Histocompatibility Testing

The following sections are included:

• CONTENTS

The following sections are included:

• Introduction

• Origin and maintenance of polymorphisms: classical models

• Primary evidence for selection

• HLA polymorphism is old

• Haplotypes and linkage disequilibrium

• Selective mechanisms

• HLA and foetal maternal immunity

• Conclusions

• References

The following sections are included:

• Introduction

• Function of MHC class I and class II molecules

• Structure of MHC class I and class II molecules

• Antigen binding by MHC class I and class II molecules

• MHC class I

• MHC Class II

• Synthesis and export

• Genomic organisation of the MHC

• Polymorphism

• Influence of polymorphism on peptide binding

• Interactions with other molecules

• References

The following sections are included:

• The beginning of nomenclature: serology

• The molecular revolution

• Nomenclature in the future

• References

The following sections are included:

• The methods

• The sources of antibodies
  • Antisera
  • Monoclonal antibodies

• The place of serology today and in the future

• References

The following sections are included:

• Introduction

• Clinical relevance
  • Organ transplantation
  • Bone marrow transplantation
  • HLA-AB typing for platelet transfusion
  • HLA-B27 typing
  • HLA typing for disease association studies
  • HLA and cancer research

• Methods
  • Antisera
  • Lymphocytes
  • Complement
  • Incubation time and temperature

• Typing equipment
  • Dyes and microscopes

• Interpretation
  • Cross-reactivity
  • Splits and associated antigens
  • HLA-Class II typing
  • Homozygosity versus non/low expression
  • HLA nomenclature: Serology - DNA equivalents

• References

The following sections are included:

• Introduction

• Development of HLA-specific antibodies
  • Pregnancy
  • Transplantation
  • Blood transfusion
  • Planned immunisation

• Clinical relevance of HLA-specific antibodies
  • Solid organ transplantation
  • Bone marrow transplantation
  • Transfusion reactions

• Non-HLA antibodies
  • Autoantibodies
  • Monocyte/endothelial cell antibodies
  • Epithelial cell antibodies
  • Anti-idiotype antibodies

• Screening for HLA-specific antibodies
  • Procedures for identifying antibodies in the patient's sera
  • Procedures for screening sera to be used as reagents
  • Citrated blood
  • Heparinised blood
  • Screening sera with DTT
  • High PRA

• Summary

• References

The following sections are included:

• Introduction

• Structures of sHLA molecules and their biological role
  • Structure of soluble HLA class I and class II antigens

• Soluble HLA molecules in healthy individuals

• Clinical and biological relevance of sHLA
  • Infectious disease
  • AIDS
  • Autoimmune disease
  • Cancer
  • Transplantation
  • Pregnancy
  • Immunoregulation

• Methodology
  • Determination of sHLA concentration
  • Protocol for the determination of total β2m-associated sHLA-I
  • Determination of sHLA allotypes
  • Protocol for the determination of sHLA-A2 molecules
  • Determination of sHLA-II
  • Protocol for the determination of sHLA-DR α/β heterodimer complexed with peptides
  • Biochemical analysis of sHLA molecules
  • Protocol for SDS-PAGE and ID-IEF analysis
  • SDS-PAGE
  • ID-IEF
  • Western blotting

• References

The following sections are included:

• Introduction

• Clinical relevance

• Methods
  • Preparation of lymphocytes
  • Separation of T and B cells
  • Collection and storage of sera for crossmatching
  • Complement dependent cytotoxicity (CDC) crossmatch
  • Standardisation of the flow cytometer
  • Flow cytometry crossmatch
  • Crossmatching by ELISA
  • External Quality Assurance

• References

The following sections are included:

• Probe-hybridisation techniques

• Electrophoresis-based techniques

• DNA conformational analysis techniques

• References

The following sections are included:

• Introduction

• Clinical relevance

• Methods
  • Design of PCR primers and PCR primer mixes
  • General information on PCR-SSP using Phototyping methods
  • DNA extraction
  • Dispensing primer mixes
  • Setting up PCR-SSP using TDMH buffer
  • Setting up PCR-SSP using heparin-contaminated DNA
  • PCR amplification program
  • Electrophoresis
  • Gel photography
  • Interpretation of results

• Notes
  • Testing control solutions
  • Testing allele-specific primers
  • Testing TDMH

• Troubleshooting
  • All reactions have failed (no allele, no control-specific amplicons)
  • Generally weak reactions
  • Too many allele-specific amplicons in one locus
  • Too many allele-specific amplicons in all loci
  • No allele-specific amplicons at one locus
  • Individual reaction failure
  • Allele-specific bands present but no controls
  • Control-specific amplicons but no alleles
  • Part of the typing has worked well, but the remainder has failed

• Updating the typing system

• Materials
  • DNA preparation
  • Ingredients for primer mixes
  • PCR ingredients
  • Setting up PCR reactions
  • Gel electrophoresis

