THE COMBINED EFFECTS OF FORMALIN FIXATION AND THE COMBINED EFFECTS OF FORMALIN FIXATION AND
INDIVIDUAL STEPS IN TISSUE PROCESSING ON IMMUNO-
RECOGNITION
DENNIS OTALI, ROB STOCKARD, DENISE OELSCHLAGER,
UPENDER MANNE, AND WILLIAM E. GRIZZLE
SUPPORTED IN PART BY THE EARLY DETECTION
RESEARCH NETWORK AND THE COOPERATIVE HUMAN
TISSUE NETWORK
• VARIABLES AFFECTING
IMMUNORECOGNITION IN IHC
– Antigen-Antibody
– Secondary Detection System– Secondary Detection System
– Tissue
– Fixation
– Tissue processing
– Shrinkage of Cells
Purpose of Study
• Antigen masking in immunohistochemistry (IHC) has mainly been attributed to fixation
• Very little literature on effects of tissue • Very little literature on effects of tissue processing on IHC staining
• The purpose of this study was to determine whether the effects of fixationand tissue processing on IHC staining could be additive
Tissue Processing Model
• Removal of free water with graded
ETOHs 70%-1hr, 80%-1hr, 90%-1hr,
absolute-3hr.absolute-3hr.
• Development of hydrophobic
environment- with xylene, 2hr.
• Infiltration with paraffin within
hydrophobic environment
Hypothesis
• Fixation and tissue processing modify conformation of Ag, preventing Immunodetection of some Ags some Ags
• Ki67 detection on frozen tissues fixed in NBF, but not FFPE.
• PCNA can be detected in FFPE
Can epitope masking be reversed?
Antigen retrieval
Heating temp pressure cooker
pH-EDTA pH=8
Methodology• Slide preparation
– slides labeled/identified
– Coated with polylysine
– Autoclaved
• Cell model
– Cell culture – DU145 & SKOV3
– Wash off media – fresh tissue
• Fixation • Fixation
– No fixation, 5m, 6h,24h, 48h 72h
• Tissue Processing
– No fixation with each of other steps in TP e.g. 70%ETOH
. IHC with and without AR for Ki-67 and PCNA; staining at the same
time.
. Photography
. Evaluation
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 min Fixation Ki67
Staining Intensity0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
Percentage of Cells (%)
60
80
100
NBF
NBF+AR 60
80
100
NBF
NBF+AR
6 hours Fixation Ki67
5 min Fixation Ki67
24 hours Fixation Ki67
THE
EFFECT
OF 10%
NBF
FIXATION
ON
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
40
0 1 2 3 4
0
20
40
0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
Staining Intensity Staining Intensity
Staining Intensity
48 hours Fixation Ki67 72 hours Fixation Ki67
ON
DU145
CELLS
WITH
AND
WITHOUT
AR Ki67
Staining score vs time in 10% NBF, Ki67 DU145
3
4NBF - AR
NBF + AR
Fixation Time
Score
0
1
2
3 NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Percentage of Cells (%)
40
60
80
100
NBF
NBF+AR
No NBF Fixation 70% ETOH
KI67
40
60
80
100
NBF
NBF+AR
5 min NBF +70% ETOH
Ki67
Staining score vs time in 10% NBF, 70% ETOH, Ki67 DU145
Effect of 10% NBF +70%ETOH on
DU145 Cells
STEP 1 Formalin for various timepoints
STEP 2 70% ETOH for 1 hr
STEP 3 80% ETOH for 1 hr
STEP 4 90% ETOH for 1 hr
STEP 5 100% ETOH for 3 hr
STEP 6 Xylene for 2 hr
STEP 7 Paraffin for 2 hr
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
Staining Intensity0 1 2 3 4
0
20
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
6 hours NBF + 70% ETOH
Ki67
Staining Intensity Staining Intensity
24 hours NBF + 70% ETOH
Ki67
Staining Intensity
48 hours NBF + 70% ETOH
Ki67
72 hours NBF + 70% ETOH
Ki67
70% ETOH, Ki67 DU145
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Percentage of Cells (%)
