Common Stains in Cell path

Question 1

Haematoxylin and Eosin

Answer 1

“General Morphology” to show nuclei (blue/black) and everything else in shades of red/pink.

H&E gives the diagnosis in over 70% of reporting

Often followed by “special stains” or ICC for complete diagnosis.

Principle staining:

Chemical attraction - Charges on dye/tissue attracted to each other (opposites)
Nuclei Acids, -ve charges, Basophilic
Reacts with haematoxylin, +ve charge, basic dye
Blue

RBCs, Cytoplasm, Muscle, Collagen etc, +ve charges, Acidophilic
Reacts with eosin, -ve charge, acidic dye
Red

Question 2

Elastin

Answer 2

Millers Elastin/ van Gieson
Elastin-Black
Collagen-Red
Fibrin-Yellow

Question 3

Fibrin/ Collagen

Answer 3

MSB
 "RBCs-Yellow
Fibrin-Red
Muscle-Red
Collagen-Blue"

Question 4

Fibrin/ Collagen”

Answer 4

Masson trichrome
“Muscle & Erythrocytes-Red
Collagen-Blue”

Question 5

Fibrin/ Collagen

Answer 5

Phosphotungstic acid and Haematoxylin
“Fibrin-Blue
Collagen-Orange/Red
Muscle-Blue”

Question 6

“Collagen III

(reticulin)”

Answer 6

“Reticulin methods
(silver impregnation)”
“Reticulin-Black
“

Question 7

Collagen IV

Answer 7

Periodic acid Schiff/ Haematoxylin
“Basement membrane-Pink
Nuclei-Blue”

Question 8

Amyloid (fibrous protein) cause of amyloidosis

Answer 8

“Congo red

Sirius red
Thioflavin T

Crystal violet”

” Amyloid-Red; also Congo red gives
apple green birefringence (cross-polarizers)
Amyloid-Red
Thioflavin-T is fluorescent dye (fluorescent microscopy)
Amyloid-Blue”

Question 9

Van Gieson’s stain

Answer 9

Two acid dyes (acid fuchsin and picric acid) combined in a single solution
Principle staining:
Picric acid, small molecules, penetrate all the tissue rapidly, but are only firmly retained in the close
textured red blood cells and muscle.
Acid fuchsin, larger molecules, displaces picric acid molecule from collagen fibres, which has larger
pores and allow larger molecules to enter.
Differentiates Muscle from Collagen (Yellow/Red).
Also identifies Bile (bottle green)

Question 10

Trichrome stains

Answer 10

Three acid dyes (e.g. picric acid, acid fuchsin and methyl blue can be used)
All work on similar principle – Chemical attraction (Acid/Base), Density/permeability of tissues, Molecular size of dye.
Three dyes are usually used separately and sequentially
Combinations/matching to tissues gives different colours
Differentiates Muscle/Collagen
Also demonstrates Fibrin
Some variations have 3 colours, some have 2 (not as useful)
E.g. Picro Mallory, Azan, Masson Trichrome, Masson Goldner, MSB etc

Question 11

Martius Scarlet Blue (M.S.B.) stain

Answer 11

Modification of the Masson Trichrome procedure to demonstrate Fibrin

Three acid dyes to replace eosin: martius yellow, brilliant crystal scarlet (red), aniline blue

Based on chemical attraction: acid dye/basophilic tissue, density of tissue, permeability of tissue/molecular size of dye

Principle MSB staining:
Step 1: Martius yellow quickly goes in, and is tightly held in RBSs, but quickly washed out of
collagen/muscle cytoplasm
Step 2: Brilliant crystal scarlet (red) files cytoplasm/muscle/collagen but cannot enter RBCs as they are saturated with Martius yellow
Step 3: Phosphotungstic acid acts as a dye excluder/differentiator, it competes with the red dye and takes red dye out of the tissue. The reaction is stopped when collagen in only faintly stained (cytoplasm/muscle will still be red)
Step 4: Collagen simply needs to be filled with aniline blue and any excess rinsed off
Optional: haematoxylin counterstain but this will need to go first in the sequence

Question 12

Silver Impregnation

Answer 12

Impregnation of tissue to enhance the contrast in tissues (no staining with dye!)
Black, fine deposits of silver and silver oxide
Very sensitive method but expensive and technique can be unreliable
Silver salt is reduced to metallic silver in reduction reactions at sensitive sites (e.g. aldehyde groups)
Three different ways of producing metallic silver

Argentaffin reaction – argentaffin or enterochromaffin cells of the gut only due to strong reducing pigment.