• Specialist equipment

• Suppliers Addresses

• References

The following sections are included:

• Introduction
  • HLA-A, -B and -C primers
  • HLA-DRB primers
  • Protocols

• Clinical relevance
  • Renal transplantation
  • Bone marrow transplantation
  • Bone marrow registries

• Methods
  • Materials
  • PCR
  • Electrophoresis of PCR products
  • Dot blotting of membranes
  • Dehybridisation of membrane
  • 3′ end-labelling of SSOP
  • Prehybridisation, hybridisation and stringent SSPE washes
  • Chemiluminescence detection

• Tips, Quality Control And Troubleshooting
  • Note on the use of tetramethylammonium chloride (TMAC)

• Equipment

• References

The following sections are included:

• Introduction

• Dideoxy-mediated chain termination DNA sequencing
  • Use of PCR fragments as initial template material
  • Streptavidin/biotin linkage to isolate single stranded DNA
  • DNA sequencing enzymology improvements
  • Fluorescent dye labelling
  • Automated sequencing machine development
  • Sequence analysis software

• Establishment of the SBT concept

• Development of DNA-based sequencing-based typing
  • Class II
  • Class I
  • Refinement of SBT strategies leading to the current methods
    • Ambiguous allele combinations
    • PCR and sequencing primer design problems
    • Allele drop out
    • Methods still too slow, complicated and expensive
  • HLA-DPB
  • HLA-DPA
  • HLA-DRB
  • HLA-DQB
  • HLA-DQA
  • HLA-C
  • HLA-B
  • HLA-A

• Future role and direction of SBT

• References

The following sections are included:

• Introduction

• Single strand conformation polymorphism (SSCP)

• Methods for SSCP

• Heteroduplex analysis

• Methods for Heteroduplex analysis
  • PCR amplification
  • Polyacrylamide gel electrophoresis

• Reference strand mediated conformation analysis (RSCA)

• Limitations of DNA conformational techniques and how these are resolved by RSCA
  • Complex banding patterns
  • Inability to identify all variants
  • Subjectivity of data analysis
  • Intra and intergel variability
  • Limitations relating to distance travelled by DNA in polyacrylamide gels
  • Limitations related to quantity of loaded DNA
  • Discrimination of homoduplexes from heteroduplexes with only one or two mismatches

• Methods for RSCA
  • PCR amplification
  • Preparation of FLRs
  • Hybridisation reaction
  • Preparation of top and bottom marker duplexes for HLA-A typing
  • Preparation of top and bottom marker duplexes for HLA-B typing
  • Post-hybridisation treatment
  • Preparation of non-denaturing polyacrylamide gel
  • Electrophoresis
  • Analysis of results

• Acknowledgements

• References

The following sections are included:

• Introduction
  • Microsatellites

• HLA region microsatellites

• Clinical and research relevance

• Methods
  • DNA extraction
  • PCR amplification and labelling of primers
  • Standard PCR mixes
  • PCR amplification conditions
  • Electrophoresis
  • Final composition of samples
  • Composition of migration buffer
  • Electrophoresis conditions

• Interpretation
  • Variants

• Some difficulties and pitfalls in microsatellite analysis

• Conclusion

• References

The following sections are included:

• Introduction

• On-line sequences

• On-Line Sequence Alignments

• Other On-line Resources

• Shareware and freeware computer applications
  • GeneDoc
  • MACAW
  • ProMSED2
  • Other software

• Appendices
  • Internet Sites
  • Software

• Contact E-mail addresses

• References

HLA compatibility between a transplant recipient and a potential donor is one of the main factors that contributes to the successful outcome of the transplant. Major and minor differences in the HLA antigens between donor and recipient cells can be detected by T cells with a high degree of sensitivity.

The following sections are included:

• Introduction

• Mixed Lymphocyte Reaction (MLR) / Mixed Lymphocyte Culture (MLC)
  • Introduction
  • Clinical relevance
  • Methods
  • Internal & external QC
  • Specialist equipment or reagents required

• Modifications of Mixed Lymphocyte Cultures
  • Mixed epidermal cell-lymphocyte reaction
  • Skin explant assay
  • T-cell activation molecule gene expression in MLC
  • Addition of cytokines to the MLC

• Cell Mediated Lympholysis (CML)
  • Introduction
  • Clinical relevance
  • Methods

• Cytotoxic T Lymphocyte Precursor (CTL-p) frequency analysis
  • Introduction to limiting dilution analysis (LDA)
  • Introduction to CTL-p
  • Clinical relevance
  • Methods
  • Internal & external QC

• Modified CTL-p assays

• Helper T Lymphocyte Precursor (HTL-p) frequency analysis for the enumeration of IL-2 secreting-cell frequencies
  • Introduction
  • Clinical relevance
  • Methods
  • Internal & external QC
  • Specialist equipment or reagents required

• Combined CTL-p & HTL-p assays

• Helper T Lymphocyte Precursor (HTL-p) frequency analysis for the enumeration of IL-4 secreting cell frequencies

• Cytokine Profiles
  • Introduction
  • Clinical relevance
  • Methods to determine cytokine protein secretion profiles (IL-2, IL-4, IL-10 and IFN_γ)
  • Specialist equipment or reagents required

• References

In this speciality there are primary differences in donor origin, the procurement, supply, storage and histocompatibility requirements of donor material and the urgency and availability of substitute and alternative patient treatments. These make the distinction between organ allocation (the case for all cadaveric donor derived solid organs, except corneas) and donor selection apparent.

