dr. guadalupe manriquez – special stains clinical utility: an overview

Special Stains Clinical Utility: An Overview

Dr. Guadalupe Manriquez

Principal Pathologist - Ventana Medical Systems

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Introduction to Special Stains

Special Stains Panels

Simple Stains

Complex Stains

Case Studies

4

Why the term Special Stains?

•  Classified as “Special” because it is not a routine stain that is performed on a tissue sample.

•  The routine stain for tissue sections is the H & E (Hematoxylin and Eosin) stain.

•  The term usual is often used to distinguish it from immunoperoxidase stains.

•  The stains are based on dye chemistry.

What are Special Stains?

§  Special stains are important tools to Pathologists for detecting, identifying, and diagnosing tissue abnormalities.

§  Special stains are based on chemical reactions that use various colored dyes and reagents.

§  Many special stains rely on the chemical processes of oxidation and reduction.

§  Many special staining techniques are dependent on the use of acid, bases, and buffers for optimal performance.

Why are Special Stains Used?

Special stains are used to identify tissue processes by identifying:

•  Tissue structures

•  Cellular structures

•  cellular products

•  cellular artifacts

What are Special Stains Used for?

Stains for Mucins

§ PAS

§ Diastase Kit

§ Light Green for PAS

§ Alcian Blue for PAS

§ Alcian Blue

§ Mucicarmine

Stains for Connective Tissue

§  Elastic

§  Jones H&E (Silver)

§  Jones Light Green (Silver)

§  Trichrome Blue

§  Trichrome Green

§  Reticulin (Silver)

What are Special Stains Used for?

Stains for Microorganisms

§ AFB

§ Alcian Yellow

§ Steiner (Silver)

§ GMS (GMS)

Other Stains

§  Giemsa

§  Iron

§  Congo Red



Simple Stains

Complex Stains

Case Studies

10

Why are Special Stains run in Panels?


Stains for infectious agents such as bacteria, fungi (aspergillums & mucormycosis), and spirochetes are used when there is no morphologic clue as to the suspected organism.

–  Granulomatous inflammation points us to mycobacterium or fungal infections like coccidiodomycosis (Valley Fever) and histoplasmosis.

–  Panel includes Gram stain, Steiner, Warthin-Starry or Dieterle, and GMS

Panel Stains Micro-Organism PAS, PAS Diastase, GMS, Giemsa, AFB, Mucicarmine, Fite, Steiner, Alcian Yellow Connective Tissue Trichrome, Reticulin, Elastic, Fibrin, PTAH, Movat's Pentachrome Carbohydrate PAS, PAS Diastase, PAS Alcian Blue, Mucicarmine Hemat/Bone Marrow PAS, PAS Diastase, Iron, Giemsa, Reticulin Liver Trichrome, PAS, Reticulin, Iron, Orecin Kidney Trichrome, PAS, Jones Methanamine

11

Liver Panel

§  Panel includes an evaluation of: §  Microscopic intracellular glycogen and iron §  Changes in the reticulin (skeletal framework) §  Deposition of extracellular collagen denoting irreversible

tissue damage

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Kidney Panel §  Two basement membrane stains are used:

§  PAS to evaluate the collapse of glomerular capsule, lipid deposits, thickening of the basement membrane, and mesangial matrix

§  Jones Silver to evaluate vessels, deposits and location of deposits relative to the endothelium

§  Trichrome can be used to evaluate collagen deposition and irreversible tissue damage.



Simple Stains

Complex Stains

Case Studies

Stains for Polysaccharides or Mucopolysaccharides

§  PAS Hematoxylin, PAS Diastase

§  PAS Light Green, PAS Alcian Blue

§  Alcian Blue pH 2.5

§  Mucicarmine

Periodic Acid Schiff (PAS))

§  Stains Mucopolysaccharides (Mucins) §  Mucins are made up of carbohydrates + protein.

–  Two Types of Mucins §  Neutral Mucins §  Simple non-sulfated and complex-sulfated acid mucins

§  Stains Carbohydrates –  Carbohydrates are often called sugars or starches and are made up of different

types: §  monosaccharides - glucose, fructose §  disaccharides - sucrose "table sugar” (glucose fructose) §  polysaccharides - glycogen "body fuel”

–  Diastase digests glycogen - this is used to identify glycogen with certainty and/or eliminate it from the field of view.