Argyrophil reaction – external reducer needed e.g. formalin (Reticulin fibres, neuroendocrine cells, neurons & axons).

Ion-exchange reactions (e.g. phosphates/carbonates of mineralized bone in von Kossa stain).

Question 13

Reticulin Stain

Answer 13

Acidified Potassium Permanganate- Oxidising agent which produces aldehyde groups

Bleach w 1% Oxalic acid- Decolouriser background stain

Iron Alum- Mordant

Ammoniacal silver nitrate- Silver in a solution which can be easily reduced

Formalin- Reducing agent. Causes deposition of metallic silver

Sodium thoisulphate- Excess silver in unprecipitated state is removed

Silver impregnation normally used (identifies Type 3 collagen) Reticulin fibres
Also demonstrated with PAS
Useful to note Cirrhosis patterns in liver

Classic method for neuroendocrine cells – Grimelius argyrophil reaction
Silver impregnation (no counterstain needed)

Question 14

Von Kossa Stain

Answer 14

Silver impregnation technique on undecalicified sections of bone tissue
None calcium specific stain (reacts with phosphates/carbohydrates in mineralized bone)
Allows for the identification of e.g. lesions, metabolic bone disease
Alternatives to identifying calcium: Alizarin Red (picture), Masson Goldner

Question 15

Elastic Fibres

Answer 15

Stain red/pink with H&E, but special stains are needed for more exact studies.
ID important in blood vessels
Common methods are Orcein, Verhoeff’s, Weigert’s (many modifications), Aldehyde fuchsin

Question 16

Amyloid

Answer 16

Amyloid is an abnormal protein, amino acid sequences similar or identical to body proteins.
Amyloid protein very variable in its amino acid composition, all have a β-pleated sheet.
Very resistant to enzyme degradation => accumulates between cells in many organs
These amyloid proteins form fibrils.
Some types of amyloid are considered to be pathological e.g. β-Amyloid (Alzheimer’s disease)
Difficult to demonstrate. Many tinctorial stains lack specificity (lots of false positives) and Amyloid variety aa compositions => may stain different.
Preferred method in most laboratories: Congo Red.
Congo Red allows “Dichroism “ with polarised light – apple green birefringence (needed for diagnosis)

Question 17

Carbohydrates

Answer 17

Classical stain: Periodic acid-Schiff (PAS) – based on release of aldehydes by periodic acid, Schiff reaction to recolour (chromophore restored).

Oxidation Carbohydrates to dialdehydes

Dialdehydes react with Schiff Reagent [restores a modified chromophore]

Schiff’s reagent consists of a solution of basic fuchsin (pararoaniline) that has been decolorized with sulphurous acid

On reacting with tissue, chromophoric group restored as a modified chromophore (different colour to the original dye)

Colour is enhanced in water

Carbohydrates e.g. Glycogen, Glycoproteins (membranes), Proteoglycans (mucins)
Used with Diastase as a negative control in Glycogen demonstration.
Other methods include Alcian blue (mucins), PA Silver (basement membrane – type 4 collagen)
Note combination of PAS/Alcian blue – acid/neutral mucins – useful for GIT diagnosis

Question 18

Evaluation of Mucins

Answer 18

Neutral and acid mucins (e.g. submandibular gland, pancreas)
Alcian blue/PAS sequential staining technique
Alcian blue (pH 2.5) stains all acid mucins deep blue
PAS stains all mucins (neutral and acid) but Alcian blue will prevent or block PAS reaction selectively
Indicative of certain pathological conditions (e.g. mucin metabolism- Hunter’s disease or Hurler’s disease, accumulation in/around cells, severe abnormalities after birth)

Question 19

Neutral Lipids

Answer 19

Demonstrated by “lysochrome” principles – elective solubility when dye leaves the solvent and “stains” the lipid
Classic methods – Oil Red O, Sudan dyes (note Osmium Tetroxide stains lipids black - fixation)
Aqueous mountant needed, as neutral lipid will dissolve in dehydrating alcohols

Question 20

Pigments

Answer 20

Perls histochemical reaction for Haemosiderin (iron pigment)
Masson Fontana for Melanin (silver impregnation)
PAS for Lipofuscin (wear and tear pigment)

Question 21

Metachromatic stains

Answer 21

Colour shift - Certain tissue structures (chromotropes) can stain a different colour to the colour
of the dye solution.
Particularly the thiazine group dyes (e.g. toluidine blue, methylene blue, azure A)
Spacing between the acid groups (shorter distance more polymerization of the dye stronger metachromatic)
The purple-to-red red staining of mast cell granules with toluidine blue is called metachromasia.
The regular blue staining of other structures orthochromasia.

