This document discusses histocompatibility in kidney transplantation. It describes the discovery of human leukocyte antigens (HLAs) in 1958 and how they are encoded on chromosome 6. It then summarizes HLA Class I and Class II loci, how they present antigens, and the techniques used for HLA typing, including comparing CDC, FCXM, SPI, SAB, and epitope matching methods. The document also discusses HLA-specific allosensitization, antibody detection and risk assessment, and the virtual crossmatch.

1. Histocompatibility in Kidney Transplantation
DR SCIENTHIA SANJEEVANI

2.  first human leukocyte antigens (HLAs) were discovered in 1958 by Jean Dausset,
Rose Payne, and Jon van Rood. characterized using antibodies
 in sera obtained from multiparous women andfrom patients after multiple blood
transfusions

3. THE HLA SYSTEM
 recognized as the human equivalent of the major histocompatibility complex
(MHC), previously identified in inbred rodents, the products of which control the
recognition of self and foreign antigen
 encoded on the short arm of chromosome 6
 most intensively studied region of the human genome.
 The region spans over 4 mega bases and contains in excess of 250 expressed
genes, making it the most gene dense region characterized to date.
 about 28% ofthese genes encode proteins with immune-related functions.

5.  complex multigene family consisting of more than 10 loci.
 HLA types are codominantly inherited on a maternal and paternal haplotype and
transmitted as a single Mendelian trait

6. HLA CLASS I
 span 2 megabases at the telomeric end of the 6p21.3 region of chromosome 6.
 This region encodes the classical transplantation antigens (HLA-A, -B, and -C) that are expressed on virtually all
nucleated cells.
 Genes of the HLA class I loci encode the 44 kD heavy chains which associate with intracellular peptides present
within the cytoplasm
 α1 and α2 chains are highly polymorphicand fold to form a peptide-binding cleft that accommodates peptides of 8–
10 amino acids in length that are mostly derived from endogenous proteins present within the cell cytoplasm.
 major areas of amino acid polymorphism line the sides and base of the cleft and thereby govern the peptide-
binding repertoire of the HLA molecule
 α3 domain proximal to the cell membrane is highly conserved and acts as a ligand for CD8 expressed on T
lymphocytes.
 The tertiary structure is stabilized on the cell surface by non-covalent association with β2-microglobulin, a non-
polymorphic 12 kD protein encoded on chromosome 15

7. CLASS I LOCI
HLA-H, -J, -K, and -L pseudogenes
HLA-N, -P, -S, -T, U, -V, -W, -X, -Y, and -Z gene fragments that are not
transcribed or translated.
HLA-G expressed on placental trophoblast cells, implicating a
possible involvement in fetal– maternal development
HLA-E, -F, and -G Limited polymorphism and are known to act as ligands
natural killer (NK) cell inhibitory receptors (e.g., CD94)

8. HLA Class II
 consists of three main loci: HLA-DR, -DQ, and -DP.
 The glycoprotein products are heterodimers with non-covalently associated alpha and beta chains of molecular weight
approximately 33 kD and 28 kD, respectively.
 Both chains have two extracellular immunoglobulin- like domains, a transmembrane region and a cytoplasmic tail
 The membrane distal domains α1 and α1 form a peptide binding , accommodating peptides of 10–20 amino acids that
are derived predominantly from ingested (endocytosed or phagocytosed) extracellular (exogenous) proteins
 The α1 domains of HLA-DR, -DQ, and -DP are highly polymorphic and so govern the peptide binding repertoire. They
are constitutively expressed on cells with immune function such as B lymphocytes, activated T lymphocytes, and antigen
presenting cells (monocytes, macrophages, and cells of dendritic lineage).
 The conserved membrane proximal domain associates with CD4 on T lymphocytes with predominantly helper/inducer
function and thereby confers HLA class II restriction and forms the basis of cellular and humoral immunity to circulating
pathogens such as bacteria.

