curs 2 - imunologia transplantului+tr [read-only]
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Immunology of
Transplantation
Prof. Ileana Constantinescu MD PhD
Centre for Immunogenetics and Virology
Reference Centre in Immunology of Transplantation for Romania
Fundeni Clinical Institute
Bucharest
VIROLOGICAL ASSESSMENT
Both donor and recipient are tested for: VHB, VHD, VHC, HIV 1/2, CMV, EBV, HSV 1 si 2, VZV, HTLV 1/2 , rubella virus, toxoplasma gondii and chlamydia.
MethodsIndirect diagnostic tests (serological)Direct diagnostic tests, molecular biology tests (PCR, RT-PCR).
• IMMUNOGENETICSThe purpose of tissue typing is to identify the expression of MHC on
cells. More than one method may be required to give a complete
picture.
HLA Typing by molecular biology methods – PCR
SSOP- sequence-specific oligonucleotide probe
hybridization (medium resolution )
SSP – sequence-specific primers (high resolution)
SBT – allele SEQR (the highest available resolution)SBT – allele SEQR (the highest available resolution)
Anti-HLA antibody detection and identification- AHG CDC
- ELISA
Cross- match- CDC
- ELISA
Sample of cells or tissue
Combine DNA with sequence-specific primer fix for each allele
Amplify by PCR
Assay Report
Sample ID: 455FM59
Patient Name: F.M. – Kidney donor(mother) for recipient F.I.
Entered on: 1/22/2002
Account: admin LiPA HLA-A/v.1.4/001102
AssayResult
ALLELE GROUP TYPING:ALLELE GROUP TYPING:
A*02A*24
Assay Report
Sample ID: 456FI38
Patient Name: F.I. – Kidney recipientEntered on: 1/22/2002
Account: admin LiPA HLA-A/v.1.4/001102
AssayResult
ALLELE GROUP TYPING:ALLELE GROUP TYPING:
A*02 A*24
ALLELE GROUP TYPING:
Assay Report
Sample ID: 455FM59
Patient Name: F.M. Entered on: 1/24/2002
Account: admin LiPA HLA-B/v.1.4/001102
AssayResult
ALLELE GROUP TYPING:
B*18B*35
ALLELE GROUP TYPING:
Assay Report
Sample ID: 456FI38
Patient Name: F.I. Entered on: 1/24/2002
Account: admin LiPA HLA-B/v.1.4/001102
AssayResult
ALLELE GROUP TYPING:
B*18B*39
ALLELE GROUP TYPING:
Assay Report
Sample ID: 455FM59
Patient Name: F.M. Entered on: 1/21/2002
Account: admin LiPA HLA-DRB/v.5.4/001102
AssayResult
ALLELE GROUP TYPING:
DRB1*07
DRB1*11
ALLELE GROUP TYPING:
Assay Report
Sample ID: 456FI38
Patient Name: F.I.Entered on: 1/21/2002
Account: admin LiPA HLA-DRB/v.5.4/001102
AssayResult
ALLELE GROUP TYPING:
DRB1*11
DRB1*13
DQB1* DQB1*
Assay Report
Sample ID: 455FM59
Patient Name: F.M. Entered on: 1/21/2002
Account: admin LiPA HLA-DQB/v.2.6/001102
AssayResult
DQB1*03
DQB1*03
DQB1* DQB1*
Assay Report
Sample ID: 456FI38
Patient Name: F.I. Entered on: 1/21/2002
Account: admin LiPA HLA-DQB/v.2.6/001102
AssayResult
DQB1*03
DQB1*06
DNA
80 ng for Class I
40 ng for Class II
Importance of DNA
Quality
100 ng Genomic DNA 1% Agarose Gel
Assign-SBT Resolves AmbiguitiesSequences are arranged in “layers”
…Master sequence
Patient result
HLA SBT
Resolve heterozygous sequence ambiguities
- Separate alleles by SSP-PCR- Sequence hemizygous PCR product- Resolve ambiguity- High throughput - High throughput - Uniform Protocols- Pre-formulated reagents- All Sequencing platforms
Add resolution to typings obtained by lower resolution methods (e.g. SSP, SSOP)
Take advantage of low resolution data to select appropriate reagents
HLA Antibody Detection
HLA antiserum screening is an important work effort in clinical HLA laboratories.
The result is used to determine the degree of humoral alloimmunization,expressed as percent panel reactive antibody (%PRA).(%PRA).
The antibody specificity can accurately predict donor incompatibility and the development of chronic allograft rejection.
