Histotechnology HTL Exam - Comprehensive Review

Alcian Blue demonstrates

Acid mucins

ATPase Enyme Histochemistry demonstrates

ATPase in muscles

Bielschowsky's Silver Technique demonatrates

Dendrites, axons, neurofibrils

Brown & Brenn gram stain demonstrates

bacteria

Congo red demonstrates

amyloid

Cresyl Echt Violet demonstrates

Nissl substance

Geimsa stain demonstrates

mast cells or H. pylori

Gomori Burtner demonstrates

melanin

Gomori's Aldehyde Fuchsin

demonstrates

elastic and mast cells

Gordon & Sweet's demonstrates

reticulin

Grocott's Methenamine Silver demonstrates

fungi (eg. Pneumocystis)

Hematoxylin & Eosin shows

general stain

Immunohistotochemistry

Anything an antibody can be made for

Luxol fast blue stain shows

myelin

Toluidine blue stain shows

mast cells or H. pylory

Masson's Trichrome shows

collagen

Oil Red O technique shows

neutral fats (simple fats)

Papanicolaou technique shows

cytology smears

Periodic Acid Schiff's shows

neutral mucin, glycogen

Perl's Prussian Blue shows

Hemosiderin

Southgate's mucicarmine

epithelial acid mucins

Verhoeff's Van Gieson

elastic

Van Kossa shows

calcium

Ziehl Neelsen shows

Acid fast bacilli (T.B.)

Nuclei in Gomori's Aldehyde Fuchsin technique

yellow - no specific stain for nuclei

Mast cells in a Gomori's Aldehyde Fuchsin technique

purple - GAF not specific for elastic

Gram positive bacteria if over-differentiated after crystal violet

red

Nuclei in the Brown and Brenn Gram stain if over differentiated with picric acid/acetone

yellow

Fungi in the Periodic Acid Schiff (PAS) technique

magenta - chitin binds with Schiffs

Fungi in the Grocott's Methenamine Silver technique

black - silver impregnation

Neutral mucins in a PAS technique if potassium permanganate is used as the oxidizing agent

light pink - colour of background - CHO overoxidized from aldehydes to acid state

Cytoplasm in an H&E if over differentiated with acid alcohol

pink - only affects nuclei

Epithelial acid mucins in the Southgate's mucicarmine technique

red

Cytoplasm in an H&E if the blueing agent is not properly removed

very pale pink

Nuclei in the Congo Red technique

blue - stained with aluminum hematoxylin

Calcium in the Von Kossa technique

black - silver impregnation

Glycogen, after diastase in the Periodic Acid Schiff technique

pale pink - background colour

Mycobacterium after Ziehl Neelsen technique

red - AFB stain with carbol fuchsin

Mycobacterium after Ziehl Neelsen technique

red - AFB stain with carbol fuchsin

Streptococcus after Ziehl Neelsen technique

blue - colour of background

Neutral lipids for Oil Red O cut on a cryostat

red to orange

Neutral lipids for Oil Red O cut from wax block

not demonstrated - removed

Hemosiderin after a Perl's Prussian Blue

blue

Nuclei after Masson's trichrome using an aluminum Hematoxylin

red - same as cytoplasm

Fine elastic fibres in Verhoeff's Van Gieson (VVG) after leaving in Van Gieson reagent too long

not black/not demonstrated - iron hematoxylin removed by the acid

Cytoplasm if underdifferentiated in the VVG technique

grey to black

Reticulin fibres if iron alum is skipped in the Gordon and Sweet's

not demonstrated

Melanin if potassium permanganate was used as an oxidizing agent in the Gomori Burtner technique

not demonstrated - removed

Melanin in the Luxol fast blue if overdifferentiated with lithium carbonate

pink

Nissl substance using the Cresyl echt violet technique

violet

Carboxylated mucins after Alcian Blue technique ph 1.0

no demonstration - not blue

Mast cells using Geimsa or Toluidine Blue

violet - both metachromatic dyes

Control slide for Alcian Blue

small intestine

Control slide for Bielschowsky's Silver Technique

grey matter

Control slide for Brown & Brenn gram stain

infected tissue

Control slide for Congo Red

known amyloid slide

Control slide for Cresyl Echt Violet

grey matter

Control slide for Gomori Burtner

skin

Control slide for Gomori's Aldehyde Fuchsin

aorta or skin

Control slide for Gordon & Sweet's

spleen or liver

Control slide for Grocott's Methenamine Silver

known fungi slide

Control slide for Masson's Trichrome

kidney

Control slide for Hematoxylin & Eosin

small intestine

Control slide for Luxol Fast Blue

cerebellum

Control slide for Masson's Trichrome

kidney

Control slide for Oil Red O

fatty liver

Control slide for Periodic Acid Schiff's(PAS) glycogen

liver

Control slide for PAS for neutral mucins

small intestine

Control slide for Perl's Prussian Blue

known hemosiderosis slide

Control slide for Verhoeff's Van Gieson

aorta or skin

Control slide for Ziehl Neelsen

known TB lung

A pathologist is reading the gross description report and it indicates that the patient sample they are examining should be uterus but under the microscope they see tissue from a breast on the slide. What could be the reason for the pathologist not receiving the correct specimen with the gross description report? (3)

The Pathologist Assistant opened two specimen containers at a time and grabbed the wrong specimen while grossing.

