Dr. Janette Kwok
December 2006
A score and five years ago, Professor Gibson, the then Head of Pathology Department, the University of Hong Kong, made an important decision in establishing the Tissue Typing Service in Hong Kong. He enrolled Dr Brian Hawkins to establish the Tissue Typing Services at Queen Mary Hospital in 1981. It was then part of the Hospital Pathology Services of the Department of Pathology, the University of Hong Kong. In addition to providing laboratory services, it actively participated in research. Since the take over of the hospital services by the Hospital Authority, the Tissue Typing Laboratory became a division under the Department of Pathology and Clinical Biochemistry of Queen Mary Hospital. It is a centralised service to the entire territory within Hong Kong. The service was primarily utilised for renal transplantation until bone marrow transplantation (BMT) became available in the early 1990s. Since then the service has taken on a centralised role for all related and unrelated BMT.
The Laboratory supports transplantation programs in several ways. We identify organ and stem cell recipient and donor human leukocyte antigens (HLA) for matching purposes; we screen waiting recipient sera for anti-HLA antibodies; and we perform crossmatches between recipient sera and donor cells just before a potential transplant procedure.
In the early days, typing for the HLA was done using serological techniques, namely the microlymphocytotoxicity test. Several improvements were made as to the efficiency and reliability of these techniques. One of the major drawbacks of serology, however, is the need of viable cells. Blood cells drawn from deceased donors may have a grossly impaired viability and a reduced expression of antigens on their cell surface. This makes serological typing difficult and prone to errors. In the mid-1980s, molecular typing was first introduced in many laboratories. It dealt with the so-called restriction fragment length polymorphism method, a tedious method not suitable for prospective typing. Only with the introduction of the polymerase chain reaction (PCR) was the suitability of molecular techniques with respect to perspective typing achieved. In 1991, the Tissue Typing Laboratory introduced the DNA-based technology, improving the typing resolution of BMT donors and recipients.
Perhaps no other clinical laboratory has undergone the marked changes in technology that the clinical histocompatibility laboratory has in the past decade. Serum screening has moved from HLA antigen targets on cell panels constructed from volunteers and commercially available frozen cells to flow cytometric detection of antibodies bound to microspheres coated with partially purified HLA antigens and to ELISA-based methods. Crossmatch has transitioned from microlymphocytotoxicity-based assays assessing complement mediated cell lysis due to recipient anti-HLA antibodies coating donor cells to more sensitive flow cytometric-based tests. Finally, HLA antigen detection by serological assays is being gradually replaced by nucleic acid-based methods for identification of HLA alleles.
HLA allele identification could reasonably be proposed as the most complex contemporary problem in molecular diagnostics. First, more than 1,600 alleles are now known to be present in worldwide populations at 12 expressed Class I and II loci. The encoded polypeptides of these alleles differ from each other by one or more amino acid substitutions by what are effectively missense mutations. No other human genetic loci are as polymorphic as the HLA loci. For example, the HLA-B locus currently has more than 800 known alleles. Secondly, the steady description of new alleles plagues laboratories as allele calls made in the past for patients and donors become out of date. Thirdly, laboratories are asked to provide allele identification at various levels of resolution for different clinical situations. High resolution allele level typing is required for unrelated bone marrow transplantation while serological or low resolution typing is adequate for renal transplantation.
Tissue Typing Laboratory is committed to providing quality services to our clients. The laboratory participates in external quality assurance programs to ensure our service quality attains international standards. The laboratory has obtained the accreditation from the American Society for Histocompatibility and Immunogenetics (ASHI) since 2002 and the American College of Pathologists (CAP) since 2004.
Terminology
Tissue Typing or HLA typing means to determine antigens within the HLA system. HLA genes are the blueprints to show how the cells should organize the amino acids to make proteins. The HLA genes are on chromosome 6. The HLA genes are passed down from parents to child. Each person gets two copies of each gene, one copy from the father and one from the mother. A child will not match either parent exactly, but will be half of a match. As each parent has two copies of each gene, there are four possible combinations of genes that could be passed down to their children.
In many organ transplantations, especially for kidneys, the survival of the graft depends on the number of HLA shared between donor and recipient. Its importance is just next to the blood group compatibility. In a so called mismatched transplant, the donor and recipient do not share the HLA antigens. Transplanted organs come from living persons or from deceased.
Crossmatch is a test which indicates if there is a specific immune reactivity between donor and recipient. It is performed by mixing a small amount of the patient’s serum with the potential donor’s white cells. If the patient has antibody to the donor’s HLA, the donor’s cells will be injured and this is referred to as a “positive crossmatch”. A patient can develop antibody after a pregnancy, a transfusion, or a transplant. A positive crossmatch is a contraindication to transplant.
Antibody screening is a test procedure which will reveal if the patient has antibody to other HLA. It is performed by mixing the patient’s serum with cells from 60 different individuals, in separate tests.
Panel Reactive Antibody or PRA, is the amount of HLA antibody present in a patient’s serum. If, for example, the patient’s serum reacts with 30 out of 60 HLA, then the patient’s PRA is 50%. The PRA is calculated for each screening serum sample. In addition to determining the PRA, we need to know how specific the antibody is. Some patients have one or two antibody specificities, while others have numerous specificities. Since HLA antibody can “come and go”, it is important to test the PRA regularly on a periodic basis.
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