Positive-crossmatch

kidney transplantation

경북대학교병원 신장내과
장
김찬덕
 HLA Sensitization
ƒ Sensitization
S iti ti
: presence of pre-formed antibodies against HLA in the blood

ƒ Transplantation across a positive crossmatch can result in

hyperacute or acute AMR.

ƒ Sources of HLA sensitization

: previous transplants, transfusions, pregnancies etc.

: Among patients receiving their first kidney transplant with no

known history of blood transfusions, approximately 20% of
nulliparous
p women and 13% of men were sensitized ((PRA>10%). )
 Kidney waiting list: peak PRA
OPTN/SRTR data as of May 4, 2009

30%

25%

20%

15% PRA 10-79%

PRA ≥ 80%
10%

5%

0%
2004 2005 2006 2007 2008 KNUH
 Rates of KT according to PRA
(UNOS 2001-2008)

80%

60%

40% LD
20% DD
DD
0%
LD
PRA 0
0-10%
10%
PRA 10-80%
PRA > 80%

LD: Living Donor

DD: Deceased Donor
 Methods for anti
anti-HLA
HLA Ab screening

Flow- Flow
Flow- Multiplex
CDC
(Complement- cytometry ELISA cytometry platform
(Complement-
dependent) beads Luminex
independent)

ƒ Sensitive for detection of IgG HLA Ab

ƒ Specificity is higher by exclusion of non-HLA Ab & auto Ab
ƒ Less
L influenced
i fl d by
b the
th presence off therapeutic
th ti anti-lymphocytic
ti l h ti Ab

Girnita et al. Pediatr Transplantation 2006

Tissue-typing methods to screen transplant candidates
for anti-HLA antibodies

Marfo K et al. CJASN 2011;6:922-936

Complement-Dependent lymphocytotoxicity
(CDC) test

72 well
 Interpretation
p of CDC
Score Interpretation % Dead cells
1 Negative 0-10
0 10
2 Doubtful negative 11-20
4 Weak positive 21-50
6 Positive 51-80
8 Strongly positive 81-100

Viable cell – no eosin uptake (bright)

Non viable cell – eosin dye uptake
Flow cytometric
y crossmatch ((FCXM))

Secondary Ab Serum + Cells

Fluorecently labelled
Incubate at 37C for 30 min

Wash x 3
Recipient anti-HLA Ab
Add anti-IgG FITC, CD3 PE

Donor HLA molecule Incubate at 4C for 30 min

Donor cell Wash

A l
Analyze by
b FCM
 T cell FCXM :
Anti-IgG FITC Histogram
Example

Counts

anti-IgG FITC

Sample(Patient)/Control MFI ratio : Positive

FCM T cell MFI ratio cutoff: 2.0
FCM B cell MFI ratio cutoff: 3.0
Principle of Luminex
Luminex-PRA
PRA

PE- anti-IgG
g

Alloantibody in serum

Pooled-multiple antigens (eg. Class I) ; screen

Several antigens (eg. CREG, individual Ag) ; screen, ID
Single antigen (eg. B2705) ; ID, virtual CM

Luminex bead
 Panel Reactive Ab (PRA)
Donor specific antibody (DSA) HLA A1
Recipient serum

+ Complement (CDC)
FCM, ELISA, Luminex

PRA = 9/72 = 12.5 %

 Approach to sensitized patients
: Who needs desensitization ?

ƒ AHG-CDC CXM (+) : All vs. ≤ 1 : 16

ƒ AHG-CDC CXM (-), flow cytometry CXM (+)

ƒ AHG-CDC CXM (-), Flow CXM (-), DSA (+)

ƒ AHG-CDC CXM (-), Flow CXM (-), DSA (-), High PRA,
Retransplantation
 Algorithmic approach to sensitized patients
CDC T (+) & B (+)
Pts with a living donor More than 3 DSAs
More than 1 strong
DSA (MFI > 5,000)
5 000)
: Do not attempt
Pts have
Pts have desensitization
no DSA
DSA &
& CMX (- CDC T (-) & B (+)
CMX (+)
) FCMX T or B (+)
Total MCS < 300
DSA (MFI < 5,000)
: No desensitization
Transplant
Internal & ATG & High
g dose
national IVIG (2g/kg)
kidney paired CDC T or B (+)
exchange FCMX T or B (+)
programs Total MCS > 300
DSA (MFI > 5,000)
: Desensitization
MCS: median channel shift PP, IVIG, rituximab
MFI: mean fluorescence intensity Einstein/Montefiore transplant center
 Treatment options for sensitized patients
Removal of antibodies byy Plasmapheresis
p ((PP)) or
1
1.
Immunoadsorption (IA)
2. Inhibition of antibody production

a. Anti-B cell agents: rituximab (anti-CD20)

b. Plasma cell inhibitors: bortezemib (proteosome inhibitor)

