Gomori Reticular Fiber Stain –Effect of Varying Developing Reagents

By: Lawrence Reynolds

Advanced Histotechnology II

CLB-465-M1

Professor Daniel Packert

April 2016

Overview� Background

� Reticulin / Purpose of Staining

� Types of Reticulin Stain

� Procedure for Gomori’s Reticulin Silver Stain

� Uses of Gomori’s Reticulin Silver Stain

Purpose of Research (Effect of Varying Developing � Purpose of Research (Effect of Varying Developing Reagents on Gomori Reticular Fiber Stain)

� Equipment / Materials / Techniques & Methods

� Staining Reagents & Research Study Procedure

� Data Interpretation / Results

� Discussion / Conclusion

Reticulin (type III collagen)

� Reticulin → Scleroprotein in

connective fibers

� Reticular fibers →

� Support structure for organs (e.g., liver & spleen)

� Cannot be visualized in H&E

� Argyrophilic (stains black w/

silver impregnation)

� Low affinity for silver salts

Purpose of Staining

� Fixed tissue sections lack contrast

� Stain binding allows for visualization

� Reticulin silver stains → demonstrate reticulin

fibers

� How?

� Pre-treat reticulin fibers w/ sensitizing agent

� Follow-by silver impregnation

� Reducing agent brings silver to visible form

Types of Reticulin Stains

� Gordon & Sweet’s Method

� Gomori’s Reticular Fiber Stain

Uses → Gomori’s Reticulin Silver Stain

� Interpret liver biopsies

� Demonstrate liver’s micro-architecture

� Changes in normal reticular fiber patterns

� Dx: liver disease (cirrhosis), hepatic necrosis, cancer

� Reticulin fibers surround damaged hepatocytes

� Collapse in empty space left behind

Uses → Gomori’s Reticulin Silver Stain

� Reticulin crowding → focal hepatocyte loss

� Reticulin collapse → hepatocye necrosis

� Thickening hepatic cell plates → hepatic regeneration

Overview → Gomori’s Reticulin Silver Stain

1. Oxidation w/ Potassium Permangenate

2. Sensitization w/ Ferric Ammonium Sulfate

3. Silver Impregnation w/ Ammoniacal Silver Nitrate

4. Reduction (a.k.a. Developing) w/ Formaldehyde4. Reduction (a.k.a. Developing) w/ Formaldehyde

5. Toning w/ Gold Chloride

6. Remove Unreduced Silver w/ Na Thiosulfate (HYPO)

7. Counterstain w/ Eosin

Steps → Gomori’s Reticulin Silver Stain

1. Oxidation w/ Potassium Permangenate

� Enhances staining of reticulin fibers

� Hexose sugars of reticulin fibers become visible by:

� Oxidation of its adjacent glycol groups to aldehydes

� Resulting active sites readily take up silver

� Potassium metabisulphate removes excess KMnO4

Steps → Gomori’s Reticulin Silver Stain

2. Sensitization w/ Ferric Ammonium Sulfate

� Metallic salts deposited around reticulin (metallic impregnation)

Metal-Organic compound formed

� Why? Reticular fibers → low affinity for silver salts

� NH4Fe(SO4)2 later replaced by Ag from diamine silver

impregnation solution

Steps → Gomori’s Reticulin Silver Stain

3. Silver Impregnation w/ Ammoniacal Silver Nitrate

� Metal-Organic Compound now replaced by SILVER

� 4 Silver atoms deposited @ each reactive sugar

residue site in reticulinresidue site in reticulin

� Aldehyde group reduces diamine silver to metallic silver

Steps → Gomori’s Reticulin Silver Stain

4. Reduction w/ Formaldehyde (a.k.a. Developing)

� Develops deposited silver to visible silver

Using formaldehyde reducer:

� Residual silver diamine ions on reticulin →

� Further reduced to visible metallic silver form

Steps → Gomori’s Reticulin Silver Stain

5. Toning w/ Gold Chloride

� Bound metallic silver replaced by metallic gold

3Ag⁰ + AuCl3 � Au⁰ + 3AgCl⁰ ⁰

� Reticulin fibers color change (brown → black)

� More stable compound formed

� Improved clarity / Increased contrast of section

Steps → Gomori’s Reticulin Silver Stain

6. Remove Unreduced Silver w/ Na Thiosulfate (HYPO)

� Unreactive silver removed from tissue

� Prevents further reduction by later exposure to light

7. Counterstain w/ Eosin

� Depends on :

� Type of tissue stained & pathologist preference

Purpose of Research

� Determine effect of different

reducing reagents on Gomori’s

reticular silver stain

Equipment / Materials� Leica RM2125 microtome

� Fisher Scientific water bath

� Pre-embedded liver blocks� Pre-embedded liver blocks

� Wax blocks cut w/ microtome @ 4 microns

� Poly Scientific staining kit

Gomori’s Silver Staining Reagents:� Potassium permanganate 0.5% aq.

� Potassium metabisulfite 2% aq.

� Ferric ammonium sulfate 2% aq.

� Ammoniacal silver nitrate solution

� Formalin 20% non buffered

Oxidation

Sensitization

Silver impregnation

Reduction� Formalin 20% non buffered

� Gold chloride 0.2% aq.

