Curs 2 - Imunologia transplantului+TR

8/18/2019 Curs 2 - Imunologia transplantului+TR

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Immunology ofTransplantation

Prof. leana Constantinescu MD PhD

Centre for Immunogenetics and VirologyReference Centre in Immunology of Transplantation for Romania

Fundeni Clinical Institute

Bucharest


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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 , r!e""a #irs, to$op"as%a &ondii and ch"a%'dia(

Methods

Indirect dia&nostic tests )sero"o&ica"*

Direct dia&nostic tests, %o"ec"ar !io"o&' tests

)+C, T-+C*(


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• IMM!"#$!$TIC%The purpose of tissue typing is to identify the e'pression of M(C

on cells. More than one method may )e re*uired to gi+e a complete

picture.

(,- Typing )y molecular )iology methods PCR

%%"P/ se*uence/specific oligonucleotide pro)e

hy)ridi0ation 1medium resolution 2

%%P se*uence/specific primers 1high resolution2%BT allele %$3R 1the highest a+aila)le resolution2

-nti/(,- anti)ody detection and identification / -(# CDC

/ $,I%-

Cross/ match / CDC

/ $, %-


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Sa%p"e of ce""s or tisse

Co%!ine D. 0ith seence-

specific pri%er fi$ for each

a""e"e

%p"if' !'

+C


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-ssay Report

%ample ID4 566FM67

Patient !ame4 F.M. 8idney donor1mother2 for recipient F.I.$ntered on4 9:;;:;


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-ssay Report

%ample ID4 56=FI>?

Patient !ame4 F.I. 8idney recipient$ntered on4 9:;;:;


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ALLELE GROUP TYPING:

B*18B*35

-ssay Report

%ample ID4 566FM67

Patient !ame4 F.M.$ntered on4 9:;5:;


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B*18B*39

-ssay Report

%ample ID4 56=FI>?

Patient !ame4 F.I.$ntered on4 9:;5:;


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DRB1*

07

DRB1*

11

-ssay Report

%ample ID4 566FM67



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DRB1*

11

DRB1*

13

-ssay Report

%ample ID4 56=FI>?

Patient !ame4 F.I.$ntered on4 9:;9:;


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DQB1*

03

DQB1*

03

-ssay Report

%ample ID4 566FM67



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DNA

80 ng for Class I

40 ng for Class II


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Importance of D!-

3uality

100 ng Gen!"# DNA 1$ Ag%&'e Ge(


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Assign-SBT Resolves AmbiguitiesSequences are arranged in

“layers”…Master sequene


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Patient result


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(,- %BT

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 loer resolution methods !e"g" SSP# SS$P%

&a'e advantage of lo resolution data to select appropriate reagents


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(,- -nti)ody Detection

)LA %n"'e&+! '#&een"ng "' %n "!,&%n -&. e//& "n #("n"#%( )LA(%&%&"e'

Te &e'+( "' +'e ee&!"ne e eg&ee / +!&%(

%(("!!+n"%"ne,&e''e %' ,een ,%ne( &e%#"e %n"!(PRA%"

Te %n" ',e#"/"#" #%n %##+&%e( ,&e"# n& "n#!,%""("%n e ee(,!en / #&n"# %((g&%/ &ee#"n

)ethods: A)G ;D;

