HLA in Transplantation and Transfusion: Comprehensive Study Notes

HLA and MHC: Definitions and Concepts

Key Definitions

• HLA (Human Leucocyte Antigens): Cell-surface proteins encoded by MHC genes, serving as molecular identity markers for cells.

• MHC (Major Histocompatibility Complex): A gene region on chromosome 6 (6p21.3) encoding HLA proteins.

• Histocompatibility: The immune system's ability to distinguish self from non-self, crucial for transplantation compatibility.

• Alloantigen: An antigen that varies between individuals of the same species; HLA antigens are the primary alloantigens in transplantation.

Exam Fact: HLA proteins are products of MHC genes on chromosome 6p21.3.

HLA Structure: Class I vs Class II

Structural and Functional Comparison

Feature	HLA Class I	HLA Class II
Genes	HLA-A, HLA-B, HLA-C	HLA-DR, HLA-DQ, HLA-DP
Structure	Alpha chain (a1, a2, a3) + beta2-microglobulin (not MHC-encoded)	Dimer: alpha (a1, a2) + beta (b1, b2)
Peptide Groove	Closed ends; binds 8-10 amino acids	Open ends; binds 10-30 amino acids
Peptide Source	Intracellular (endogenous)	Extracellular (exogenous)
Presents to	CD8+ cytotoxic T cells	CD4+ T helper cells
Tissue Distribution	All nucleated cells	Antigen-presenting cells (APCs) only
Key Polymorphism Domain	a1 and a2 domains	b1 domain

• Mnemonic: Class I x CD8 (1x8=8); Class II x CD4 (2x2=4).

MHC Gene Organization

• Class I region (telomeric): HLA-A, HLA-B, HLA-C

• Class II region (centromeric): HLA-DR, HLA-DQ, HLA-DP

• Class III region: Complement components (C2, C4, factor B), TNF (not HLA molecules)

Inheritance, Polymorphism, and Nomenclature

Genetic Principles

• Mendelian Co-dominant Inheritance: Both parental alleles are expressed.

• Haplotype: A set of HLA alleles inherited together from one parent.

• Linkage Disequilibrium: Certain allele combinations are inherited together more frequently than expected by chance.

HLA Polymorphism

• HLA is the most polymorphic system in the human genome, with polymorphism concentrated in the peptide-binding region (PBR).

• High frequency of non-synonymous mutations in the PBR, affecting peptide binding and immune recognition.

• Significant ethnic variation in allele frequencies.

• As of 2019: >18,000 Class I alleles; >7,000 Class II alleles.

HLA Nomenclature

Element	Meaning	Clinical Importance
HLA- + Gene (e.g. A)	HLA prefix + gene name	Identifies gene locus
* (asterisk)	Separator between gene and allele fields	Distinguishes DNA from serology
Field 1 (e.g. 02)	Allele group (serological type)	Matches old serology
Field 2 (e.g. 01)	Specific HLA protein	Most important for transplant matching
Field 3	Synonymous DNA substitution	Usually not clinically relevant
Field 4	Non-coding region difference	Usually not clinically relevant
Suffix N	Null allele (not expressed)	Functionally absent
Suffix L	Low expression	Reduced cell surface levels

• Example: HLA-A*02:01:01:02N

HLA Typing Methods

Overview of Typing Techniques

Method	Resolution	Time	Key Features
CDC (serology)	Low	4-5 hrs	Antibody + complement + live cells; detects complement-fixing antibodies only
PCR-SSP	Low to High	~2.5 hrs	Sequence-specific primers; agarose gel; rapid; does not require live cells
PCR-SSO / Luminex	Intermediate	Several hrs	Bead hybridization; automated; high throughput
Sanger SBT	High	~2 days	Direct sequencing; phase ambiguity; expensive
NGS	Very High	~3 days	Gold standard for HSCT; phased alleles; no prior type needed

CDC Typing (Complement Dependent Cytotoxicity)

1. Isolate lymphocytes from blood.

2. Add cells to wells with alloantisera of known HLA specificity; incubate at 22°C for 30 min.

3. Add rabbit complement; incubate at 22°C for 60 min.

4. If antibody binds HLA, complement activation leads to cell lysis.

5. Add dyes (ethidium bromide, acridine orange, ink); read under UV microscope.

• Scoring: 1 (≤10% dead, negative), 2 (10-20%), 4 (20-40%), 6 (40-80%), 8 (80-100%, strongly positive)

Advantages and Disadvantages of CDC

Advantages	Disadvantages
Simple equipment; low cost; detects IgM; up to 100 cells per panel; DTT eliminates IgM interference	Only detects complement-fixing antibodies; low sensitivity; time-consuming; requires live cells; subjective scoring

PCR-SSP

• Developed by Olerup & Zetterquist (1992).

• Multiple parallel PCRs with sequence-specific primers; results visualized on agarose gel.

• Does not require live cells; higher resolution than CDC; rapid and objective.

NGS (Next Generation Sequencing)

• Provides high-resolution, unambiguous, phased allele assignment.

• Gold standard for haematopoietic stem cell transplantation (HSCT).

HLA Antibody Detection

Clinical Importance

• Pre-formed donor-specific antibodies (DSA) can cause hyperacute rejection.