-The Pathologist Assistant gave the wrong requisition to the wrong specimen after grossing.

-The Technologist while embedding opened two cassettes at one time and put the wrong tissue in the wax mold with the wrong cassette on top.

_______counteracts the shrinking of the cells that occurs from picric acid in Bouins fixative

Acetic acid

What information would you find on a tissue cassette?

Block number

Year and surgical number

Number of tissue pieces

Before processing decalcified tissue, what is the next step?

Neutralize acid

The grossing area receives a liver biopsy that appears to be fatty. The pathologist wants to preserve the lipids in the tissue. What is the best fixative to use for lipid studies?

Osmium Tetroxide

A technologist completed an H +E stain on breast tissue and decided to check the slide under the microscope to verify if the stain worked correctly. The technologist noticed that all the lipids were destroyed in the tissue. What fixative was used in the grossing area that could of destroyed all the lipids on the slide?

100% Ethanol

Which fixative is the best in providing maximum preservation of glycogen in the tissue?

Picric Acid 95-100% Ethyl Alcohol

Problems associated with Picric Acid

Yellow discolouration of tissue

Hemolyzes RBCs

Dissolves Iron

Explosive when dry

Problems associated with 95-100% Ethyl Alcohol

Flammable Poisonous Excessive hardness & shrinkage

Government regulated

Describe the choice of fixative to use and three reasons why you chose that fixative for immunological studies.

B5 (or Zinc Formalin)

- Enhances immunoreactivity

- Coagulant fixative

- gives greater permeability

- better antibody penetration

- more intense staining

- Enhances nuclear detail, and good morphology

- Enhances staining of both nuclei & cytoplasm

Upon microscopic examination, an H & E stained section of routinely processed spleen shows small brown to black granules evenly distributed throughout the tissue.

Suggest a possible causative agent and describe how this artifact can be removed.

Formalin pigment - Acid Formaldehyde Hematin (AFH)

Formaldehyde / formalin, acid pH, hemoglobin (blood rich tissues)

1) Bring section to absolute alcohol

2) Place section in saturated picric acid for 5 min to 2 hours depending on the amount of pigment present

3) Wash for 15 to 20 min in running tap water

4) Proceed to desired stain

surface decalcification

Rough in to expose the tissue

- Surface of the tissue is placed in 1% HCl for 15-60 minutes

- This allows only several sections to be cut

a. Disposal of used 10% NBF

Wear appropriate PPE - mask, gloves and safety glasses. Work in a chemical fume hood. Mix with formalex in a ratio of 1:5 with formalin. Leave for a minimum of 1 hour (when a precipitate has formed, the fixative has been "neutralized"). This solution can now safely be poured down the sink with running tap water.

The 3 ingredients in B5 fixative are:

Mercuric chloride, Sodium acetate, Formaldehyde

Which fixative is classified as a non-additive

95% Ethyl alcohol

Which fixative is considered a substitute for B5?

zinc formalin

Commercial stock formaldehyde solution contains:

40% formaldehyde

Formalin pigment (AFH), may be removed from tissue by: (acidic ph and blood cause formalin pigment)

Alcoholic picric acid

Non-coagulant fixatives

Creates a gel that makes penetration by subsequent solutions difficult. Example:

10% NB Formalin

Formaldehyde, Acetic acid

additive fixatives

(non "A" words)

Formalin, Mercury, Osmium, Glutaraldehyde

Non-additive fixatives

("a" words)

Alcohol, acetic acid, Acetone

Goagulants (ZAPAM)

Zinc, Alcohol, Picric acid, Acetone, Mercury

Tissue is fixed in order to:

Stop autolysis

Which fixative is considered the "routine fixative"?

10% NB Formalin

Best secondary fixative for cytoplasmic (trichrome) staining

Bouin's

After fixing tissue in Bouin's solution the excess picric acid is removed by washing in:

70% Ethanol

Which fixative makes lipids insoluble?

Osmium tetroxide

A dye appears colorless because its chromophore was reduced. The reaction is reversible.

Leuco compound

Basophilic tissue has a ___________ charge.

Negative