3. Inhibition of complement cascade: eculizumab (anti-C5a)

4. IVIG has multiple effects on different immune pathways

Splenectomy (removes a major source of lymphocytes, including

5. antibody-secreting B cells, B cell precursor cells,
and plasma cells)
Proposed mechanisms of action of IVIG
on cellular immunityy

Anti-idiotypic effect
 The Use of Immunoglobulin Therapy for Patients
Undergoing SOT:
An Evidence-Based Practice Guideline
ƒ Presented by Canadian Blood Services & the national
Advisory Committee
ƒ 791 literature
lit t citations
it ti & 45 reports
t reviewed
i d

When transplantation will involve use of a kidney from a

living donor to whom the patient is sensitized, IVIG is
recommended to decrease donor-specific
p sensitization.

1 Give
1. Gi IVIG 2 g/kg
/k monthly
thl for
f 4 months
th bbefore
f T
Tx

2 PP followed by IVIG 100 mg/kg: perioperative period

2.

Transfus Med Rev. 2010 Jan;24 Suppl 1:S7-S27

 Rituximab®: anti-CD20 monoclonal Ab
™ Postulated mechanisms of action
- Complement-dependent cytotoxicity
- Ab dependent cell –mediated
cytotoxicity
- Induction
I d ti off apoptosis
t i
™ Depletion of CD20-positive cells dose
reduce some Ab responses.
responses

Plasma cells PP
IVIG

Splenectomy
Pre B cells
& B cells Rituximab
 High dose IVIG vs. PP+low dose IVIG+rituximab
Group Regimen
Group 1 (n=32) PP, low-dose IVIG (100 mg/kg), rituximab (375 mg/m2 x1)
Group 2 (n=13) High-dose IVIG (2.1~3 g/kg)
Group 3 (n=16) PP, low-dose IVIG (100 mg/kg), rituximab (375 mg/m2 x1),
Pre KT ATG,
Pre-KT ATG Post-KT
Post KT DSAs monitoring

Achieving a negative CMX Humoral rejection rates

Group 1 84 % Group 1 37 %
Group 2 38 % Group 2 80 %
Group 3 88 % Group 3 29 %

ƒ 3/8 nott responding

di tot hi
high-dose
hd IVIG did respond
d when
h switched
it h d to
t
PP/low-dose IVIG/rituximab.

ƒ Multiple PP treatments leads to more reproducible desensitization and

lower humoral rejection rates than a single high-dose of IVIG.
Stegall et al. AJT 2006
 Rituximab + IVIG
ƒ 76 (31 LD/45 DD) HLA-sensitized pts
y
ƒ Flow cytometry y PRAs more than 30%
(75% of patients were 80%)
ƒ Positive pretransplant T
T-cell
cell FCMXs or had DSAs
ƒ Only pretransplant B-cell FCMXs (+) were eliminated
Induction:
Alemtuzumab or
IVIG: 2.0 g/kg (maximum 140 g) ATG or
Rituximab: 1 g Daclizumab
Maintenance:
Tacrolimus/MMF/
-30
30 -15
15 -1
1 Pds

Acceptable CMX: Negative CDC

CXM KT
T-cell FCMX < 250 MCS (negative < 50 MCS)
Vo et al. Transplantation 2010
Rituximab + IVIG

Vo et al. Transplantation 2010

 KNUH desensitization protocol
for cross-matching
cross matching (+)
FK506 0
0.05
05 mg/kg po bid
MMF 500~750 mg po bid
PDS 0
0.25
25 mg/kg po bid PDS IV

PP+IVIG (0
(0.1g/kg)
1g/kg)
HD HD HD HD HD HD HD
14
-14 Fr Sa Su Mo Tu We 7
-7 Fr Sa Su Mo Tu 1
-1 KT D4

Rituximab
500 mg Crossmatching Simulect IV
 CMC protocol for cross-matching (+)
FK 0.1 mg/kg/day
MP 125 mg/day
MMF 1.5 g
g/day
y
Plasmapheresis
IVIG (0.2g/kg)