ELI


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Class (,- -nti)ody

-nalysis

*&+ ,ui'Screen

> HA Class + Ab Screen

> Pooled platelets !minimumof .// donors%

> Highly specific !no Class ++interference%

> 0le1ible formats# easy to

use> Screen up to 2/ samples

per tray in 3"4 hrs

> 5inScreen softare

*&+ ,ui'-+6 Class +

> HA Class + antibody

specificity> Percent Panel Reactive

!(PRA%

> Panel of 2/ donors

> Solubilized Class + antigen

from platelets

> Sensitive capture assay

> Softare analysis pac'age

including CR7* analysis


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Class (,- -nti)ody

-nalysis

*&+ 8-Screen

> HA Class ++ Ab Screen

> Soluble HA from 789&ransformed cells

> Affinity purified

> 0le1ible format - strip ells

> Highly specific !no Class +

interference%> Screen 2/: samples per

tray in 3"4 hrs

> 5inScreen softare

*&+ ,ui'-+6 Class ++

> HA Class ++ Ab specificity

> Percent panel reactive!(PRA%

> Panel of ./ cell lines

> Affinity purified Class ++

HA from 789

transformed cell lines

> Sensitive capture assay

> Softare analysis pac'age


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!at"ent sa#$le %!at"ent sa#$le &

!at"ent sa#$le '

!at"ent sa#$le 4

!at"ent sa#$le (

Negat")e ontrol

!os"t")e ontrol

*lan+ ,ell

IgG IgM *-Sreen NA,


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- ti) d % i


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-nti)ody %creening

-lgorithm

Ne- ,%"en' /+(( -&.=+,?(- ',e#"/"#" %n PRAELI


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Ant".o/ Mon"tor"ng Sste#

ELISA assa /es"gne/ to /etet/onor reat")e IgG ant".o/"es "n

re"$"ent sera


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1se/ for I##unolog"al#on"tor"ng of /onor-s$e"f"

2LA alloant".o/"es "ntrans$lant $at"ents t3at #a

lea/ to earl graft loss or

3ron" reet"on


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Retros$et")e Cross#at3

!ros$et")e Cross#at3

!ost-trans$lant

I##unolog"al Mon"tor"ng


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Detets onl 2LA /onor

s$e"f" ant".o/"es

IgG s$e"f" - 5"ll not /etet

IgM 6autol#$3ototo7"

ant".o/"es


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Detets non-o#$le#ent

."n/"ng ant".o/"es

Detets Class II s$e"f"

2LA ant".o/"es "n $resene

of strong Class I ant".o/


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%st ste$9 lsate $re$arat"on

ta+es a.out %( #"nutes after

"solat"on of ells


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LY


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LY


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LY


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&n/ ste$9 ELISA

ta+es a.out ' to 4 3rs -

/e$en/"ng on nu#.er of /onors


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Negat")e

Control

Lsate

Control

!os"t")e

Control

Class I Class II

Re"$"ent

Sa#$les


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,3at are "t:s .enef"ts;


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No "nterferene 5"t3 t3era$eut"?resue@ "##unosu$$resents


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D"st"ngu"s3 .et5een Donor an/non-Donor 2LA A.s



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No "nterferene 5"t3I)IG$3eres"s $rotools



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1$ to 44 $at"ent sera $er $late

5"t3 one /onor

1$ to B /onors 5"t3 4

re"$"ents ea3 $er $late


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,3at are t3e .enef"ts of us"ngEl"sa an/ 53at /o ou requ"re to

run t3e assas;


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M"rot"ter $late .ase/ ELISA

le7"."l"t )ersat"le sna$-"n str"$sCon)en"ene sreen"ng on a s"ngle

tra for a )ar"et of ant".o/"es

or#at f"ts #ost o##er"all

a)a"la.le #"ro$late rea/ers


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Current Microplate

Readers

6yne1 )R;# )R


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8idney pancreas transplantation

@hole organ transplantation

>P%n#&e%' &%n',(%n%"n %(ne PTA

>

> )LA ,"ng: A B DRB1

> ;"# %n""e'> ;&''!%#


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Transplantation of

pancreatic isletsL%nge&%n' #e((' %&e %&gee

"&(g"#%( %''e''!en / n&

%n &e#","en)LA ,"ng: A B DRB1

;"# %n""e'

#&''!%#

8idney transplantation in


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8idney transplantation in

children

U'+%( e n& "' ne / e ,%&en'

T"''+e ,"ng: A B DRB1

;"# %n""e'

#&''!%#


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5.5

Curs 2 - Imunologia transplantului+TR

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