the big section

Question 1

Special stains
The term special stains traditionally referred to any staining other than an …..
It covers a wide variety of methods that may be used to visualize particular tissue structures, elements, or even microorganisms not identified by …… staining.
Other methods of staining use immunohistochemistry or in …. ………… to target specific proteins or …/… sequences. These methods were sometimes also included as members of the “special stains” family.
However they are quite different in method and purpose and are now typically separated into a third category know as “advanced stains”.

Answer 1

The term special stains traditionally referred to any staining other than an H&E.
It covers a wide variety of methods that may be used to visualize particular tissue structures, elements, or even microorganisms not identified by H&E staining.
Other methods of staining use immunohistochemistry or in situ hybridization to target specific proteins or DNA/RNA sequences. These methods were sometimes also included as members of the “special stains” family.
However they are quite different in method and purpose and are now typically separated into a third category know as “advanced stains”.

Question 2

Can be used as a panel of stains or a single stain
Examples of special stains:

Infection: Periodic …. ……..(PAS), Gr…., Gra,,,, Giemsa, Zn

Polysaccharides: …. ………. …………

Pigments: …….,……,…….
Inflammation: ……,……..

Mast cells: ……,,,,,,…….

Answer 2

Can be used as a panel of stains or a single stain
Examples of special stains:

Infection: Periodic Acid Schiff (PAS), Grocott, Gram, Giemsa, Zn

Polysaccharides: PAS, Alcian Blue, Mucicarmine

Pigments: Perls, Masson Fontana, Aldehyde/Fuchsin

Inflammation: Trichrome, Elastic

Mast cells: Giemsa, Toluidine blue

Question 3

Validation of staining Methods are validated by use of control sections:

Positive control Known to contain ……… / structure validates that the method is working

Negative control Re,,,,, or ,,,,,,,,,,,,,,, of target

Perl’s control: …….. granular deposition of Prussian blue ,,,,,,,,,, pigment hem,,,,,,,
around h……….; control tissue – liver

PAS control: ………. stained …………. cells in the tubular glands; control tissue – colon

Answer 3

Validation of staining Methods are validated by use of control sections:

Positive control Known to contain substance / structure validates that the method is working

Negative control Removal or deactivation of target

Perl’s control: Blue granular deposition of Prussian blue stained pigment hemosiderin around hemosiderin control tissue – liver

PAS control:Magenta stained secretory cells in the tubular glands; control tissue – colon

Question 4

For all unknown (test) tissue with a known ……….. should be added to the test …….. and should be stained at the same ……. : this includes ………… agents

Answer 4

For all unknown (test) tissue with a known positive should be added to the test slide and should be stained at the same time: this includes infectious agents

Question 5

Periodic Acid Schiff

PAS staining is mainly used for staining structures containing a ,,,,,,,, proportion of c…………. such as glycogen, g………, ………… connective ti……., mucus and basement membranes. Often used to stain kidney ………, liver biopsies, certain g………. storage diseases in striated muscles and suspected ……… infections.

Answer 5

PAS staining is mainly used for staining structures containing a high proportion of carbohydrates such as glycogen, glycoproteins, proteoglycans typically found in connective tissues, mucus and basement membranes. Often used to stain kidney biopsies, liver biopsies, certain glycogen storage diseases in striated muscles and suspected fungal infections.

Question 6

Pas staining

This is without a question the most versatile and widely used technique for the demonstration of ……. or glycoc………….
The first histochemical use of this technique was by McManus for the demonstration of mucin. Subsequently other studies have demonstrated the ability of the PAS technique to demonstrate other carbohydrate-containing molecules, e.g. gly…….and certain glycopr……….

Answer 6

This is without a question the most versatile and widely used technique for the demonstration of carbohydrates or glycoconjugates.
The first histochemical use of this technique was by McManus for the demonstration of mucin. Subsequently other studies have demonstrated the ability of the PAS technique to demonstrate other carbohydrate-containing molecules, e.g. glycogen and certain glycoproteins.