Si l
Simulect

-14 -7 0
KT
Cross-matching
Cross matching Crossmatching
rituximab을 이식 일주일전과 이식 하루전에 375 mg/m2로 2차례 투여
Plasma pheresis는 column을 이용하여 IgG를 선택적으로 제거하는 방법을 이용
 SNUH rescue protocol
p
Mycophenolate mofetil
Tacrolimus
Steroids
Anti- Anti-
CD20 CD20

PP PP PP PP PP PP PP
IVIG IVIG IVIG IVIG IVIG IVIG IVIG

-15 -14 -7 OP +7 +14 +21

9 Rituximab
Rit i b (375mg/m
(375 / 2 BSA),
BSA) att D-15,
D 15 -11
9 MMF (750mg bid), started at D-15
9 Tacrolimus (0.05mg/kg bid, target level 10-12 ng/ml), started at D-15
9 PP/Low dose IVIG (100mg/kg), 3 times/wk, started at D-14

9 Crossmatch and PRA identification,

identification after 2ndd , 5tth PP/IVIG
9 CD 19, 20 count, at D-20, -1 24
 Acute AMR incidence & allograft survival

ƒ Low incidence of hyperacute rejection

ƒ Favorable renal allograft survival rates
ƒ Acute AMR remains a common occurrence: 20–50%

Lefaucheur et al. AJT 2008

Thielke et al. Transplantation 2009
Magee
g et al. Transplantation
p 2008
Gloor et al. AJT 2010
Haririan et al. AJT 2009
Vo et al. NEJM 2008
 Long-term outcomes in highly-HLA
sensitized pts receiving desensitization
ƒ 218 HS patients
ti t underwent
d td
desensitization
iti ti & ttransplantation
l t ti
ƒ Desensitization: combinations of HD IVIG, rituximab, plasmapheresis

Yrs of follow up 1 3 5 7 9
Patient No 218 118 60 34 6
Functioning 197 94 40 18 4
Failed 16 16 13 10 1
DWFG 3 6 6 4 1
Scr (mg/dL) 1.35 1.39 1.53 1.49 1.35
Graft survival 92.5% 94.5% 75.5% 64.3% 80.0%
Patients survival 98.6% 94.9% 90.0% 88.2% 83.3%

DWFG: Death With Functioning Graft

Cedars Sinai Medical Center, LA, AJT 2009
 이식 환자의 자가 부담금액

1200

1000

800
용 (만원)

본인부담
600
비용

보험급여
선택진료
400

200

0
Donor Recipient DXM (+) Recipient

Donor : 재원 8일 (다인실 입원).

R i i t : 재원 21일 (중환자실 4일,
Recipient 4일 1인실 10일 입원).
입원)
DXM(+) Recipient : 재원 28일 (중환자실 4일, 1인실 10일 입원, 혈장교환 6회)
 Desensitization Cost
Rituximab: 요양급여 인정기준

• 혈장반출술 치료에 반응하지 않는 ABO 혈액형 부적합 신이식 환자의 거부반

응 예방에 375mg/m2 으로 1회
• 면역글
면역글로불린/혈장반출술
불린 혈장반출술 치료에 반응반응하지 않 않아 PRA>50%
> 또는
는 교차반응
반응 검
사 양성 환자의 거부반응 예방에 375mg/m2 으로 1회
• 면역글로불린/혈장반출술 등의 치료에 반응하지 않는 신이식 후 급성 체액성
거부반응에 375mg/m2 으로 1회

BSA 1.7m2의 경우 700mg × 1회 = 190만원

Plasmapheresis: 사례별 심사로 요양급여 인정가능

1 volume × 6회 = 5만원 × 6회 = 30만원

Immunoglobulin: 본인 100%
0.1g/kg × 6회 = 11만원 × 6회 = 66만원
 Baseline Characteristics of Direct Cross-Match (+)
R i i t in
Recipients i KNUH

Cause of Etiolog No. of

AHG FCM MFI PRA Desensitizat KT date y of HLA
ion ESRD Mismatch

정O환
정 환 ((-)) ((-)) Class I ((82%)) PRA ((+)) 2009.9.24 DM 5MM

신O옥 (+) T-cell (+) 14.3 Class I (46%) DXM (+) 2009.3.5 CGN 2MM

강O자 (-) T,B-cell (+) 13.3/7.6 Class I (21%) DXM (+) 2009.2.12 PKD 4MM

김O옥 ()
(-) T cell (+)
T-cell 97
9.7 Class I (2
(2.1%)
1%) DXM (+) 2008 9 17
2008.9.17 CGN 5MM