20
40
60
80
100
NBF
NBF+AR
0 min Fixation to 90%ETOH
20
40
60
80
100
NBF
NBF+AR
5 min NBF to 90%ETOH
Staining score vs time in 10% NBF, 70- 90% ETOH, Ki67 DU145
Effect of 10% NBF +90%ETOH on
DU145 Cells
STEP 1 Formalin for various timepoints
STEP 2 70% ETOH for 1 hr
STEP 3 80% ETOH for 1 hr
STEP 4 90% ETOH for 1 hr
STEP 5 100% ETOH for 3 hr
STEP 6 Xylene for 2 hr
STEP 7 Paraffin for 2 hr
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
Staining Intensity0 1 2 3 4
0
20
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
6 hours NBF to 90%ETOH
Staining Intensity Staining Intensity
Staining Intensity
24 hours NBF to 90%ETOH
48 hours NBF to 90%ETOH 72 hours NBF to 90%ETOH
70- 90% ETOH, Ki67 DU145
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 hr24 hr
48 hr
72 hr
Percentage of Cells (%)
20
40
60
80
100
NBF
NBF+AR
No NBF 70% ETOH to ABS
Ki67
20
40
60
80
100
NBF
NBF+AR
5 min NBF 70% to ABS
ETOH Ki67Staining score vs time in 10% NBF, 70% to absolute ETOH, Ki67 DU145
4
Effect of 10% NBF Fixation -
100%ETOH on DU145 cells
STEP 1 Formalin for various timepoints
STEP 2 70% ETOH for 1 hr
STEP 3 80% ETOH for 1 hr
STEP 4 90% ETOH for 1 hr
STEP 5 100% ETOH for 3 hr
STEP 6 Xylene for 2 hr
STEP 7 Paraffin for 2 hr
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
Staining Intensity0 1 2 3 4
0
20
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
Staining Intensity Staining Intensity
Staining Intensity
6 hours NBF 70% to ABS
ETOH Ki6724 hours NBF 70% to ABS
ETOH Ki67
48 hours NBF 70% to ABS
ETOH Ki6772 hours NBF 70% to ABS
ETOH Ki67
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in 6 hr24 hr48 hr72 hr
NO-FIX 5 MIN NO-FIX-AR 5 MIN-AR
STAINING KI-67
STEP 70% ETH-XYLENESTAINING KI-67-AR
STEP 70% ETH-XYLENE
6 HR 24 HR
48 HR 72 HR
6 HR-AR 24 HR-AR
48 HR-AR 72 HR-AR
Percentage of Cells (%)
20
40
60
80
100
NBF
NBF+AR
0 min Fixation to XYL
20
40
60
80
100
NBF
NBF+AR
5 min Fixation to XYL
Staining score vs time in 10% NBF, 70% ETOH to xylene, Ki67 DU145
Effect of 10% NBF Fixation -
XYLENE on DU145 Cells
STEP 1 Formalin for various timepoints
STEP 2 70% ETOH for 1 hr
STEP 3 80% ETOH for 1 hr
STEP 4 90% ETOH for 1 hr
STEP 5 100% ETOH for 3 hr
STEP 6 Xylene for 2 hr
STEP 7 Paraffin for 2 hr
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
Staining Intensity0 1 2 3 4
0
20
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
6 hours Fixation to XYL
Staining Intensity Staining Intensity
Staining Intensity
24 hours Fixation to XYL
48 hours Fixation to XYL 72 hours Fixation to XYL
70% ETOH to xylene, Ki67 DU145
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 hr24 hr
48 hr
72 hr
0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 min Fixation to Wax
Staining Intensity0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
5 min Fixation to Wax
Staining Intensity0 1 2 3 4
Staining Intensity
Staining Intensity0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
Percentage of Cells (%)
0
20
40
60
80
100
NBF
NBF+AR
0 1 2 3 4
0
20
40
60
80
100
NBF
NBF+AR
6 hours Fixation to Wax
Staining Intensity Staining Intensity
Staining Intensity
24 hours Fixation to Wax
48 hours Fixation to Wax 72 hours Fixation to Wax
Staining score vs time in 10% NBF, 70% -absolute ETOH, xylene to paraffin wax,
Ki67 DU145
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
DU145/SKOV3 comparison
10% NBF fixation Ki67
