*3.3.2. Polymerase chain reaction*

The Polymerase Chain Reaction (PCR) is a revolutionary system for investigating the DNA nucleotide sequence of a particular region of interest in any individual [16]. Very small amounts of DNA can be used as a starting point, such that it is theoretically possible to tissue type using a single hair root. Sequencing DNA has been transformed from a long and laborious exercise to a technique that is essentially automatable.

The first step in this technique is to obtain DNA from the nuclei of an individual. The double stranded DNA is then denatured by heat into single stranded DNA. Oligonucleotide primer sequences are then chosen to flank a region of interest. The oligo- nucleotide primer is a short segment of complementary DNA, which will associate with the single stranded DNA to act as a starting point for reconstruction of double stranded DNA at that site.

If the oligonucleotide is chosen to be close to a region of special interest like a hypervariable region of HLA-DRB then the part of the DNA, and only that part, will become double stranded DNA, when DNA polymerase and deoxyribonucleotide triphosphates are added. From one copy of DNA it is thus possible to make two. Those two copies can then, in turn, be denatured, reassociate with primers and produce four copies. This cycle can then be repeated until there is a sufficient copy of the selected portion of DNA to isolate on a gel and then sequence or type.

There are a number of PCR based methods in use. For example:

**•** *Sequence Specific Priming (SSP) -* In this test, the oligonucleotide primers used to start the PCR have sequences complimentary to known sequences which are characteristic to certain HLA specificities. The primers, which are specific to HLA-DR15, for example, will not be able to instigate the PCR for HLA-DR17. Typing is done by using a set of different PCR's, each with primers specific for different HLA antigens.

#### **3.4. Sequence Specific Oligonucleotide (SSO) Typing**

By this method, the DNA for a whole region (e.g. the HLA DR gene region) is amplified in the PCR. The amplified DNA is then tested by adding labeled (e.g. Radioactive) oligonucleotide probes, which are complementary for DNA sequences, characteristic for certain HLA antigens. These probes will then "type" for the presence of specific DNA sequences of HLA genes.

which pathogens use to evade this immune response is to down regulate their MHC cell surface expression. Natural Killer cells are able to detect altered expression of MHC through a number of cell surface receptors leading to target cell lysis [19]. These recep‐ tors include the killer immunoglobulin like receptors (KIR), which are also expressed on some effector T cells. In humans, the KIR gene cluster is located on chromosome 19. KIR genes are both polygenic and polymorphic [20]. The KIR gene cluster codes for 15 ex‐

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The ligands for KIR receptors are HLA class I molecules [21]. These include HLA-C lo‐ cus antigens with either Asn (Group 1 HLA-C antigens) or Lys (Group 2 HLA-C anti‐ gens) at position 80, the HLA-Bw4 epitope and some HLA-A antigens. KIR receptors binding to HLA class I are either inhibitory or are stimulatory with the overall effect of NK cell interaction with the target cell dependent on the balance between these inhibito‐ ry and stimulatory signals. It is thought that the inhibitory KIR's bind class I with great‐ er affinity than the corresponding activating KIR with the effect that under normal circumstances the inhibitory signal prevails. The 'missing self' hypothesis holds that NK cell alloreactivity occurs when the ligand for inhibitory KIR receptors is down regulated or 'missing', leading to activation. This however requires that KIR receptors engage their cogent HLA class I molecules during maturation to acquire effector function. NK cells that express only inhibitory KIRs for absent HLA class I molecules are hypo responsive

Inhibitory KIR receptors possess long cytoplasmic tails with immunoreceptor tyrosine based inhibitory motifs (ITIMs). Activating KIR receptors have short cytoplasmic tails that pair with adaptor molecules with immunoreceptor tyrosine based activating motif (ITAMs). The nomenclature for KIR receptors therefore includes an 'L' (long tail) for inhibitory KIR's and an 'S' (short tail) for activating KIR's. The nomenclature also includes 'P' for pseudo genes. The inhibitory and activating KIR receptors share sequence and structural similarities in their extracellular domains. KIR's have either 2 or 3 extracellular immunoglobulin domains and this is reflected in their nomenclature as either '2D' or '3D', giving KIR receptors nomenclature such as KIR2DL1, KIR2DS2 and KIR3DL1, where the final digit indicates the order in which

The KIR genes assemble into haplotypes with two haplotypes described, 'A' and 'B'. The 'A' haplotype has only one activating KIR (2DS4), while the 'B' haplotype has a higher number of

The major histocompatibility complex class I related chain was first described in the 1990's [22]. The genes are located centromeric to the HLA class I B gene. The only two MIC genes which are expressed are MICA and MICB. MICA and MICB share a significant amount of sequence homology with HLA class I and have some similarity in their conformation. MICA and MICB antigens have α1, 2 and 3 domains like classical HLA antigens but do not associate with β2 microglobulin and do not bind peptide for presentation to T cells. Instead, MIC antigens serve

activating KIRs and generally possess more KIRs than the 'A' haplotype.

as ligands for the NKG2D receptor on NK cells and on some T cells.

pressed KIR genes and 2 pseudo genes.

in the non transplant setting.

the genes were described.

**4.2. MICA/B**

#### **3.5. Panel Reactive Antibodies (PRA)**

PRA has been used to measure patient HLA sensitisation ever since pre-formed donor specific HLA antibodies were associated with hyperacute rejection in renal transplantation in the 1960's [17]. As traditionally defined, PRA refers to the percentage of an antibody screening panel with which the patient's serum reacts. A kidney patient with a PRA > 85% is considered highly sensitised. This measure of PRA however relies on the composition of the panel which may not necessarily reflect the antigen frequencies in the donor population. This measure of PRA is not therefore a good reflection of the chances of the patient finding a compatible donor. Variations in cell panels, both commercial and in house, result in wide variations in recorded PRA for patients on the waiting list.

The calculated PRA (cPRA) was introduced to overcome this problem [18]. The cPRA can be calculated in a number of different ways, but relies on the identification of a potential recipi‐ ent's anti-HLA antibody profile. This has been made much easier by the wide adoption of solid phase assays such as Luminex. Luminex assays, especially those involving the use of single antigen beads (SABs) allow fine specificity definition and allow the strength of the reactions (MFI) to be used to assess immunological risk and help decide whether or not specificity should be listed. The cPRA is then calculated by defining a set of unacceptable mismatches for that recipient, and weighting those mismatches according to the frequency of the antigen in the donor population. This could be based on the frequency of different HLA antigens in the most recent 10,000 deceased donors.The cPRA therefore gives a measure of the chances of a patient finding a compatible donor in the donor pool.

cPRA removes some of the variability between laboratories using different panels and allows a PRA value to be assigned which reflects the patients' transplantability.