Question 7

Pas staining

When applied to tissue obtained from a …… biopsy, a PAS stain will normally show large amounts of ……….. in hep………..
In renal pathology, a PAS stain is used diagnostically to demonstrate G………… basement membranes.

Answer 7

When applied to tissue obtained from a liver biopsy, a PAS stain will normally show large amounts of glycogen in hepatocytes.
In renal pathology, a PAS stain is used diagnostically to demonstrate Glomerular basement membranes.

Question 8

PAS stain – oxidation = periodic acid

Any carbohydrate  especially with a 
6 (or 5) member ring structure.
Simple or complex sugars
Glu........., gl........., m.......
↓

Oxidise with HIO4 = periodic acid
↓↓↓

Open (oxidised ring structure
Two reactive ………… (CHO) groups

Answer 8

Any carbohydrate  especially with a 
6 (or 5) member ring structure.
Simple or complex sugars
Glucose, glycogen, mucin
↓

Oxidise with HIO4 = periodic acid
↓↓↓

Open (oxidised ring structure
Two reactive aldehyde (CHO) groups

Question 9

PAS stain – oxidation = periodic acid

The PAS technique is based upon the reactivity of free ……… groups within car……….. with the Schiff reagent to form a bright red/magenta end product.
The initial step in the PAS technique is the oxidation of hyd…….. groups attached to adjacent ………. atoms, 1,2-glycols, within the c………….
The result is the formation of two free aldehyde groups and the cleavage of the adjoining carbon-to-carbon bond. This is produced by treatment of the sections with a dilute solution of periodic acid (……), most protocols using a …-….. solution for 5–10 minutes.

Answer 9

The PAS technique is based upon the reactivity of free aldehyde groups within carbohydrates with the Schiff reagent to form a bright red/magenta end product.
The initial step in the PAS technique is the oxidation of hydroxyl groups attached to adjacent carbon atoms, 1,2-glycols, within the carbohydrate.
The result is the formation of two free aldehyde groups and the cleavage of the adjoining carbon-to-carbon bond. This is produced by treatment of the sections with a dilute solution of periodic acid (HIO4), most protocols using a 0.5–1.0% solution for 5–10 minutes.

Question 10

PAS stain – Schiff reaction (CHO +ve)

Basic fuc…… + sulph…….. acid → leu… dye= schiff

Schiff reagent + aldehyde groups → magenta reaction product

NB this is a standard test for ……… in organic chemistry – works on solutions in a test tube

Answer 10

Basic fuchsin + sulphurous acid → leuco dye (colourless) = Schiff reagent

Schiff reagent + aldehyde groups → magenta reaction product

NB this is a standard test for aldehydes in organic chemistry – works on solutions in a test tube

Question 11

Pas stain Schiff reaction (CHO +ve)

The intensity of the colour which develops following reaction with Schiff reagent is dependent upon the ……… concentrations of reactive g…… structures.
Schiff reagent is prepared from basic fuc….. which is not a specific dye but a mixture of triaryl…….. dyes such as pa……….., ro……… and new fuc……..
A number of methods for the synthesis of Schiff reagent have been described since Schiff’s original in 1866 but in all, an aqueous solution of sul………. acid is produced.
Schiff reagent reacts with the free ald……… generated from 1,2-glycol groups in periodic acid-treated carbohydrates. The initial monosaccharide-Schiff reagent con……. is a colourless intermediate reaction. The loosely bound sulfonate of the central carbon is removed in the following aqueous ……… The re-establishment of the quinoid structure of the triarylmethane molecule results in the deposition of a deep red/magenta coloration at the site of the carbohy…..-Schiff reagent complex.

Answer 11

The intensity of the colour which develops following reaction with Schiff reagent is dependent upon the tissue concentrations of reactive glycol structures.
Schiff reagent is prepared from basic fuchsin which is not a specific dye but a mixture of triarylmethane dyes such as pararosaniline, rosaniline and new fuchsin.
A number of methods for the synthesis of Schiff reagent have been described since Schiff’s original in 1866 but in all, an aqueous solution of sulfurous acid is produced.
Schiff reagent reacts with the free aldehydes generated from 1,2-glycol groups in periodic acid-treated carbohydrates. The initial monosaccharide-Schiff reagent conjugate is a colourless intermediate reaction. The loosely bound sulfonate of the central carbon is removed in the following aqueous rinse. The re-establishment of the quinoid structure of the triarylmethane molecule results in the deposition of a deep red/magenta coloration at the site of the carbohydrate-Schiff reagent complex.

