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