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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.


Methods
Indirect diagnostic tests (serological)
Direct diagnostic tests, molecular biology tests
(PCR, RT-PCR).










IMMUNOGENETICS
The 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)

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:

A*02



A*24

Assay Report

Sample ID: 456FI38

Patient Name: F.I. Kidney recipient
Entered on: 1/22/2002

Account: admin LiPA HLA-A/v.1.4/001102


AssayResult
ALLELE GROUP TYPING:

A*02

A*24
ALLELE GROUP TYPING:

B*18


B*35

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*18

B*39
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:

DRB1*
07

DRB1*
11

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*
11

DRB1*
13
Assay Report

Sample ID: 456FI38

Patient Name: F.I.
Entered on: 1/21/2002

Account: admin LiPA HLA-DRB/v.5.4/001102


AssayResult
DQB1*
03
DQB1*
03

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*
06
Assay Report

Sample ID: 456FI38

Patient Name: F.I.
Entered on: 1/21/2002

Account: admin LiPA HLA-DQB/v.2.6/001102


AssayResult
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 Ambiguities
Sequences 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
- 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).

The antibody specificity can accurately predict donor incompatibility
and the development of chronic allograft rejection.


Methods: AHG CDC
ELISA screening Class I and Class II
- identification Class I and Class II
GTI QuikScreen

HLA Class I Ab Screen
Pooled platelets (minimum
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

GTI Quik-ID Class I

HLA Class I antibody
specificity
Percent Panel Reactive
(%PRA)
Panel of 40 donors
Solubilized Class I antigen
from platelets
Sensitive capture assay
Software analysis package
including CREG analysis

GTI B-Screen

HLA Class II Ab Screen
Soluble HLA from EBV
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
GTI Quik-ID Class II

HLA Class II Ab specificity
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
Patient sample 5
Negative control
Positive control
Blank Well
IgG IgM B-Screen NAW
Antibody Screening
Algorithm

New patients full work-up
Flow specificity and PRA
ELISA specificity and PRA
Current 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
donor reactive IgG antibodies in
recipient sera
Used for Immunological
monitoring of donor-specific
HLA alloantibodies in
transplant patients that may
lead to early graft loss or
chronic rejection
Retrospective Crossmatch
Prospective Crossmatch
Post-transplant
Immunological Monitoring
Detects only HLA donor
specific antibodies
IgG specific - will not detect
IgM (autolymphocytotoxic)
antibodies
Detects non-complement
binding antibodies
Detects Class II specific
HLA antibodies in presence
of strong Class I antibody
1st step: lysate preparation

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 -
depending on number of donors
Negative
Control
Lysate
Control
Positive
Control
Class I Class II
Recipient
Samples
Antibody Monitoring
System
What are its benefits?
Employs three sets of Controls
Reagent Control
Negative Control
Lysate Control
Lysates can be frozen at 80 C
for future use
Antibody Monitoring
System
No interference with therapeutic
rescue immunosuppresents
Distinguish between Donor and
non-Donor HLA Abs
Antibody Monitoring
System
No interference with
IvIG/pheresis protocols
Antibody Monitoring
System
Antibody Monitoring
System
Up to 44 patient sera per plate
with one donor
Up to 6 donors with 4
recipients each per plate
Antibody Monitoring
System
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

Dynex MRX, MR7000, MR5000
LabSystems Multiscan MS, EX, RC
BioTek EL-800, ELx-800, MicroQuant
New Dynex Opsys
Low cost
Simple to maintain
Reliable
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 targeted
Virological assessment of both , donor
and recipient
HLA typing: A, B, DRB1
Cytotoxic antibodies
crossmatch
Kidney transplantation in
children
Usualy the donor is one of the parents
Tissue typing: A, B, DRB1
Cytotoxic antibodies
crossmatch
5.5
2010