Question 12

The intensity of the colour which develops following reaction with Schiff reagent is dependent upon the tissue concentrations of reactive glycol structures.
Schiff reagent is prepared from basic fuchsin which is not a specific dye but a mixture of triarylmethane dyes such as pararosaniline, rosaniline and new fuchsin.
A number of methods for the synthesis of Schiff reagent have been described since Schiff’s original in 1866 but in all, an aqueous solution of sulfurous acid is produced.
Schiff reagent reacts with the free aldehydes generated from 1,2-glycol groups in periodic acid-treated carbohydrates. The initial monosaccharide-Schiff reagent conjugate is a colourless intermediate reaction. The loosely bound sulfonate of the central carbon is removed in the following aqueous rinse. The re-establishment of the quinoid structure of the triarylmethane molecule results in the deposition of a deep red/magenta coloration at the site of the carbohydrate-Schiff reagent complex.

Answer 12

The intensity of the colour which develops following reaction with Schiff reagent is dependent upon the tissue concentrations of reactive glycol structures.
Schiff reagent is prepared from basic fuchsin which is not a specific dye but a mixture of triarylmethane dyes such as pararosaniline, rosaniline and new fuchsin.
A number of methods for the synthesis of Schiff reagent have been described since Schiff’s original in 1866 but in all, an aqueous solution of sulfurous acid is produced.
Schiff reagent reacts with the free aldehydes generated from 1,2-glycol groups in periodic acid-treated carbohydrates. The initial monosaccharide-Schiff reagent conjugate is a colourless intermediate reaction. The loosely bound sulfonate of the central carbon is removed in the following aqueous rinse. The re-establishment of the quinoid structure of the triarylmethane molecule results in the deposition of a deep red/magenta coloration at the site of the carbohydrate-Schiff reagent complex.

Question 13

Pas

Pas kidney ……

Pas liver gly……… h………….

Answer 13

Pas

Pas kidney normal
Pas liver glycogenic hepatopathy

Question 14

Pas
The reactivity of schiff reagent with glyco……. within the basal la…….vmakes the PAS technique valuable by of means assessing basement membrane ………….

Answer 14

The reactivity of schiff reagent with glycoproteins within the basal lamina makes the PAS technique valuable by of means assessing basement membrane thickness

Question 15

Pas
The reactivity of Schiff reagent with gly……….. within the ba…. lam…….. makes the PAS technique a valuable means of assessing basement membrane thi……..

Increased basement membrane thickness, particularly in the glom…… capill………… of the kidney, occurs in a number of pathological conditions.

The detection of mucins or glycogen by the PAS technique may aid the differential diagnosis of t………..

Answer 15

The reactivity of Schiff reagent with glycoproteins within the basal lamina makes the PAS technique a valuable means of assessing basement membrane thickness.

Increased basement membrane thickness, particularly in the glomerular capillaries of the kidney, occurs in a number of pathological conditions.

The detection of mucins or glycogen by the PAS technique may aid the differential diagnosis of tumours.

Question 16

Pas
Neutral mucins are stained deep red/magneta with the pas. This is seen in the go…… cells which contain neutral mu…………

Answer 16

Neutral mucins are stained deep red/magenta with the PAS.