19. COMPARISON OF HLA TYPING TECHNIQUE

20. HLA-SPECIFIC ALLOSENSITIZATION
Routes of Sensitization
 blood transfusion
 Pregnancy
 previous organ transplantation-poorly HLAmatched kidneys can result in allosensitization to the
mismatched donor HLA.
 idiopathic (natural) HLAspecific antibodies result from cross-reactivity with infectious agents,
reactive with specific epitopes expressed on denatured HLA proteins but absent on HLA proteins in their
native form do not react with native antigen in cell-based assays and are considered non-harmful to a
transplanted kidney

21. Antibody Detection and Specificity
Definition
 In order to perform effective antibody screening, serum samples should be obtained regularly
from patients on the transplant waiting list to provide a historical and contemporary
assessment (within the last 3 months) of antibody specificity
 If a potential sensitization event occurs, additional samples are required, e.g., 14 and 28 days
following a transfusion with blood products

22. Risk Assessment for Antibody-Mediated
Rejection
Immunological
Risk
CDC-XM (IgG) FC-XM Luminex-SAB
DSA
HIGH POSITIVE Positive Positive (MFI
>5000)
Intermediate Negative Positive Positive (MFI
>2000)
Low Negative Negative Positive (MFI
2000

23. The CDC crossmatching technique
• Pioneered by Terasaki and colleagues in the 1960s
• scored on the percentage of dead cells
 0-no dead cells
 2- 20% lysis (generally taken as the cut-off for a
positive result)
 8 -all cells having lysed ,strongest possible
reaction
 TITRED CROSSMATCH
 dilution usually performed to 1 in 2, 4, 8, 16, 32,
64 and so on.
 high titre of high avidity DSAb, it may be that
many dilutions are required for the test to
become negative (e.g. 1 in 128)
 Low titres- negative crossmatch could be
achieved with a desensitization protocol

25. Interpretation of CDC-XM results
T cell XM B cell XM Explanation
Negative Negative No significant DSA
Very low titre DSA
Non-complement fixing DSA
Positive Positive HLA antibodies
Non-HLA antibodies IgG
Autoantibodies
IgM antibody
Recent treatment with
thymoglobulin/
alemtuzumab
Negative Positive DSA to HLA class II only
Low titre HLA class-I DSA
Treatment with rituximab

27. The Flow-Cytometry Crossmatch (FCXM)
 1990’s
 depends on donor lymphocytes
being incubated with recipient
serum and flourocein dye
 semi-quantitative and more
objective

30. Solid Phase Immunoassay (SPI)
 commercial kits of purified recombinant HLA molecules coated on a microtitre
plate or synthetic beads
 specific for HLA antibodies
 Can be done by
 ELISA-purified recombinant HLA molecules coated on a microtitre plate
more sensitive than CDCXM
 LUMINEX- synthetic beads, more sensitive than CDCXM AND FCXM

32. SAB
 relevant beads are all coated with a single cloned antigen.
 most precise and specific in detecting DSA against a specific antigen

34. Epitope matching
 limited area of the HLA molecule comprising a 15-25 amino acid sequence
 Can be private and public
 public epitopes result in numerous cross-reactions during HLA testing that result in
false positive results
 can be done both in the laboratory (in-vitro) and from a computed based system (in-
silico)
 highly specific SAB system is the commonest in-vitro epitope based matching system
 The most widely used computer-based algorithm is the ‘HLA Matchmaker
algorithm’- allows donor and recipient matching based on epitopes and has been
used especially in relation to transplanting highly sensitized recipients.

35. The Virtual Crossmatch (VXM)
 Advancement from wet XM
 The antigens against which DSA are detected are referred to as Unacceptable
Antigens (UA), which can trigger HAR
 compares the recipient’s UA against the HLA screening of the potential donor,
by a virtual matching process rather than an actual laboratory ‘wet’ crossmatch

36. Panel Reactive Antibodies
 Testing recipient sera against a panel of donor lymphocytes from a sample of
donors representing the potential local donor population
 recipient’s %PRA indicates the probability of having a positive crossmatch against
a given donor from that population and thereby the chances of receiving a
crossmatch negative kidney