C4d staining and morphology in

protocol biopsies

Michael Mengel*

For the ESPRIT-Group

European Study Group For Protocol Biopsies In Renal

Transplantation

*Disclosure: M.M. received travel grants form Wyeth and Roche

Multicentric Study• 3 European TX-Centers• Protocol- und Indication Bx• Classification according up-dated Banff • unstained paraffin slides from all centers• C4d positive > 25% of PTC stained

– 25-50% focal positive– >50% diffuse positive

• C4d negative < 25% of PTC stained• correlation with morphological and clinical

parameters

Incidence of C4dTX-center

type of biopsy Bxn Bx C4d (%)

>50%PTCC4d (%)

25-50%PTCC4d (%)negative

center 1indication Bx

48 5 (10.4) 7 (14.6) 36 (75.0)

center 2indication Bx

99 15 (15.2) 8 (8.1) 76 (76.7)

center 3indication Bx

230 26 (11.3) 17 (7.4) 187 (81.3)

total indication Bx 377 46 (12.2) 32 (8.5) 299 (79.3)

center 1protocol Bx

128 1 (0.8) 2 (1.6) 125 (97.6)

center 2protocol Bx

94 6 (6.4) 4 (4.3) 84 (89.3)

center 3protocol Bx

329 4 (1.2) 7 (2.1) 318 (96.7)

total protocol Bx 551 11 (2.0) 13 (2.4) 527 (95.6)

total 928 57 (6.1) 45 (4.8) 826 (89.1)

Inzidence of C4d

10,414,6

75,0

0,8 1,6

97,6

15,2

8,1

76,7

6,4 4,3

89,3

11,37,4

81,3

1,2 2,1

96,7

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

80,0

90,0

100,0

C4d diffusepositive

Indication

C4d focalpositive

Indication

C4dnegative

Indication

C4d diffusepositiveProtocol

C4d focalpositiveProtocol

C4dnegativeProtocol

C4d stain

% o

f ca

ses

center 1

center 2

center 3

Indication biopsies: C4d clinico-pathological correlationclinic C4d (%)

>50%PTCC4d (%)25-50%

C4d (%)<25%

univariatep value

multivariatep value

Sex(%females) 24 (52.2) 12 (37.5) 106 (35.5) ns ns

recipient age >60 years 6 (13.0) 4 (12.5) 46 (15.4) ns ns

Bx < 3 months after TX 34 (73.9) 17 (53.1) 202 (67.6) ns ns

prior cellular rejection to Bx 6 (13.0) 8 (25.0) 51 (17.1) ns ns

no induction therapy 3 (23.1) 5 (33.3) 49 (40.8) ns ns

induction therapy with anti-IL2 6 (46.2) 6 (40.0) 56 (46.7) ns ns

induction therapy with ATG/OKT 4 (30.8) 4 (26.7) 15 (12.5) ns ns

prior TX 11 (32.4) 7 (23.3) 37 (14.5) 0.02 ns

PRA >30% 12 (26.1) 5 (15.6) 22 (7.4) <0.0001 0.02

morphologyno cellular rejection 8 (24.2) 11 (36.7) 107 (43.1) ns ns

borderline 7 (21.2) 4 (13.3) 44 (17.7) ns ns

cellular tubular interstitial rejection (Banff IA+IB)

7 (21.2) 9 (30.0) 54 (21.8) ns ns

cellular vascular rejection(Banff IIA+IIB)

10 (30.3) 6 (20.0) 40 (16.1) ns ns

vascular rejection(Banff III)