This is seen in the goblet cells which contain neutral mucins

Question 17

Pas
Positive staining - normal

Basement membrane, fungi, glycogen (removed after diastase or amylase predigestion), mucins (neutral and acid-simple non-sulfated and acid-complex sulfated types), surfactant

Positive staining - disease

Basement membrane containing ……….. (cylin……..), clear cell tumors, renal cell carcinoma (PAS+ glycogen removed with ………….), parasites

Negative staining

……….. (acid-simple mesenchymal and acid-complex connective tissue types)

Answer 17

Positive staining - normal
Basement membrane, fungi, glycogen (removed after diastase or amylase predigestion), mucins (neutral and acid-simple non-sulfated and acid-complex sulfated types), surfactant
Positive staining - disease
Basement membrane containing tumours (cylindroma), clear cell tumors, renal cell carcinoma (PAS+ glycogen removed with diastase), parasites
Negative staining
Mucins (acid-simple mesenchymal and acid-complex connective tissue types)

Question 18

PAS

The PAS technique is also a sensitive and relatively fast means of demonstrating viable f…….. in tissue sections due to the presence of ………… acid-reactive p…………………. in the …………… or walls of many fungal species.
Common fungal species which are PAS reactive include Can……. albicans, Histopla………. capsulatum, Cryptococcus and Blastomyces

Answer 18

The PAS technique is also a sensitive and relatively fast means of demonstrating viable fungi in tissue sections due to the presence of periodic acid-reactive polysaccharides in the capsules or walls of many fungal species.
Common fungal species which are PAS reactive include Candida albicans, Histoplasma capsulatum, Cryptococcus and Blastomyces

Question 19

Alcian Blue technique

Alcian blue is a large co………… molecule

It is composed of a central copper-containing phthaloc………… ring linked to four isothiouronium groups via thioether bonds.

Proteoglycan/hyaluronic acid components of connective tissue and cartilage with al………. blue at pH of …… .

Acidic ………… mucins, e.g. the sialomucins and sulfomucins of the large intestine are reactive at pH 2.5.

Neutral mucins, e.g. in the gastric mucosa and Brunner’s glands are not reactive with alcian blue.

Combined alcian blue-PAS technique is used to differentiate neutral mucins from acidic mucins.

Answer 19

Alcian blue is a large conjugated dye molecule
It is composed of a central copper-containing phthalocyanine ring linked to four isothiouronium groups via thioether bonds.
Proteoglycan/hyaluronic acid components of connective tissue and cartilage with alcian blue at pH of 2.5.
Acidic epithelial mucins, e.g. the sialomucins and sulfomucins of the large intestine are reactive at pH 2.5.
Neutral mucins, e.g. in the gastric mucosa and Brunner’s glands are not reactive with alcian blue.
Combined alcian blue-PAS technique is used to differentiate neutral mucins from acidic mucins.

Question 20

Alcian Blue technique

Alcian blue is a large conju…….. dye molecule initially used for the dyeing of textile fibres. It is composed of a central ……….-containing phthalo…………….. ring linked to four isothiouronium groups via thioether bonds.

The exact mechanisms by which alcian blue stains carbohydrates are unknown, but it is widely believed that the cationic isothiouronium groups bond via …………..c linkages with polyanionic molecules within tissues.

The su…….. and carb………. groups of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid are ionized at a pH of …. and therefore carry a negative charge. This accounts for the staining of the proteoglycan/hyaluronic acid components of connective tissue and cartilage with alcian blue at this pH.

Similarly, the acidic epithelial mucins, e.g. the sialomucins and sulfomucins of the large intestine are reactive at pH ………… Neutral mucins, e.g. in the gastric mucosa and Brunner’s glands are not reactive with alcian blue.

Answer 20

Alcian Blue technique

Alcian blue is a large conjugated dye molecule initially used for the dyeing of textile fibres. It is composed of a central copper-containing phthalocyanine ring linked to four isothiouronium groups via thioether bonds.

The exact mechanisms by which alcian blue stains carbohydrates are unknown, but it is widely believed that the cationic isothiouronium groups bond via electrostatic linkages with polyanionic molecules within tissues.

The sulfate and carboxylate groups of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid are ionized at a pH of 2.5 and therefore carry a negative charge. This accounts for the staining of the proteoglycan/hyaluronic acid components of connective tissue and cartilage with alcian blue at this pH.

Similarly, the acidic epithelial mucins, e.g. the sialomucins and sulfomucins of the large intestine are reactive at pH 2.5. Neutral mucins, e.g. in the gastric mucosa and Brunner’s glands are not reactive with alcian blue.