Question 22

Immunological

Answer 22

Based on “Antigen – Antibody” reaction.

Use of “labelled” antibodies that seek out corresponding antigen – labelling allows visualisation

Question 23

Cytopathology

Answer 23

Cervical smear - Pananicolaou stain (lecture Gynae Cytology week 10)

Question 24

Haematology

Answer 24

Blood smear - Based on Romanowsky staining – Combination of Methylene Blue/Eosin
(Leishman, May Grunwald, Giemsa, Wright etc)

Question 25

Bacteria

Answer 25

Grams stain (gram-negative bacteria, gram-positive bacteria)
			 	-  Ziehl Neelsen – mycobacterium (Tuberculosis)

Question 26

Fungi

Answer 26

• Fungi - Grocott (Slv), PAS – Particularly important when monitoring immunodeficiency

Question 27

Name the type of Pigments found in tissues, and consider special stains used to identify them.

Answer 27

Exogenous and Endogenous

Exo: Carbon, Asbestos, Drugs in liver samples

Endo: Haemosiderin, Melanin,

Methods: Haemosiderin- Perls Stain
Melanin- Fontana Masson

Question 28

Name special stains to identify Carbohydrates and Lipids

Answer 28

The oil red O (ORO) stain can identify neutral lipids and fatty acids in smears and tissues. Fresh smears or cryostat sections of tissue are necessary because fixatives containing alcohols, or routine tissue processing with clearing, will remove lipids. The ORO is a rapid and simple stain. It can be useful in identifying fat emboli in lung tissue or clot sections of peripheral blood.

Neutral - These can be found in glands of the GI tract and in prostate. They stain with PAS but not with Alcian blue, colloidal iron, mucicarmine, or metachromatic dyes.

Question 29

What is Birefringence and give examples of this being used in CP practise

Answer 29

Sodium urate crystals are also birefringent on polarization
Using a red plate, the crystals show negative birefringence (yellow color) when the crystal’s long axis is aligned in the direction of the slow wave. At 90 degrees to this, the crystals will be blue.
Have 2 different refractive indices

Question 30

Consider Iron storage in tissues and how it can be demonstrated with Histochemistry

Answer 30

Haemosiderin

Perls Prussian Blue

Question 31

Describe the Silver impregnation technique, and give examples of this being used in CP practise.

Answer 31

Melanin is normally found in the skin, eye, and substantia nigra. It may also be found in melanomas.

The commonly used Fontana-Masson (“melanin stain”) method relies upon the melanin granules to reduce ammoniacal silver nitrate (but argentaffin, chromaffin, and some lipochrome pigments also will stain black as well)

Question 32

Consider the use of frozen sections in CP practise and explain the use: Why? and When?

Answer 32

The quality of the slides produced by frozen section is of lower quality than formalin fixed paraffin embedded tissue processing.

In the performance of Mohs surgery, it is a simple method for real-time margin control of a surgical specimen. If a tumor appears to have metastasized, a sample of the suspected metastasis is sent for cryosection to confirm its identity. This will help the surgeon decide whether there is any point in continuing the operation.

Rarely, cryosections are used to detect the presence of substances lost in the traditional histology technique, for example lipids. They can also be used to detect some antigens masked by formalin.

Question 33

Perls’ Prussian blue staining.

Answer 33

The principle is that ferric ions (Fe3+) from protein bound tissue deposits react with ferrocyanide ions and form potassium ferric ferrocyanide (or Prussian blue)[ FeCl3 + K4Fe(CN)6 = KFeFe(CN)6¯ + 3KCl]. The Prussian blue staining is usually counterstained with a dye such as Neutral Red. Result is ferric iron (deep blue) and nuclei (red)

Question 34

Overview Iron Overload (Hemosiderosis; Hemochromoatosis)
[By James Adam Hamilton, MD, Assistant Professor of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine]

Answer 34

Typical adults lose about 1 mg iron (Fe) per day in shed epidermal and GI cells;

> Menstruating females lose on average an additional 0.5 to 1 mg/day from menses.