1 (3.0) 0 3 (1.2) ns ns

ptc inflammation 42 (91.3) 20 (62.5) 123 (41.3) <0.0001 <0.0001

glomerular inflammation 24 (53.3) 7 (22.6) 50 (16.8) <0.0001 ns

acute tubular damage 42 (91.3) 25 (78.1) 158 (53.0) <0.0001 ns

glomerular thrombosis 1 (2.2) 1 (3.1) 6 (2.6) ns ns

arteriolar necrosis 1 (2.2) 0 5 (1.7) ns ns

protocol biopsies: C4d clinico-pathological correlation

clinic C4d (%)>50%PTC

C4d (%)25-50%

C4d (%)<25%

univariatep value

multivariatep value

Sex(%females) 3 (37.5) 1 (10.0) 190 (40.3) ns ns

recipient age >60 years 6 (13.0) 4 (12.5) 46 (15.4) ns ns

Bx < 3 months after TX 3 (27.3) 4 (30.8) 219 (41.6) ns ns

prior cellular rejection to Bx 2 (25.0) 2 (20.0) 98 (20.2) ns ns

no induction therapy 3 (60.0) 2 (22.2) 96 (22.0) ns ns

induction therapy with anti-IL2 2 (40.0) 7 (77.8) 281 (64.3) ns ns

induction therapy with ATG/OKT 0 0 60 (13.7) ns ns

prior TX 4 (80.6) 1 (11.1) 53 (12.4) <0.0001 ns

PRA >30% 1 (12.5) 1 (10.0) 26 (5.5) ns ns

morphologyno cellular rejection 6 (60.0) 7 (63.6) 286 (71.7) ns ns

borderline 3 (30.0) 3 (27.3) 89 (22.3) ns ns

cellular tubular interstitial rejection (Banff IA+IB)

1 (10.0) 1 (9.1) 23 (5.8) ns ns

cellular vascular rejection(Banff IIA+IIB)

0 0 1 (0.3) ns ns

vascular rejection(Banff III)

0 0 0 - ns

ptc inflammation 6 (85.7) 3 (37.5) 12 (3.3) <0.0001 <0.0001

glomerular inflammation 2 (28.6) 2 (25.0) 10 (2.8) <0.0001 ns

acute tubular damage 4 (57.1) 3 (37.5) 157 (43.5) ns ns

glomerular thrombosis 0 0 0 - ns

arteriolar necrosis 0 0 0 - ns

30 60 90 120 150

Time of survival in months

0,0

0,2

0,4

0,6

0,8

1,0

Gra

ft S

urv

ival

C4dC4d negative

C4d positive

C4d negative-censored

C4d positive-censored

Kaplan Meier Analysis

24 C4d+ protocol Bx

matched with

72 C4d- protocol Bx

for: Age, Sex, time point of Bx, Banff grade

F.R., male, born 1944Renal-Transplantation 31.08.01

Analgesic nephropathy, stone kidneys

Hemodialysis 11/1988

1. Transplantation 5/1990

Transplant failure 1/1999 with CAN

2. Transplantation 8/2001

Relapsing peptic ulcers (bleeding), Hypertension (4

antihypertensive drugs), Hyperlipoproteinemia, Coronary heart

disease

K 1

K 1-C4d+

K 2

K 2-C4d+

Indication

K 3

F.R., male, born 1944Renal-Transplantation 31.08.01

0,5

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

Biopsies

Predni

CSA

MMF

RR Med

Pravastatin

ASS

U- Protein(g/L)

S-Creatinine(µmol/L)

1,3

0,9

0,08 0,20,4 0,3

(4)

Summary • Incidence for C4d in protocol bx significantly lower

• Incidence varies between centers

• Incidence is higher with presensitized patients

• focal C4d stain correlates with AHR morphology

• C4d in protocol bx less often with cellular rejection

• cases of “subclinical” humoral rejection

prognostic relevance:

No acute graft losses

Harbinger of chronic humoral rejection??

Hannover-GermanyHermann HallerAnke SchwarzWilfried GwinnerMichael Mengel

ESPRIT-Group

European Study Group For Protocol Biopsies In Renal Transplantation

Antwerp-BelgiumMarc De Broe

Jean-Louis Bosmans

Johannes Bogers

Barcelona-SpainDaniel Seron

Francesco Moreso

Marta Carrera