Methods: AHG – CDCELISA – screening Class I and Class II
- identification Class I and Class II
Class I HLA Antibody
AnalysisGTI QuikScreen
• HLA Class I Ab Screen• Pooled platelets (minimum
of 300 donors)
GTI Quik-ID Class I
• HLA Class I antibody specificity
of 300 donors)• Highly specific (no Class II
interference)• Flexible formats, easy to
use• Screen up to 40 samples
per tray in 2.5 hrs• WinScreen software
• Percent Panel Reactive (%PRA)
• Panel of 40 donors• Solubilized Class I antigen
from platelets• Sensitive capture assay• Software analysis package
including CREG analysis
Class II HLA Antibody
AnalysisGTI B-Screen
• HLA Class II Ab Screen• Soluble HLA from EBV
Transformed cells
GTI Quik-ID Class II
• HLA Class II Ab specificity• Percent panel reactive Transformed cells
• Affinity purified• Flexible format - strip wells• Highly specific (no Class I
interference)• Screen 40+ samples per
tray in 2.5 hrs• WinScreen software
• Percent panel reactive (%PRA)
• Panel of 30 cell lines• Affinity purified Class II
HLA from EBV transformed cell lines
• Sensitive capture assay• Software analysis package
Patient sample 1
Patient sample 2
Patient sample 3
Patient sample 4
IgG IgM B-Screen NAW
Patient sample 5
Negative control
Positive control
Blank Well
Antibody Screening
Algorithm
New patients – full work-up� Flow specificity and PRA� ELISA specificity and PRA
Current patients – Negative or PositiveCurrent patients – Negative or Positive� Negatives screened monthly or quarterly� Any neg-pos refluxed to Ab ID� Positives screened monthly by ELISA� Specificity and PRA tracked � Ambiguous specificity refluxed to Flow
Antibody Monitoring System
ELISA assay designed to detect ELISA assay designed to detect donor reactive IgG antibodies in
recipient sera
Used for Immunological monitoring of donor-specific
HLA alloantibodies in transplant patients that may transplant patients that may
lead to early graft loss or chronic rejection
• Retrospective Crossmatch• Prospective Crossmatch• Post-transplant • Post-transplant
Immunological Monitoring
• Detects only HLA donor specific antibodies
• IgG specific - will not detect • IgG specific - will not detect IgM (autolymphocytotoxic) antibodies
• Detects non-complement binding antibodies
• Detects Class II specific • Detects Class II specific HLA antibodies in presence of strong Class I antibody
1st step: lysate preparation
takes about 15 minutes after takes about 15 minutes after isolation of cells
LYSATE PREPARATION
LYSATE PREPARATION
LYSATE PREPARATION
LYSATE PREPARATION
2nd step: ELISA
takes about 3 to 4 hrs -takes about 3 to 4 hrs -depending on number of donors
NegativeControl
LysateControl
Class I Class II
RecipientControl
PositiveControl
RecipientSamples
Antibody Monitoring System
What are it’s benefits?What are it’s benefits?
Employs three sets of ControlsReagent Control
Negative Control
Lysate ControlLysate Control
Lysates can be frozen at –80° Cfor future use
Antibody Monitoring System
No interference with therapeutic No interference with therapeutic “rescue” immunosuppresents
Distinguish between Donor and
Antibody Monitoring System
Distinguish between Donor and non-Donor HLA Abs
No interference with
Antibody Monitoring System
No interference with IvIG/pheresis protocols
Antibody Monitoring System
• Up to 44 patient sera per plate with one donorwith one donor
• Up to 6 donors with 4 recipients each per plate
Antibody Monitoring System
What are the benefits of using What are the benefits of using Elisa and what do you require to
run the assays?
Antibody Monitoring System Microtiter plate based ELISA
Flexibility — versatile snap-in strips
Convenience — screening on a single tray for a variety of antibodies
Format — fits most commercially available microplate readers
Current Microplate
Readers
Dynex MRX, MR7000, MR5000LabSystems Multiscan MS, EX, RCBioTek EL-800, ELx-800, MicroQuantBioTek EL-800, ELx-800, MicroQuant
· New Dynex Opsys
Low cost
Simple to maintainReliable
Kidney – pancreas transplantation
whole organ transplantation
• Pancreas transplantation alone (PTA)• Simultaneous pancreas-kidney (SPK)
transplantation• Pancreas after kidney (PAK) transplantation
Immunological algorythm:• HLA typing: A, B, DRB1• Cytotoxic antibodies• Crossmatch
Transplantation of
pancreatic islets
Langerhans cells are targetedVirological assessment of both , donor and recipientand recipientHLA typing: A, B, DRB1Cytotoxic antibodiescrossmatch
Kidney transplantation in
children
Usualy the donor is one of the parentsTissue typing: A, B, DRB1Cytotoxic antibodiesCytotoxic antibodiescrossmatch
5.5
2010