Periodic Acid Schiff (PAS)

§  Schiff's reagent is made from a dye called pararosanilin.

§  Treated with sulphurous acid (H2SO3).

§  Causes a loss of the color in solution.

§  When exposed to aldehydes, Schiff's reagent combines with aldehydes to give a bright red product.

§  In Histology, these aldehydes are attached to, or produced from, a tissue structure so that it ends up colored bright red.

Human bowel - PAS/ hematoxylin - 40x

Periodic Acid Schiff (PAS)

PAS Stains:

§  Nuclei blue with hematoxylin

§  Carbohydrates and mucins magenta (bright pink) with “Schiff” reagent

PAS/Alcian Blue Stain

§  Used to differentiate between neutral mucins and acid mucins on the same slide.

§  Acid mucins stain with both PAS and Alcian Blue.

§  Neutral mucins stain with PAS only.

§  Use is for testing intestinal metaplasia, Barrett’s esophagus, and gastritis.

Human bowel - PAS/ Alcian Blue - 20x

PAS / Alcian Blue Stains:

§  Acid mucins deep blue with a combination of PAS and Alcian Blue

§  Neutral mucins magenta with PAS

PAS/Alcian Blue Stain

PAS/Diastase Stain

§  Diastase is an enzyme which selectively digests (breaks down) glycogen.

§  This produces negative PAS staining of the glycogen.

§  Any remaining PAS staining after diastase digestion must be due to protein-bound neutral polysaccharides, such as mucin or fungus.

§  Diastase is used in order to remove the glycogen staining from the tissue.

§  Sometimes used to be certain that it is/was really glycogen in the first place.

PAS/Diastase Stain PAS/Diastase Stain

§  Clinical Utility:

§  This can identify glycogen with certainty: §  glycogen storage disease in liver §  excess glycogen excreted in kidney

§  Removal of normal glycogen provides greater visual clarity of: §  mucins in adenocarcinoma §  fungus and bacteria (i.e. in liver containing glycogen)

PAS positive staining Removed with diastase

PAS/Diastase Stain

PAS/Light Green Stain

§  Stains anything that will stain with PAS

§  Light green counterstain provides a different visual contrast and is preferred for certain conditions

§  Assists with differentiating between basement membranes and fungus in kidney §  fungus will stain magenta §  basement membrane will stain purple

PAS / Light Green stains:

§  Fungus magenta with Schiff

§  Background light green

§  Basement membranes purple

PAS/Light Green Stain

Human kidney - PAS/ Light Green - 20x

§  simple, non-sulfated mucins containing sialic acid and found in epithelial cells

§  complex, sulfated mucins found in epithelial cells

§  simple, mesenchymal mucins found in tissue stroma

§  complex, connective tissue mucins found in tissue stroma, cartilage, and bone

§  Stains Acid Mucins

Alcian Blue Stain (pH 2.5)

Human bowel - Alcian Blue - 10x

Alcian Blue Stain

Alcian Blue Stains:

§  Nuclei red with Nuclear Fast Red counterstain

§  Acid mucin bright blue

Mucicarmine Stain

§  Stains Mucopolysaccharides §  Stains acid mucopolysaccharides, like those stained by Alcian Blue. §  Stains some but not all neutral polysaccharides, including the mucins secreted by

adenocarcinoma type tumors. §  Stains cell walls of Cryptococcus bacteria.

§  The "Carminophilic” properties of certain mucins are due to the “dye lake” formed by the natural dye carmine & the metal salt of aluminum

–  These chemicals, when combined in a solution, have an "affinity" for mucin. §  Staining occurs in the mucin as opposed to the cell that is producing it. §  The staining occurs by absorption of reagent into the mucin. §  The more mucin that is present, the "darker" the stain will look.

Human bowel - Mucicarmine - 20x

Mucicarmine Stain

Mucicarmine stains:

§  Nuclei stain black with iron hematoxylin

§  Mucins red with carmine dye

§  collagen and background yellowish

PAS

Sarcomas (tissue stroma, cartilage, bone)

Alcian Blue

Epithelial

+

+

Mucicarmine

Epithelial adenocarcinomas

+

_

Glands of GI or prostate

_

Mucicarmine

_

Alcian Blue

+

Staining of Mucins - Clinical Significance

Iron Stain

§  Also called “Prussian Blue”

§  Elements in the tissue can reduce iron salts and make the precipitate which stains the tissue “Prussian Blue”.