Staining score vs time in NBF
Ki67 SKOV3 II
01234
0min5m
in
6hrs
24hr
sII
Time
Score
No AR
AR
Poly. (No AR)
Poly. (AR)
Staining score vs time of NBF
fixation Ki67 DU145
0
1
2
3
4
0min5m
in6h
rs24
hrs
48hr
s72
hrs
Score
No AR
AR
Poly. (No AR)
Poly. (AR)
DU145/SKOV3 comparisonDU145/SKOV3 comparison
10% NBF, 70-Xylene Ki67
Staining score vs time in NBF fixation
Ki67 70 - XYL DU145
0
1
2
3
4
0min5m
in6h
rs24
hrs
48hr
s72
hrs
Time
Score
No AR
AR
Poly. (No AR)
Poly. (AR)
Staining score vs time in NBF fixation
Ki67 70-XYL SKOV3
0
1
2
3
4
0min 5min 6hrs 24hrs
Time
Score
No AR
AR
Poly. (No AR)
Poly. (AR)
Staining score vs time in 10% NBF, Ki67 DU145
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Staining score vs time in 10% NBF, 70% ETOH, Ki67 DU145
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Staining score vs time in 10% NBF, 70- 90% ETOH, Ki67 DU145
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Staining score vs time in 10% NBF, 70% to absolute ETOH, Ki67 DU145
Score
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
Staining score vs time in 10% NBF, 70% ETOH to xylene, Ki67 DU145
Score
3
4NBF - AR
NBF + AR
NBF - AR
Staining score vs time in 10% NBF, 70% -absolute ETOH, xylene to paraffin wax,
Ki67 DU145
Score
3
4NBF - AR
NBF + AR
NBF - AR
Fixation Time
Score
0
1
2 NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Fixation Time
Score
0
1
2
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Fixation Time
Score
0
1
2
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Ki67 Summary
Immunorecognition of Ki67 somewhat decreases with increase in duration in 10% NBF – most of loss is recovered with ARwith AR
• Fixation + TP (Hydrophobic environment) in combination masks most of the Ki67antigen; recovery is partial
NO FIX 5 MIN
6 HR 24 HR
NO-FIX-AR 5 MIN-AR
6 HR-AR 24 HR-AR
STAINING OF PCNA
STEP 70% ETH- XYL
STAINING OF PCNA WITH AR
STEP-70% ETH- ZYL
24 HR
48 HR 72 HR48 HR-AR 72 HR-AR
Staining score vs time in 10% NBF, PCNA DU145
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Staining score vs time in 10% NBF, 70% ETOH PCNA DU145
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Staining score vs time in 10% NBF, 70-90% ETOH PCNA DU145
Fixation Time
Score
0
1
2
3
4NBF - AR
NBF + AR
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Staining score vs time in 10% NBF, 70% to absolute ETOH PCNA DU145
3
4NBF - AR
NBF + AR
NBF - AR
Staining score vs time in 10% NBF, 70% ETOH to xylene PCNA DU145
3
4NBF - AR
NBF + AR
NBF - AR
Staining score vs time in 10% NBF, 70% -absolute ETOH, xylene to paraffin wax,
PCNA DU145
3
4NBF - AR
NBF + AR
NBF - AR
Fixation Time
Score
0
1
2
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Fixation Time
Score
0
1
2
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
Fixation Time
Score
0
1
2
NBF - AR
NBF + AR
0 min5 m
in6 h
r24 hr
48 hr
72 hr
1)PCNA REQUIRES SOME FIXATION FOR
IMMUNO-RECOGNITION
2)PCNA DOES NOT LOSE ALL IMMUNO-
RECOGNITION WITH FIXATION AND TISSUE
PROCESSING
3)MORE CONSISTENT IMMUNOSTAINING IS
OBTAINED WITH ANTIGEN RETRIEVAL.
4)AS WITH Ki-67, LOSS OF IMMUNO-
RECOGNITION OCCURS WITH BOTH FIXATION
AND TISSUE PROCESSING WITH THE MAJOR
CHANGE AT THE TRANSITION TO A
HYDROPHOBIC ENVIRONMENT.
24 h 10% NBF-no
process-Ki-67 breast
24 h 10% NBF-90%
ethanol-Ki-67 breast
24 h 10% NBF absolute
ethanol - Ki-67 breast24 h 10% NBF-xylene-
Ki-67 breast