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 sequencespecific primer fix for each

allele

Amplify by
PCR

Assay Report
Sample ID: 455FM59
Patient Name: F.M.
Entered on: 1/22/2002

Kidney donor(mother) for recipient F.I.

LiPA HLA-A/v.1.4/001102

Account: admin

AssayResult
ALLELE GROUP TYPING:

A*02

A*24

Assay Report
Sample ID: 456FI38
Patient Name: F.I. Kidney recipient
Entered on: 1/22/2002

LiPA HLA-A/v.1.4/001102

Account: admin

AssayResult

ALLELE GROUP TYPING:

A*02

A*24

Assay Report
Sample ID: 455FM59
Patient Name: F.M.
Entered on: 1/24/2002

LiPA HLA-B/v.1.4/001102

Account: admin

AssayResult
ALLELE GROUP TYPING:

B*18

B*35

Assay Report
Sample ID: 456FI38
Patient Name: F.I.
Entered on: 1/24/2002

LiPA HLA-B/v.1.4/001102

Account: admin

AssayResult
ALLELE GROUP TYPING:

B*18

B*39

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

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

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

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 Ambiguities

Sequences are arranged in
layersMaster 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

Class I HLA Antibody

Analysis
GTI Quik-ID Class I

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

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

Class II HLA Antibody

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

IgG

Patient sample 1
Patient sample 2
Patient sample 3
Patient sample 4
Patient sample 5
Negative control
Positive control
Blank Well

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

Class I

Class II

Negative
Control
Lysate
Control
Positive
Control

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

Antibody Monitoring System

Distinguish between Donor and
non-Donor HLA Abs

Antibody Monitoring System

No interference with
IvIG/pheresis protocols

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

Current Microplate
Readers
Dynex MRX, MR7000, MR5000
LabSystems Multiscan MS, EX, RC
BioTek EL-800, ELx-800, MicroQuant

New Dynex Opsys

Low cost
Reliable
Simple to maintain

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