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