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.$ntered on4 9:;5:;
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ALLELE GROUP TYPING:
B*18B*39
-ssay Report
%ample ID4 56=FI>?
Patient !ame4 F.I.$ntered on4 9:;5:;
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ALLELE GROUP TYPING:
DRB1*
07
DRB1*
11
-ssay Report
%ample ID4 566FM67
Patient !ame4 F.M.$ntered on4 9:;9:;
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ALLELE GROUP TYPING:
DRB1*
11
DRB1*
13
-ssay Report
%ample ID4 56=FI>?
Patient !ame4 F.I.$ntered on4 9:;9:;
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DQB1*
03
DQB1*
03
-ssay Report
%ample ID4 566FM67
Patient !ame4 F.M.$ntered on4 9:;9:;
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DNA
80 ng for Class I
40 ng for Class II
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Importance of D!-
3uality
100 ng Gen!"# DNA 1$ 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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Ant".o/ Mon"tor"ng Sste#
,3at are "t:s .enef"ts;
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Ant".o/ Mon"tor"ng Sste#
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
Ant".o/ Mon"tor"ng Sste#
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No "nterferene 5"t3I)IG$3eres"s $rotools
Ant".o/ Mon"tor"ng Sste#
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Ant".o/ Mon"tor"ng Sste#
1$ 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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Ant".o/ Mon"tor"ng Sste#
,3at are t3e .enef"ts of us"ngEl"sa an/ 53at /o ou requ"re to
run t3e assas;
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Ant".o/ Mon"tor"ng Sste#
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