Question 21

PAS D - Periodic Acid Schiff with Diastase digestion

After diastase digestion gly…………, proteoglycans, hyaluronic acid will be negative with PAS

Presence of glycogen will be evidenced by loss of staining after e……….. treatment when compared to the u…………. sections

Answer 21

PAS D - Periodic Acid Schiff with Diastase digestion

After diastase digestion glycogen, proteoglycans, hyaluronic acid will be negative with PAS

Presence of glycogen will be evidenced by loss of staining after enzyme treatment when compared to the untreated sections

Question 22

So, if to combine AB and D-PAS?What can we demonstrate?

Neutral and Acidic Mucins

Why we cannot demonstrate glycogen in this reaction?

Because glycogen will be digested by di………, leaving only ……….. to react with ………… reagent

Answer 22

So, if to combine AB and D-PAS?What can we demonstrate?

Neutral and Acidic Mucins

Why we cannot demonstrate glycogen in this reaction?

Because glycogen will be digested by diastase, leaving only mucins to react with schiff reagent

Question 23

Fungal Infections

Fungi are widespread in nature and humans are regularly exposed to spores from many species.

The most common are the superficial my…….. (dermatophytic mycoses).

Other infections, are related to dimini……..shed or compromised im. systems (systemic mycoses).

Fungi in tissues mainly display primitive forms – ase……… imperfect, which appears as sp……….l yeast or sp……. forms.

Some can produce appearances of t……. hyphae.

These features are important morphologically to identify different types of fungi.

Answer 23

Fungal Infections

Fungi are widespread in nature and humans are regularly exposed to spores from many species.
The most common are the superficial mycoses (dermatophytic mycoses).
Other infections, are related to diminished or compromised immune systems (systemic mycoses).
Fungi in tissues mainly display primitive forms – asexual imperfect, which appears as spherical yeast or spore forms.
Some can produce appearances of tubular hyphae.
These features are important morphologically to identify different types of fungi.

Question 24

Fungal Infections

Fungi are widespread in nature, and humans are regularly exposed to the spores from many species.

The most commonly encountered fungal diseases are the supe………. myco………… which affect the subcu. or horny layers of the skin or hair shafts, and cause conditions such as athlete’s foot or ringworm.

These dermatophytic fungi belong to the Micro………., Trichophyton and Epidermophyton groups and may appear as ………….. or mycelial forms within the ke……….

They are seen quite well in the ……… stain, but are demonstrated better with the Gr……..and P…. stains.

Answer 24

Fungal Infections

Fungi are widespread in nature, and humans are regularly exposed to the spores from many species.
The most commonly encountered fungal diseases are the superficial mycoses which affect the subcutaneous or horny layers of the skin or hair shafts, and cause conditions such as athlete’s foot or ringworm.
These dermatophytic fungi belong to the Microsporum, Trichophyton and Epidermophyton groups and may appear as yeasts or mycelial forms within the keratin.
They are seen quite well in the H&E stain, but are demonstrated better with the Grocott and PAS stains.

Question 25

Fungal Infections
As with other infections, the increase in the number of patients with dimi……… or compromised immune systems has increased the incidence of sy…….. mycoses, representing opportunistic attacks by fungi, frequently of low virule……., but often fatal if untreated.

When fungi grow in tissue they may display primitive ase…….. (imperfect) forms which appear as either sph…….. yeast or spore forms. Some may produce vegetative growth which appears as tubular hyphae which may be septate and branching.

These features are important morphologically for identifying different types of fungi. A mass of interwoven hyphae is called a fungal mycelium. Only rarely, when the fungus reaches an open cavity, the body surface, or a luminal surface such as the bronchus, are the spore-forming fruiting bodies called sporangia or conidia, produced.

