyte antigen (HLA) type is like a barcode on the immune system’s cells. It allows the body to determine self from non-self so it can fight invading organisms.

This barcode plays such a critical role in human defences that each individual has developed their own almost unique one. So it can’t be compromised. 

Unfortunately this makes transplantation difficult. Unless a donor has essentially the same HLA barcode as a recipient, the donor cells can attack the body, or the recipient’s cells can attack the donor's cells.

For successful stem cell transplants, we need to find donors with the same HLA type. And for this we need a very large pool of donors to select from.

 

How do you carry out HLA typing?

HLA is a group of highly polymorphic genes in a region called the major histocompatibility complex, found on chromosome six.

HLA typing involves identifying these gene products.  We analyse the DNA sequence of each gene, which allows us to define specific alleles and determine a person’s HLA type.

DNA can come from a blood sample, buccal swab or saliva.

We use a state-of-the-art method known as Third Generation Sequencing for our HLA typing.

There are several purification stages and quality checks along the way, and the whole process takes six days from receiving the DNA samples to the final results.

The process starts with a ‘block’ of 96 DNA samples – 95 patient or donor samples and one control sample – extracted and prepared by the Lab Management Team so that the samples are all of the same concentration and quality.

 

Amplification: PCR

The TGS team starts by adding DNA based molecules called primers to the samples. The primers recognise each of the six different HLA genes we want to look at (known as HLA-A, -B, -C, -DP, -DQ and -DR) and include a short piece of DNA known as a barcode so that the individual genes can be linked back to the person the sample came from. The samples are amplified using a process called PCR, or polymerase chain reaction, creating billions of copies of each individual’s HLA genes, all labelled with that person’s barcode.

 

Pooling

Through a two-stage process, all the HLA-A, -B and –C copies from half of the 96 people are put into one tube, all of the HLA-DP, -DQ and -DR copies from those same 48 people are put into another. Then the same is done for the remaining 47 people. So we now have just four so-called ‘libraries’, each containing people’s DNA samples for three different genes.

 

Circularisation

Next, a special horseshoe-shaped piece of DNA (called a SMRT Bell adapter) is bound onto every fragment of DNA in each pooled sample. This creates thousands of tiny circles of DNA, each encompassing a single copied HLA gene. It’s this circular shape that allows each gene to be read more than once.

 

Loading into a SMRT cell

Finally, the DNA circles are bound onto tiny magnetic beads and loaded into a SMRT cell. This is a small chip - around a centimetre across - with 150,000 tiny wells at the bottom. The SMRT cell is put on a magnet so that the magnetic beads on the DNA circles drag them down into the wells. The aim is to get exactly one circle of DNA – containing one single HLA gene from one person – into each well. Fluorescent chemicals called dNTPs, which are the building blocks of DNA, and a special enzyme called DNA polymerase, which can make copies of DNA, are added to the SMRT cell.

 

Sequencing

The SMRT cells are put into an RSII sequencing machine, which is effectively a large box full of extremely sensitive video cameras. DNA polymerase makes copies of each circle of DNA using the fluorescent dNTPs. As each individual DNA base (A, C, T or G – the four different chemicals that make up all of our DNA) is copied, a flash of light is released; a different colour depending on which base it is.

By filming the light flashes in every single one of the 150,000 tiny wells in the SMRT cell in real time and recording the order in which the different colours happen, the machine can ‘read’ the underlying order of the DNA ‘letters’ (bases) in that circle of DNA. This includes the particular HLA gene and the unique molecular ‘tag’ belonging to the person it came from. Because there are many copies of each person’s HLA genes in the SMRT cell, and the enzymes can keep going round and round the DNA circles and copying them, there may be tens or even hundreds of individual ‘reads’ from each gene. This increases the accuracy of the process, as these multiple sequences can be compared to make sure they’re all the same and correct any accidental errors.

An RSII machine can run 16 of these SMRT cells at once, each containing 48 DNA samples for three different HLA genes – that’s hundreds of copies of 2280 genes from 380 different people per run.

All that data goes over to the bioinformatics team for analysis, so we can identify which genes come from which individuals, and the exact sequence of each of their six compatibility genes. 

 

 

How many people work in the laboratories?

We have 60 scientific, technical and administrative staff working in our laboratories.

The Histocompatibility laboratories have a number of key departments:

• The Client team is the primary point of contact with transplant centres and other service users.
   
• The Laboratory Management team is responsible for turning a swab, spit or blood sample into a DNA sample. This includes sample reception, DNA extraction using the Tecan machine, quantification and normalisation and preparation of the final blocks of DNA samples ready for HLA typing. This team also currently performs blood ABO grouping, virology screening, and general maintenance of the laboratories. 
   
• The Services team  uses Sequence Specific Primers (PCR-SSP) and PCR Sequence Based Typing (PCR-SBT) methods to HLA type samples which don’t require the ‘6 loci’ typing that TGS provides. This includes requests from other registries through EMDIS (European Marrow Donor Information System), urgent samples such as 24-hour cords, and samples that don’t contain enough cells to be processed through TGS. This team also completes commercial work including disease associations and HFE testing, with the future aim of taking on serology, virology screening (e.g. CMV, Hep B, Hep C and HIV) and ABO blood grouping.
   
• The Solid Organ team provides a tissue typing and crossmatching service to support renal, liver and islet cell transplants at the Royal Free Hospital, screening potential transplant recipients and monitoring patients post-transplant for HLA alloantibodies.
   
• The TGS team performs HLA typing on clinical samples, phenotype project samples and potential donors joining the register. The team utilises Single Molecule Real-Time (SMRT) DNA sequencing to carry out typing of the HLA-A,-B,-C,-DRB1,- DQB1 and -DPB1 genes. Using this cutting edge technology, the team is able to type HLA to an allelic-level resolution. The importance in providing this level of typing is to ensure that every patient can be matched with the most suitable donor available.

Our laboratories are accredited by Clinical Pathology Accreditation (UK) Ltd, the European Federation for Immunogenetics, the Human Tissue Authority and the Care Quality Commission.