§  Mixed together, Reagent A and B create a solution that can be reduced by the iron in tissue and produce a reaction product that is blue.

Iron Stain

Iron Stain stains:

§  Iron deposits blue

§  Nuclei red with Nuclear Fast Red

Human Liver – Iron stain - 40x

Metachromatic Stains

§  Metachromasia – a “coat of many colors”

§  Some of the dyes used in histology can change color and can also be more than one color at the same time on different parts of the tissue section. §  The Giemsa Stain is an example of this

§  Elements in tissue have different electrical charges as do dyes.

§  Some dyes change color if they combine with tissue that has a different electrical charge.

§  Certain conditions can affect this such as pH and temperature.

§  Concentration of the dye in the tissue matters. When you wash out some of the dye, the color can change again. Therefore, dehydration is important.

§  It stains blood elements many shades of red to blue.

§  It also stains some bacteria (Helicobacter pylori) dark blue.

Giemsa Stain stains:

§ Blood cells many colors - including, staining the granules in eosinophils red

§ H. Pylori organisms blue

!

Giemsa

Acid Fast Bacilli (AFB)

§  AFB includes the organisms which cause Tuberculosis (TB) and leprosy, as well as other infectious disease processes.

§  "Acid fast" means that the organisms are stained, and then resist decolorization with acid solutions. These acid decolorizing solutions remove the red dye (Basic Fuchsin) from all other tissue elements, except for the acid fast bacteria.

§  Lots of different organisms stain positive with AFB Stain. This is always pathologic. AFB positive (or negative) results require careful consideration when forming a diagnosis.

§  Not all AFB organisms stain. §  Some of the "New" TB & Acid-fast Bacteria are mutating quickly and may not be as

“acid-fast” as the older, more familiar strains. §  Some of these bugs are hard to stain using conventional methods. Unfortunately, these

are the most important ones to diagnose & treat quickly.

AFB stains:

§ AFB stains acid fast bacilli red

§ Background blue

AFB Stain

Human lung – AFB stain - 20x

Human lung – AFB II stain - 20x

AFB II Stain

AFB stains:

§ AFB stains acid fast bacilli red

§ Background blue/teal

AFB Stains:

§ Microorganisms stain positive in purple

§ Analine blue stains background light blue

AFB III

Elastic Tissue is connective tissue, in the form of fibers, which can stretch and contract. They help an organ like stomach or bladder change its shape as needed. Skin has elastic tissue yet wrinkled skin doesn't have as much.

Elastic tissue can be stained both progressively and regressively.

§  Regressive staining, as in the Verhoeff Van Gieson technique, stains everything and then is removed from almost everything except the elastic fibers by a differentiating solution of solvent. This can give a very dark black elastic fiber stain.

§  Progressive staining uses a resorcin-fuchsin dye complex to stain elastic fibers selectively. The elastic fibers are dark purple in color.

Elastic Stain

Elastic Stain

Elastic Stain stains:

§  Elastic Fibers in arterial wall purple/black

§  Muscle yellow

§  Collagen red

Human Artery – Elastic stain - 10x

Trichrome Stain

§  The Trichrome stain got its name because there are three colors visible in the stained slide when the technique is properly performed.

§  Red from an acidic dye colors the muscle cells and the cytoplasm of liver cells, among other tissue components.

§  Blue from an aniline dye colors connective tissue and collagen. Green from fast green also colors connective tissue and collagen.

§  Black is the color formed by iron hematoxylin, and stains the cell nucleus.

Trichrome Stain

Trichrome stains:

§ Nuclei black

§ Cytoplasm and blood cells red

§ Collagen blue

Human liver – Trichrome stain - 20x

Trichrome III Blue

Trichrome stains:

§ Nuclei black


§ Collagen blue

Human liver – Trichrome stain - 20x

Trichrome stains:

§ Nuclei black


§ Collagen green

Human Kidney – Trichrome stain - 20x

Trichrome III Green

Congo Red

§  Modified Highman’s technique

§  Used to selectively stain diseased tissues that have amyloidosis or amyloid deposits.

§  Recent publications indicate the utility of identifying amyloid in association with plaque buildup (Alzheimer’s patients).

§  Amyloid may appear as circular or ribbon-like structures throughout the tissue.

§  Nuclear staining may vary in intensity due to tissue section and thickness.

§  Amyloid will stain pink to light red with a blue contrasting nuclear stain without a polarized lens.