• Post-transplant monitoring for new DSA predicts chronic or antibody-mediated rejection.

• Relevant for platelet refractoriness, TRALI, and FNHTR investigations.

Detection Methods

Method	Antibody Detected	Sensitivity	Gold Standard?
CDC	Complement-fixing only	Low	No
ELISA	IgG only	Medium	No
Flow cytometry	IgG and IgM	High	No
Luminex SAB	IgG (and IgM)	Very High	Yes (UK gold standard)

• Luminex SAB: Uses color-coded beads, each coated with a single recombinant HLA protein; detects specific antibody specificities; results as Mean Fluorescence Intensity (MFI).

Crossmatching and Rejection

Crossmatch Techniques

• CDC Crossmatch: Recipient serum + donor cells + complement; lysis indicates DSA presence.

• Flow Cytometry Crossmatch: More sensitive; uses fluorescent antibodies to detect IgG binding to donor T and B cells.

Types of Rejection

Type	Timing	Mechanism	Prevention
Hyperacute	Minutes-hours	Pre-formed IgG DSA + complement → thrombosis, graft infarction	Negative crossmatch before transplant
Acute cellular	Days-weeks	T cell-mediated allorecognition	Immunosuppression, HLA matching
Antibody-mediated	Days-months	New or pre-formed DSA	Monitor DSA, plasmapheresis/IVIG
Chronic	Months-years	Ongoing immune damage, fibrosis	Minimize sensitization, immunosuppression

Solid Organ Transplantation

Key Steps and Allocation

• Recipient workup: ABO typing, HLA typing (A, B, C, DR, DQ, DP), HLA antibody screen, addition to waiting list.

• Donor workup: ABO, microbiology (HIV, Hep B/C, HTLV), HLA typing, crossmatch.

• Kidney allocation prioritizes 0 mismatches at HLA-A, B, DR (000 MM) for national priority.

Effect of HLA Mismatches on Kidney Graft Survival

Mismatches (A+B+DR)	20-yr Graft Survival	Half-Life (years)
0 MM	42%	17.0
1 MM	37%	15.1
2 MM	35%	14.5
3 MM	34%	14.1
4 MM	33%	13.6
6 MM	28%	12.4

• More mismatches result in shorter graft survival.

Haematopoietic Stem Cell Transplantation (HSCT)

Principles and Matching

• Used for haematological malignancies, aplastic anaemia, immunodeficiency, haemoglobinopathies.

• Sources: bone marrow, peripheral blood, umbilical cord blood.

• High-resolution HLA typing (NGS) is essential, especially for unrelated donors (minimum 10/10 match at A, B, C, DRB1, DQB1).

• GvHD (Graft vs Host Disease): Donor T cells attack recipient tissues.

• GvL (Graft vs Leukaemia): Beneficial effect where donor immune cells eliminate residual cancer cells.

HLA in Transfusion Medicine

Key Transfusion Reactions

• FNHTR (Febrile Non-Haemolytic Transfusion Reaction): Recipient anti-HLA antibodies react with donor leucocytes; prevented by leucodepletion.

• TRALI (Transfusion-Related Acute Lung Injury): Donor anti-HLA/HNA antibodies react with recipient leucocytes; prevented by using male-only plasma.

• TaGVHD (Transfusion-Associated Graft vs Host Disease): Donor T cells engraft and attack recipient; prevented by gamma irradiation of blood products.

• Platelet Refractoriness: Most commonly caused by anti-HLA Class I antibodies; managed by providing HLA-matched platelets.

HLA and Disease Associations

Selected Disease Associations

Disease	HLA Association	Key Stats
Ankylosing Spondylitis	B*27	>95% patients; RR >150
Coeliac Disease	DQA1*05/DQB1*02 (DQ2)	99%; RR >250 (strongest known)
Narcolepsy	DQB1*06:02	>95%; RR >38
Abacavir Hypersensitivity	B*57:01	Mandatory screening before prescribing
Type 1 Diabetes (risk)	DQB1*03:02	81%; RR 14
Type 1 Diabetes (protective)	DQB1*06:02	<1% patients; RR 0.02 (protective)
Rheumatoid Arthritis	DRB1*04	81%; RR 9
Multiple Sclerosis	DRB1*15, DQB1*06	86%; RR 12

• Relative Risk (RR): Indicates how much more likely individuals with the allele are to develop the disease compared to those without.

Key Equations and Concepts

• Relative Risk (RR): $RR = \frac{\text{Incidence in exposed}}{\text{Incidence in unexposed}}$

• HLA Nomenclature Example: HLA-A*02:01:01:02N

Summary Table: HLA Typing and Antibody Detection Methods

Method	Resolution	Antibody Detected	Gold Standard?
CDC	Low	Complement-fixing only	No
ELISA	Medium	IgG only	No
Flow Cytometry	High	IgG and IgM	No
Luminex SAB	Very High	IgG (and IgM)	Yes

Additional Info

• HLA matching is critical for long-term graft survival in transplantation and for minimizing transfusion reactions.

• High-resolution typing (NGS) is increasingly standard for unrelated donor HSCT due to its accuracy and ability to resolve phased alleles.

• Prevention strategies for transfusion reactions include leucodepletion (FNHTR, TRALI) and gamma irradiation (TaGVHD).