The following sections are included:

• Introduction

• Principles of allocation
  • Clinical need or urgency
  • Waiting time
  • Evidence based
  • Random lottery
  • Biased allocation
  • Financial profit

• Histocompatibility in solid organ transplantation

• Allocation of kidneys
  • Cadaver donors
  • Living donors

• Allocation of livers

• Allocation of thoracic organs

• Allocation of corneas

• Organ allocation for multi-organ transplants

• Problems of waiting times and clinical urgency

• References

The following sections are included:

• Introduction

• Sources of stem cells
  • Bone marrow from related donors
  • Bone marrow from unrelated donors (UD-BMT)
  • Peripheral blood stem cells (PBSC)
  • Cord blood stem cells

• Clinical effect of HLA matching in stem cell transplantation
  • ID-BMT versus MUD-BMT
  • HLA Class I mismatch
  • HLA Class II mismatch

• Donor Selection Procedures

• Potential family donors

• HLA-matched unrelated donors
  • Donor search
  • High resolution confirmatory typing (CT)
  • Initial donor selection criteria
  • HLA typing of donors
  • HLA matching
  • HLA-typing methods
  • Additional selection methods: cellular assays
  • HLA and non-HLA factors

• Summary

• References

The following sections are included:

• Introduction
  • Corrected count increment (CCI)

• Non-immunological refractoriness

• Immunological refractoriness to random donor platelet transfusions

• Management of immunological platelet refractoriness
  • HLA antibody screening
  • HLA class I (A & B) typing
  • Provision of HLA-matched or crossmatch-negative platelets
    • Provision of HLA-matched platelets
    • Use of HLA- A and -B homozygous platelet donors
    • Provision of crossmatched platelets
  • Correlation between platelet support with increments following platelet transfusion

• Recruitment and selection of platelet donors

• Prevention of platelet refractoriness and HLA alloimmunisation
  • Leucodepletion
  • Ultraviolet irradiation
  • Reduced donor exposure

• Summary

• References

The extensive polymorphism of HLA makes it an excellent marker system. Some of the most common analytical aspects of HLA typing results are set out in Chapters 15 to 18. Paternity testing and genetic counselling are two additional areas, outside the scope of the present volume.

The following sections are included:

• Introduction

• History

• Definition of populations

• The genetics of the HLA system

• Summary of population genetic parameters

• Establishing reliable population genetics parameters

• Population genetics and its applications

• Population genetics for data validation

• Concluding remarks

• References

The following sections are included:

• Phenotype Frequencies

• Gene Frequencies

• Haplotype Frequencies

• Linkage Disequilibrium

• References

The following sections are included:

• Introduction

• Measuring outcomes

• Data collection and storage

• Life table analysis

• Statistical packages

• Survival curves

• Half life calculations

• Multi-variate analyses

• Interpretation and pitfalls

• References

• Supplier

The following sections are included:

• Introduction

• Basic methods of survival analysis

• Comparison of transplants with other treatment

• Autologous transplants

• Allogeneic transplants from a related donor

• Unrelated donor transplants

• Non-randomised comparisons

• Time-to-transplant bias

• Selection bias

• Endpoints

• Conclusions

• Appendix
  • Acknowledgement

• References

The following sections are included:

• Introduction
  • Basic definitions
  • Necessary preliminaries

• Study design
  • Choice of controls
  • Power and sample size

• Statistical methods
  • Two by two tables
  • Several exposure categories
    • Comparison of allele frequencies
    • Comparison of phenotypes one by one
    • Comparison of genotypes
  • Family-based case control studies
    • Ignoring matching
    • Taking into account matching
    • Multiple alleles
    • Missing parental data
    • Interpretation of results

• Appendix
  • Sample size calculations for case-control studies
  • Confidence intervals for odds ratios

• References

The following sections are included:

• Introduction

• Phenotyping

• Identification of minor H encoding genes and peptide epitopes

• Why are endogenous peptides immunogenic?

• Genotyping for minor H antigens

• Potential clinical use of minor H peptides: tolerance induction and tetramer monitoring of minor H specific T cell responses

• Summary
  • Acknowledgements

• References

The following sections are included:

• INDEX