> This iron loss is balanced by absorption of a portion of the 10 to 20 mg of iron in a typical diet.

> Iron absorption is regulated based on the body’s iron stores and is usually in balance with the body’s needs. However, because there is no physiologic mechanism to remove iron from the body, iron absorbed in excess of bodily needs (or acquired through repeated transfusion) is deposited in tissues:

> Haemochromatosis. Autosomal recessive disorder. Excessive absorption of iron. Multi-organ disorder. Liver (cirrhosis), heart (heart myopathy), pancreas (diabetes mellitus)

> Haemosiderosis. Sometimes referred to as secondary haemochromatosis. None genetic cause. Focal deposition. Alcoholism or thalassaemia (blood transfusions)

> Iron overload may result from hereditary haemochromatosis (a genetic disorder of iron metabolism) or from secondary haemochromotosis, an acquired form of the disease that is due to excess oral intake or absorption of iron or to repeated blood transfusions. Morbidity is mainly due to iron accumulation in the endocrine organs (especially the pancreas, gonads, and pituitary), liver, and heart.

> African iron overload occurs most often in sub-Saharan Africa among people who consume an iron-rich fermented drink. A genetic component is thought to contribute to the pathogenesis of African iron overload, but no gene has yet been identified.

Question 35

Paraffin Wax

Answer 35

In all staining methods using paraffin wax embedded tissue it is necessary to remove the wax embedding medium and to re-hydrate the sections prior to staining. This is an essential step as most of the dyes used are either aqueous (i.e. made up in water) or alcoholic (i.e. made up in a weak solution of alcohol) and wax embedded sections are impervious to these.

Question 36

1. What is the purpose of a ‘differentiation’ step in a staining protocol?

Answer 36

taining differentiation is the removal of washing out of the excess stain until the colour is retained only in the tissue components to be studies.

Question 37

2. Why do sections need ‘blueing’ after a haematoxylin staining?

Answer 37

The alkaline pH of the bluing solution causes the mordant dye-lake to reform in the tissue and become more permanent.

Question 38

3. What conclusions can be drawn from the H&;E staining?

Answer 38

Overall view of tissue with nuclei in blue, cytoplasm is pink

Eosin is acidophilic so stains basic structures red

Haematoxylin is a basic due, staining nucleus blue, acidic structures

Question 39

4. Which cell type(s) contain the iron stain?

Answer 39

Kuppfer Cells and other Mφ

Hemosiderin (storage iron granules) may be present in areas of old hemorrhage or be deposited in tissues with iron overload (hemosiderosis is the term used if the iron does not interfere with organ function; hemochromatosis refers to a condition of iron overload associated with organ failure).

Question 40

6. How are tissue deposits containing ferric ions called?

Answer 40

Haemosiderin

Question 41

7. What is the difference between Haemosiderosis and Haemochromatosis?

Answer 41

hemosiderosis is the term used if the iron does not interfere with organ function; hemochromatosis refers to a condition of iron overload associated with organ failure

Question 42

Perls Iron Stain

Answer 42

Perl’s iron stain is the classic method for demonstrating iron in tissues. The section is treated with dilute hydrochloric acid to release ferric ions from binding proteins. These ions then react with potassium ferrocyanide to produce an insoluble blue compound (the Prussian blue reaction). Mercurial fixatives seem to do a better job of preserving iron in bone marrow than formalin.

Question 43

Calcium Stain

Answer 43

Only calcium that is bound to an anion (such as PO4 or CO3) can be demonstrated. Calcium forms a blue-black lake with hematoxylin to give a blue color on H&E stain, usually with sharp edges.

VonKossa stain is a silver reduction method that demonstrates phosphates and carbonates, but these are usually present along with calcium. This stain is most useful when large amounts are present, as in bone.

Alizarin red S forms an orange-red lake with calcium at a pH of 4.2. It works best with small amounts of calcium (such as in Michaelis-Gutman bodies). The alizarin method is also used on the Dupont ACA analyzer to measure serum calcium photometrically.

Azan stain can be used to differentiate osteoid from mineralized bone.