§  Amyloid will stain with an apple green and yellow birefringence under polarized light. A polarized microscope is required to view the apple green birefringence when interpreting the slide.

Congo Red

Congo Red stains:

§  Amyloid pink/red


Human Kidney – Congo Red stain - 10x

Congo Red (Polarized)

Human Kidney – Congo Red stain - 10x

Congo Red stains:

§ Amyloid fluoresces to an apple-green color due to birefringence of the amyloid deposits

Alcian Yellow

§  Used to stain H. pylori

§  This stain gives a better contrast than a Giemsa

§  Not as good as Steiner

Alcian Yellow

Alcian Yellow stains:

§  Micro-organisms blue

§  Mucin yellow

§  Background blue

Human Gastric Biopsy – Alcian Yellow stain - 40x

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Gram Stain and Decolorization

§  Bacteria are classified in general by shape and Gram positive or negative staining.



Simple Stains

Complex Stains

Case Studies

Complex Stains (Silver Stains)

§  GMS (Gomori’s Methanemine Silver)

§  Reticulin Stain

§  Jones Methanamine Silver

§  Steiner

Silver Stains

§  Silver staining is a process for coating certain tissues with silver to make them opaque. Light does not pass through them, so the tissue structures look black under the microscope.

§  Silver impregnation techniques are much like developing a photograph. 1. The paper must be coated with silver. 2. The silver will be oxidized by exposure to light. 3. Then the silver deposits, or is reduced, onto the paper. 4. The resulting image is toned & fixed to hold it. 5. Until the process is complete, the image can be easily lost.

Silver Stains

§  There are two types of silver staining :

§  Argentaffin – intended for tissue that has a natural ability to attract the silver §  Ex: GMS

§  Fungus & Pneumocystis Carinii §  Endogenous staining of other tissue elements (like certain cells in the bowel). §  Some oxidation may be required. §  No chemical reducing agent is needed.

§  Argyophil – intended for tissue which must have chemical assistance to reduce the silver from solution and hold it.

§  Ex: Reticulin §  Oxidizers are always used. §  Chemical reduction of silver is required.

GMS Stain

§  Fungus and pneumocystis are micro-organisms of “opportunity”.

§  Fungus and pneumocystis frequently infect the lungs and other organs of HIV+ or immuno-suppressed patients due to chemotherapy or radiation treatments.

§  Good, positive control tissue must be used to prove the stain worked.

§  A false negative result could quickly lead to a patient’s unnecessary death due to lack of treatment for an unrecognized infection.

GMS (Fungus)

GMS stains:

§  Fungus black with silver

§  Background green

Human kidney – GMS fungus- 20x

GMS )

GMS stains:

§  Pneumocystis black with silver

§  Background green

Human lung – GMS PC- 20x

Reticulin

§  Reticulin stain is used to demonstrate Reticulin fibers.

§  Reticulin is a modification of Gordon and Sweets Stain.

§  Oxidizer, with potassium permanganate, oxides the tissue to enhance staining of reticulin fibers.

§  Decolorizer removes excess potassium permanganate.

§  Sensitizer is added to form a metal compound.

§  The metal-organic compound is replaced by the silver.

§  A reducer is applied to develop the deposited silver into visible silver.

Reticulum Stain Reticulin

Reticulin stains:

§  Reticular fibers black with silver


Human spleen – Reticulin- 20x

Human liver – Reticulin- 20x

Reticulin

Reticulin stains:

§  Reticular fibers black with silver


Jones Methanamine Silver

§  Jones stain is used for demonstration of basement membranes of the kidney.

§  Basement membranes are homogeneous, amorphous, and carbohydrate-containing proteinaceous material upon which rest epithelial and endothelial cells.

§  The principle of the silver stain depends on the local reduction and selective precipitation of silver by the aldehydic of the carbohydrates of reticulin and collagen.

§  The silver stain accentuates collagenous structures, e.g. in the glomerulus, the mesangial matrix, and the glomerular basement membrane.

Jones Methanamine Silver – H&E

Human Kidney – Jones H&E - 40x

JMS stains:

§  Basement Membranes black with silver


§  Cytoplasm pink with eosin

Jones Methanamine Silver – Light Green

JMS stains:

§ Basement Membranes black with silver

§ Background green with Light Green

Human Kidney – Jones Light Green - 40x

Steiner

§  Steiner is used to aid in the identification of causative organisms of infection such as spirochetes, Legionella pneumophilia, Helicobacter pylori and other bacteria.

