Reproducibility for C4d Reproducibility for C4d immunohistochemistry in renal allografts – immunohistochemistry in renal allografts –

results from the Banff Trialresults from the Banff Trial

Banff Initiative for Quality Assurance in Transplantation (BIFQUIT)

Samantha Chan, Jessie Climenhaga, Parmjeet Randhawa, Heinz Regele, Yaël Kushner, Robert Colvin,

and Michael Mengel

University of Alberta, Edmonton, CanadaUniversity of Pittsburgh, Pittsburgh, USA

Massachusetts General Hospital, Boston, USA

Detection of C4d is crucial for diagnosing antibody mediated rejection: reproducibility studies are limited

C4d in paraffin sections: Results

can directly influence patient

care

Sent slides requested by participating

institutions

Participants/ observers stain slides

and evaluate

Complete online or paper survey on staining methods

& C4d scoring

Mail survey + stained slides back

to organizing centre

Collect and organize slides (e.g. by

institution/participant)

C4d

Tissue microarrays (TMA)

Collect cases (n=19) for analytical spectrum

(i.e. Negative, mildly to strongly positive)

Enter survey data into data base

ALL TMA slides for score by review panel:

the ‘best stain returned was identified per consensus as the ‘reference case’

Combine with retrieved online survey data

Data Analysis

Centers contributing samples:Volker Nickeleit, Chapel Hill, USAVerena Bröcker, Hannover, GermanyParmjeet Randhawa, Pittsburgh, USA Bernard Collins, Boston, USAHeinz Regele, Vienna, AustriaMichael Mengel, Edmonton, CanadaCinthia Beskow-Drachenberg, Maryland, USASurya Seshan, New York, USA

C4d

Design of the C4d BIFQUIT trial

Reproducibility of immunohistochemical stains

• Inter-institutional variability = staining/laboratory procedure + subjectivity/interpretation by observer weighted kappa values were calculated by comparing the C4d scores provided

by each participant from their locally stained slides to the corresponding C4d scores provided by the local participant from the reference case

• Inter-laboratory variability = staining/laboratory procedure weighted kappa values were calculated by comparing the panel consensus read

for each tissue core on each slide to the panel read of the corresponding tissue core on the reference slide

• Inter-observer variability = subjectivity/interpretation by observer weighted kappa values were calculated by comparing the reads of the local

participants to the consensus reads of the panel recorded on the same TMA slide

Summary of mean kappa-values for the different components influencing the reproducibility of a C4d stain

Significance of kappa values: <0 indicating no agreement (or less agreement than expected by chance), 0–0.20 slight, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 substantial, and 0.81–1 almost perfect agreement.

NA = Not Applicable, because for the inter-observer comparison per definition the comparator has to be the panel read and cannot be a majority call

kappa >0.6 = at least moderate reproducibility

kappa >0.4 = at least fair reproducibility

Scatter plots showing the inter-laboratory and inter-observer reproducibility for each individual participant / participating laboratory using the Banff C4d schema (A) or positive vs. negative calls (B)

kapp

a va

lues

for i

nter

-obs

erve

r rep

rodu

cibi

lity

kappa values for inter-laboratory reproducibility

18%

59%

A: using the Banff C4d grading schema B: using positive negative calls

Variance in scoring between observers:• was greater with C4d1 and C4d2 cases compared to C4d0 and C4d3 cases• the panel consistently under-scored the local observers, i.e. Pathologists adjust their

scoring to the sensitivity of their local laboratory

Influence of analytical variables on C4d staining:

comparing the top 15 and bottom 15 slides ranked by kappa values for inter-laboratory reproducibility

0

5

10

15

20

25

30

35

40

45

Vent

ana

-

CCI

Bond

max

Citr

ate

Dako

EDTA Tr

is

Tris

-EDT

A

No R

espo

nse

Num

ber o

f Res

pons

es (%

)