Question 44

FORMALIN ARTEFACT

Answer 44

Formalin Brown Haemorrhagic Removed by Picrin

For Brown Haem Remember Picnics

Question 45

MALARIAL ARTEFACT

Answer 45

Think about Malaria as a blood disease, so near RBC, Brown Old Blood, Parastic

Question 46

MERCURY ARTEFACT

Answer 46

Mercury the planet is grey. These deposits are black, removed by white sodium thiosulphate. and iodine…. just remember the iodine.

Question 47

BILE PIGMENTS

Answer 47

Endogenous
BBB Bile Break Blood
Bilirubin = Rubi Red
Biliverdin = Green (in french)

Question 48

DICHROMATE ARTEFACT

Answer 48

BANANA DAQUIRI

Potassium Dichromate Fixation

D for Dichromate

Potassium is found in Bananas. Bananas are yellow.
Yellow (brown) deposits

Removed with Acid Alcohol

Question 49

MELANIN

Answer 49

M for Melanin, M for Masson, M FOR Montana → Fontana

Question 50

HAEMOSIDERIN

Answer 50

HAEMO- Blood SuiSIDE : Breakdown of red blood cells
Blood cells are broken by Macrophages, so found in macrophages
Breakdown happens in spleen and liver so found there.

Stored IN : Iron in its storage stage: Fe 3+

Counterstain is 1% neutral red, staining nucleus red

Question 51

CALCIUM

Answer 51

Von Kossa, Von in german refers to “of/ from”. So this is silver of phosphate or carbonate partnership. Useful in bone. B for bone, B for black colouring

Question 52

COPPER

Answer 52

When things oxidise, they go brown. (Think Rust)
Copper sign of oxidation enzyme failure
C O(rcein) PPE R(ubeanic)

Orcein is brown, Rubeanic is black

Question 53

URIC ACID

Answer 53

P - URI -NE Nucleotides

URI-ne Kidney diseases

Question 54

CARBON

Answer 54

Create the image of the industrial revolution and the smog. Breathing in the smog.

Carbon is seen in lungs of urban dwellers and smokers,

CARBON → NO Histological methods.

Question 55

What is fixation

Answer 55

The preservation of biological tissues from lysis and putrification
First, a fixative usually acts to disable intrinsic biomolecules—particularly proteolytic enzymes—which otherwise digest or damage the sample.

Question 56

Factors affecting fixation

Answer 56

PH 
Temperature
Osmolaroty
Size and volume of sample
duration
Time from removal to fixation

Question 57

Paraffin technique

Answer 57

In this technique, tissues are fixed, and embedded in wax. This makes the tissue hard, and much easier to cut sections from. The sections are then stained, and examined with the light microscope.

Question 58

Dehydration and clearing:

Answer 58

To cut sections, the tissue has to be embedded in paraffin wax, but wax is not soluble in water or alcohol. However, it is soluble in a paraffin solvent called ‘xylene’. Therefore, the water in the tissue needs to be replaced with xylene. To do this, first the tissue has to be dehydrated, by gradually replacing water in the sample with alcohol. This is achieved by passing the tissue through increasing concentrations of ethyl alcohol (from 0 to 100%). Finally, once the water has been replaced by 100% alcohol, the alcohol is replaced with xylene, which is miscible with alcohol. This final step is called ‘clearing’.

Question 59

Before staining taking to water

Answer 59

Unfortunately, most staining solutions are aqueous, so to stain the sections, the wax has to be dissolved and replaced with water (rehydration). This is essentially step 2 in reverse. The sections are passed through xylene, and then decreasing strengths of alcohol (100% to 0%) and finally water. Once stained, the section is then dehydrated once again, and placed in xylene. It is then mounted on the microscope slide in mounting medium dissolved in xylene. A coverslip is placed on top, to protect the sample. Evaporation of xylene around the edges of the coverslip, dries the mounting medium and bonds the coverlips firmly to the slide.

Question 60

What is a polarization?

Answer 60

a single orientation among all waves which compose light

Question 61

Polarization Perpendicular

Answer 61

No light can go through

Question 62

Sample is positioned (polarized microscopy)

Answer 62

B/n two light rays. If birefrigerent, it will split in there light into 2 waves w different delays allowing the sample to be visible.

Question 63

Thicker samples (polarized microscopy)

Answer 63

Transmitted waves are modified, changing interferences after second filter and therefore the colour of image. Allows for visualisation and characterise sample.