박O남 (-) T-cell (+) 8.1 Class I (5%) DXM (+) 2008.6.12 CGN 3MM

최O미 (-) T-cell (+) 2.8 Class I (38%) DXM (+) 2007.6.21 CGN 4MM

Class I (52%)
장O옥 (-) T,B-cell (+) 2.3/4.6 DXM (+) 2006.3.16 CGN 1MM
Class II (60%)
 Clinical Courses of Direct Cross-Match (+) Recipients
in KNUH
Immuno- Follow-
Infection Rejection
Desensitization suppressant Final Cr up
episode episode
s months
th
Tcrolimus
PP 6회 + IVIG
정O환 MMF 1.17 17mon (-) (-)
Rituximab Steroid
09.8.
Tcrolimus
PP 6회+IVIG Perigraft abscess
신O옥 MMF 1.47 24mon (-)
Rituximab Steroid
10.1~
Recurrent APN
Tcrolimus
Tc olim s
PP 6회 +IVIG 09.9.
강O자 MMF 0.86 25mon (-)
Rituximab Steroid
Septic Arthritis

Tcrolimus
PP 6회 +IVIG
김O옥 MMF 0 68
0.68 30mon (-) (-)
Rituximab Steroid
Tcrolimus
PP 5회 + IVIG
박O남 MMF 0.66 33mon (-) (-)
Rituximab Steroid
PP 6회+IVIG Tcrolimus
최O미 Rituximab MMF 0.8 44mon 08.11.APN (-)
post op PP 2회 Steroid

Tcrolimus
장O옥 PP 3회+IVIG MMF 0.83 60mon (-) (-)
Steroid
B cell– and antibody-related
y biologics
g in transplantation
p

Bortezomib
Eculizumab

Rituximab Knechtle et al. JCI 2010

 Limitation of Rituximab
Rituximab

CD19-
CD19+ CD19+
CD20-
CD20+ CD20+
CD38+
CD27-
CD27 CD27+
CD138+
B cell B cell
B cell

Mature B cell Memory B cell Plasma cell

Persistent DSA production

The Effect of Desensitization Protocols on Human
B Cell Populations In Vivo
Splenic B-Cell

Ramos et al. AJT 2007

 Eculizumab: a complement protein C5 antibody
that inhibits the formation of the membrane attack complex (MAC)

Reduction in renal C9 after treatment with eculizumab

Case report, AJT 2009

Abstract# 393: 2009 ATC meeting

Cornell LD, Burns JM, Stegall MD et al.
Prevention of endothelial cell activation with C5 inhibition in positive crossmatch KT.

Controls REC AHR Eculizumab REC AHR

C4d+ (N=18) 83% 61% C4d+ (N=10) 0% 0%
C4d (N=27)
C4d- 26% 0% C4d (N=7)
C4d- 0% 0%

REC: reactive-appearing endothelial cell, AHR: acute humoral rejection

Bortezomib blocks alloantibody production by
normal plasma cells
ATG Rituximab

Bortezomib
Perry et al. AJT 2009
 The Impact of Proteasome Inhibition on
Alloantibody Producing Plasma Cells In Vivo
Alloantibody-Producing

ƒ Bortezomib treatment decreased DSA-PCs in the marrow

(16 7±14 5 pre-treatment
(16.7±14.5 pre treatment vs
vs. 6
6.2±3.6
2±3 6 DSA-PCs/ml
DSA PCs/ml after treatment)
ƒ Bortezomib appear to potentiate DSA removal by PE in sensitized RTRs.
Diwan et al. Transplantation 2011
 Take home message
ƒ DSAs removal and inactivation through PP, IVIG, and
rituximab enable successful positive-CMX KT with good
short to intermediate term outcomes
outcomes.

ƒ Positive
Positive-CMX
CMX KT are associated with an increased
incidence of AMR, which, although reversible, has a
negative effect on long term graft survival.

ƒ New interventions aimed at the prevention of DSA-

DSA
mediated injury using complement blockade, or the
inhibition of DSA synthesis using proteasome inhibitor
were recently introduced to desensitization protocols.
Thank you for your attention!