Top 15

Bottom 15

0

5

10

15

20

25

30

35

40

45

6-6

.9

7-7

.9

8-8

.9

9-9

.9

10+

unsp

ecifi

ed

no re

spon

se

< 4

min

4 to

9 m

in

10 to

14

min

15 to

19

min

20 to

24

min

30 to

34

min

35 to

39

min

40+

min

Num

ber o

f Res

pons

es (%

)

Buffer pH Top 15

Buffer pH Bottom 15

Highest Temperature Top 15

Highest Temperature Bottom 15

0

5

10

15

20

25

30

35

40

45

Vent

ana

-

CCI

Bond

max

Citr

ate

Dako

EDTA Tr

is

Tris

-EDT

A

No R

espo

nse

Num

ber o

f Res

pons

es (%

)

Top 15

Bottom 15

0

5

10

15

20

25

30

35

40

45

50

1:10

1:15

1:25

1:30

1:40

1:50

1:80

1:10

0

1:20

0

1:40

0

1:20

00

pred

ilute

d

no

resp

onse

30-3

9 m

in

40-4

9 m

in

60 m

in

120+

min

Num

ber o

f Res

pons

es (%

)

Antibody Dilution Top 15

Antibody Dilution Bottom 15

Antibody Incubation Top 15

Antibody Incubation Bottom 15

Influence of Fixation for C4d Inter-Laboratory reproducibility

0.64 0.45 1.00 0.82 -0.23 -0.32

Below Chance

Slight

Fair

Moderate

Substantial

Almost Perfect

<0.00

0.00 – 0.20

0.21 – 0.40

0.41 – 0.60

0.61 – 0.80

0.81 – 1.00

Protocol Description

1 Standard Immediate onset of fixation for 24h in buffered, standard Formalin (4%)

2 Delay Delayed onset of fixation for 12h (storing the tissue at 4˚C) and then fixation for 24h in buffered, standard Formalin (4%)

3 Frozen Snap freezing of tissue (like simulating a frozen section procedure) and then immediate fixation for 24h in buffered, standard Formalin (4%)

4 Overfix Immediate onset of fixation for 5 days (simulation of shipment over long weekend) in buffered, standard Formalin (4%)

5 Underfix Immediate onset of fixation for only 1h (simulation of very rush processing) in buffered, standard Formalin (4%)

6 Etha Immediate onset of fixation for 24h in Ethanol (100%)

1 2 3 4 5 6

Mean kappa values

Summary and recommendationsSummary and recommendations

• The BIFQUIT trial results indicated that C4d staining on paraffin sections is of limited reproducibility.

• Refinement of the current Banff C4d scoring schema and standardization of tissue processing and staining protocols is necessary to achieve acceptable reproducibility for C4d staining on paraffin-embedded material

• Recommendations from this first BIFQUIT trial for C4d immunohistochemistry on paraffin sections:– Avoid under (<1 hour) and/or ethanol fixation of tissue specimen– Use heat induced epitope retrieval with citrate buffer at pH6-7– Incubate polyclonal anti-C4d antibody concentrated at <1:80 for >40 minutes– Better results were observed with harsher pre-treatment (e.g. autoclave epitope

retrieval), higher polyclonal anti-C4d antibody concentrations (1:10), and longer incubation times for the polyclonal anti-C4d antibody (over night / 12-16 hours)

– Simplifying the 2007 Banff C4d grading schema will reduce inter-observer variability

AcknowledgementsAcknowledgements

The panel

Astellas Pharma Canada provided an unrestricted research grant to the Banff

Working Groups

The following Institutions contributed cases to the BIFQUIT trial:Cinthia Beskow-Drachenberg, Maryland, USAVolker Nickeleit, Chapel Hill, USAVerena Bröcker, Hannover, GermanyBernard Collins, Boston, USAHeinz Regele, Vienna, AustriaSurya Seshan, New York, USA

Students helped with logistics and analysis:Samantha ChanJessie ClimenhagaVictoria SheldonAkshatha Raghuveer

Many thanks to participants in the Banff Working Group Trials!