� Sodium thiosulfate 2%

� Eosin

Reduction

Toning

Remove Unreduced Silver

Counterstain

Slide Prep Procedure

� Liver tissue biopsies

� Fixed, processed, & embedded into paraffin blocks

� Cut blocks at 4µm using micrtotome

� Place in water bath → then → on slide

� Oven melts wax for adherence to slide� Oven melts wax for adherence to slide

� Pre-staining steps to re-hydrate tissue

� Gomori Reticular Staining

� Post-staining steps to dehydrate tissue

� For adherence of cover slip

Slide Prep Procedure

� Pre-staining steps to re-hydrate tissue

1. Wash w/ xylene (2X, 10-min then 3-min) → dissolves paraffin

2. Wash w/ 100% alcohol (2X, 30-sec each) → clear out xylene

3. Wash w/ 95% alcohol (2X, 30-sec each) → prevent shock

4. Wash w/ H2O → stains are water based4. Wash w/ H2O → stains are water based

� Post-staining steps to dehydrate tissue

1. Wash w/ 95% alcohol (2X, 30-sec each)

2. Wash w/ 100% alcohol (2X, 30-sec each)

3. Wash w/ xylene (2X, 30-sec each)

Control Tissue

�Liver

� Important:

� Must know amount of reticulin to be

demonstrated in control section each timedemonstrated in control section each time

� To determine how completely the reticulin is

stained in each test sample

Thick CutSampleSample

Research Procedure� Cut many unstained slides for testing

� Stain each test slide test slide using different reducersStaining

TechniqueBlock of Liver Sample A Block of Liver Sample B

Automated→Ran 1 per Session

1 Control SlideControl Slide (run in auto stainer) 1 Control SlideControl Slide (run in auto stainer)

Manual→ 3 Test Slides Test Slides (test reducer “x”) 3 Test SlidesTest Slides (test reducer “x”)

� Compare reticular fiber clarity of testtest to control slides control slides usingusing:

� 20% non-buffered formalin &

� Special automated staining machine

Manual→ 3 Test Slides Test Slides (test reducer “x”) 3 Test SlidesTest Slides (test reducer “x”)

Manual→ 1 Control Slide Control Slide (20% formalin) 1 Control SlideControl Slide (20% formalin)

Experimental / Test Reducing Agents

Formalin 1% Non Buffered

Formalin 10% Neutral Buffered

Formalin 50%

Formalin 100%

Ammonia Hydroxide

Glutaraldehyde 3%

Potassium Chloride 0.2M

Hydroquinone

Lithium Carbonate 0.05%

Oxalic Acid 5%

Research Findings

Reducing Agent Slide Sample

Formalin 1% Non-Buffered

+++++

Glutaraldehyde 3%

++++++++++

Control-Formalin 20% NBAutostainer

+++++

Negative Control No Reducer

+

Research Findings

Reducing Agent Slide Sample

Formalin 50%

++++

Formalin 10%Neutral BufferedNeutral Buffered

++++

Control-Formalin 20% NBAutostainer

+++++

Negative Control No Reducer

+

Research Findings

Reducing Agent Slide Sample

Hydroquinone

+++

Formalin 100%Formalin 100%

++

Control-Formalin 20% NBAutostainer

+++++

Negative Control No Reducer

+

Research Findings

Reducing Agent Slide Sample

Potassium Chloride 0.2M

+

Ammonia HydroxideAmmonia Hydroxide

+

Control-Formalin 20% NBAutostainer

+++++

Negative Control No Reducer

+

Research Findings

Reducing Agent Slide Sample

Lithium Carbonate 0.05%

+

Oxalic Acid 5%Oxalic Acid 5%

+

Control-Formalin 20% NBAutostainer

+++++

Negative Control No Reducer

+

What is the goal of reducing agent?

� Enable visualization of reticular fibers by →

� Reducing silver diamine to metallic silver

3 AgCl → Ag↓ + Cl-

� Best reducing agents →

� Oxidized more readily & able to contribute H+

� ∴∴∴∴Non-buffered formalin 20% oxidized to formic acid

� → fall in pH & donation of H+

� → metallic silver precipitate binds to reticulin fiber

Comparative Amount of Reticular Fiber Visualization

Reducing Agents Reticular Fiber Visualization Comparison

Formalin 20%, Non-Buffered (control) +++++

Formalin 1%, Non-Buffered +++++

Glutaraldehyde 3% +++++

Formalin 10%, Neutral Buffered ++++

Formalin 50% ++++Formalin 50% ++++

Hydroquinone +++

Formalin 100% ++

Potassium Chloride 0.2M +

Ammonium Hydroxide +

Lithium Carbonate 0.05% +

Oxalic Acid 5% +

Reducers →Maximized Contrast of Reticulin

� Non-buffered formalin 1%

� Glutaraldehyde 3%� Glutaraldehyde 3%

� Similar to 20% non-buffered formalin (control)

Reducers →Adequate Contrast of Reticulin

� Neutral-buffered formalin 10%

� Formalin 50%� Formalin 50%

� Similar to 20% non-buffered formalin (control)

Reducers →Much Less Contrast of ReticulinHydroquinone 0.01M Formalin 100%

20% NB Formalin (control)

� Why? Perhaps in addition to reducing silver

diamine deposits along reticular fiber, they may have

reduced other components in background tissue.

Reducers →Inadequate Contrast of ReticulinAmmonium Hydroxide Potassium Chloride 0.2M

Lithium Carbonate 0.05% Oxalic Acid 5%

20% NB Formalin Negative Control

Conclusions� Histochemistry principles→ predict best reducing agents

� Buffered agents resist changes in pH (stabilize acidity)

� Less likely to contribute H+ or give up electrons

� Necessary properties of good reducing agents

� 20% non-buffered formalin (control)

� Able to slowly oxidize to formic acid

� Results in ↓ pH

� Allows for reduction of silver diamine to metallic silver

Conclusions

� Gomori’s Silver Impregnation Staining

� Used to visualize reticulin fibers in liver biopsies

� Useful in diagnosing liver diseases & malignancies

� Reticulin fibers are argyrophilicReticulin fibers are argyrophilic

� This research study →

� Provides practical variations for reticulin fiber staining

� Determines which reducing agents provide best visualization

References

Questions and Answers

Appendix