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Reproducibility for C4d immunohistochemistry in renal allografts – results 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 - PowerPoint PPT Presentation
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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!
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