§  Silver Stain that provides sharp staining of organisms with high contrast based on the impregnation of organisms with Silver

§  Developed for the detection of H. pylori, Syphilis, Lymes, Cat Scratch, and Legionnaire’s disease

§  Organisms will stain dark brown to black. Background will stain golden yellow to amber.

Steiner

Steiner stains:

§  H. pylori organisms black with silver

§  Nuclei black

§  Background golden yellow

Human Gastric Biopsy – Steiner stain - 20x

Steiner & Steiner stains:

§ Spirochete organisms black with silver

§ Nuclei black

§ Background golden yellow

Steiner

Human Lung – Steiner stain - 40x



Simple Stains

Complex Stains

Case Studies

Case #1

A 56-year-old obese man with long-standing gastro esophageal reflux (GERD) who recently received a diagnosis of Barrett's esophagus presents for a follow-up visit.

Patient initially presented with heartburn, a sour burning sensation in the back of the throat. Other symptoms include chronic cough, laryngitis, and/or nausea.

67

Endoscopic Detection of Barrett's Esophagus

§  The red, columnar-lined esophagus (arrow), and the contrast between the squamous (arrowhead) and columnar (arrow) epithelium, are characteristic of Barrett's esophagus.

68

Alcian Blue/PAS

69

§  Alcian Blue stains acidic sulfated mucosubstances found in the colon blue.

§  PAS stains neutral mucosubstances red.

§  Where there is intestinal metaplasia as in Barrett’s, the cell cytoplasm is blue to blue purple.

70

Patient Risk

§  For patients without dysplasia in whom two carefully performed endoscopic examinations a year apart have shown no evidence of disease progression, the surveillance interval may be extended up to 3 years.

§  The progression of Barrett's esophagus may involve the development of low-grade dysplasia and high-grade dysplasia before the eventual development of cancer

§  For patients with low-grade dysplasia in whom an advanced lesion has been ruled out, endoscopic surveillance twice during the initial year and annually thereafter is typically recommended.

71

Patient Risk

§  Management of acid production is effective for symptoms, but does not change the course of the disease.

§  The risk of esophageal adenocarcinoma is 30 to 40 times higher amongst patients with Barrett's esophagus then patients without this condition.

§  Multimodal endoscopic eradication therapy involves the removal of visible neoplastic lesions by means of endoscopic mucosal resection. This is followed by eradication of the remaining metaplastic epithelium with the use of mucosal ablative techniques such as photodynamic therapy, radio-frequency ablation, cryoablation, and argon plasma coagulation.

72

Statistics for GERD

§ 10-15% of patients with GERD develop Barrett’s esophagus

•  <1% of GERD patients develop adenocarcinoma

Reference:

•  Rex DK, Cummings OW, Shaw M, et al. Screening for Barrett's esophagus in colonoscopy patients with and without heartburn. Gastroenterology 2003;125:1670-1677

•  Sharma P, Falk GW, Weston AP, Reker D, Johnston M, Sampliner RE. Dysplasia and cancer in a large multicenter cohort of patients with Barrett's esophagus. Clin Gastroenterol Hepatol 2006;4:566-572

Case #2

A 39 year old male in late stage of AIDS is presented to the emergency room with fever, non-productive cough, shortness of breath (especially on exertion), weight loss, and night sweats.

73

74

Laboratory Findings

§ CD4 lymphocyte count was 180 cells/ul §  Normal range 500 cells/ul to 1,000 cells/ul

§ Patient had low oxygen saturation

75

Chest Radiography Showing Diffuse Bilateral Ground-Glass Opacities with Sparing of the Diaphragm Borders.

76

Evaluation

BAL (Brochioalveolar Lavage) was

performed which showed clustered

aggregates of organisms that

stained positively with GMS.

77

Pneumocystis jiroveci (carinii) - PCP

§ Pneumocystis pneumonia is a severe opportunistic infection found in people with AIDS. It is caused by a fungus called Pneumocystis jiroveci.

§ It can occur in other immunocompromised clinical settings like in patients being treated for malignancies, bone marrow transplants, or other severe immunocompromised states.

§ Disease is fatal if not treated with steroids and co-trimoxasole.

§ Preventative treatment is suggested for AIDS patients.

dr. guadalupe manriquez – special stains clinical utility: an overview

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