Answer 25

Fungal Infections

As with other infections, the increase in the number of patients with diminished or compromised immune systems has increased the incidence of systemic mycoses, representing opportunistic attacks by fungi, frequently of low virulence, but often fatal if untreated.
When fungi grow in tissue they may display primitive asexual (imperfect) forms which appear as either spherical yeast or spore forms. Some may produce vegetative growth which appears as tubular hyphae which may be septate and branching.
These features are important morphologically for identifying different types of fungi. A mass of interwoven hyphae is called a fungal mycelium. Only rarely, when the fungus reaches an open cavity, the body surface, or a luminal surface such as the bronchus, are the spore-forming fruiting bodies called sporangia or conidia, produced.

Question 26

Candidiasis

Candidiasis known as Yeast Infection is a medical condition as a result of an overgrowth of the fungus C………… Al…………….

Under normal circumstance, this fungus is present in the body system and is put in check by Lacto……….. bacteria which acts as part of the body im……… system.

Candida is found in parts of the body that have m……… membranes such as the m…….. (thrush), throat, the oesophagus, rec……..and the vagina (vaginal moniliasis)

Also, the skin and nails, and maybe found in body cavities such as heart-……….. or lungs vegetations.

Common symptoms:
-burning and itching in the ………… and around the vulva;
-……….discharge and can affect people with a low immunity in the body.
The overgrowth of candida can make diagnosis difficult due to chronic symptoms that it shares with a …………Infection.

Answer 26

Candidiasis

Candidiasis known as Yeast Infection is a medical condition as a result of an overgrowth of the fungus Candida Albicans.
Under normal circumstance, this fungus is present in the body system and is put in check by Lactobacillus bacteria which acts as part of the body immune system.
Candida is found in parts of the body that have mucous membranes such as the mouth (thrush), throat, the oesophagus, rectum and the vagina (vaginal moniliasis)
Also, the skin and nails, and maybe found in body cavities such as heart-valve or lungs vegetations.
Common symptoms:
-burning and itching in the vagina and around the vulva;
-whitish discharge and can affect people with a low immunity in the body.
The overgrowth of candida can make diagnosis difficult due to chronic symptoms that it shares with a Yeast Infection.

Question 27

Vaginal Candidiasis

Overgrowth of Candida can be caused by:
the use of ……… control …….. (BCP)

extensive use of …………….

consumption of refined and processed foods due to high ………. content

use of ………..
immunosuppressed ……….

Diabetes …………..

consumption of food that may trigger overgrowths such as bread and baked food (made with yeasts), or marmite.

Treatment:

Anti-fungal internal products that contain
the active ingredient clot……….. or flu…………..

Oral pill, soft gel pessary, pessary or cream.

Answer 27

Vaginal Candidiasis

Overgrowth of Candida can be caused by:
the use of Birth control Pill (BCP)

extensive use of antibiotics

consumption of refined and processed foods due to high sugar content

use of steroids
immunosuppressed women

Diabetes mellitus

consumption of food that may trigger overgrowths such as bread and baked food (made with yeasts), or marmite.

Treatment:

Anti-fungal internal products that contain
the active ingredient clotrimazole or fluconazole.

Oral pill, soft gel pessary, pessary or cream.

Question 28

Identification of Fungi

Most fungi are relatively large

Their cell walls are rich in polysa……….

Polysaccharides can be converted by o…….. to dialde……… and detected by Schiff’s reagent or hexamine-silver stains.

Fungi are weakly haema………….

Special stains are mainly used

Fluorochrome-labelled specific antibodies are available, but not widely used on fixed tissues

Answer 28

Identification of Fungi

Most fungi are relatively large

Their cell walls are rich in polysaccharides

Polysaccharides can be converted by oxidation to dialdehydes and detected by Schiff’s reagent or hexamine-silver stains.

Fungi are weakly haematoxyphilic

Special stains are mainly used

Fluorochrome-labelled specific antibodies are available, but not widely used on fixed tissues

Question 29

Identification of Fungi

Some fungi may elicit a range of host reactions from exudative, necrotizing to granulomatous whereas other fungi produce little cellular response to indicate their presence. Fortunately, most fungi are relatively large, and their cell walls are rich in poly………. which can be converted by ………… to dialdehydes and thus detected with ………… reagent or hexamine-silver stains. Fungi are often weakly haem………..and can be suspected on H&E stains.

Fluorochrome-labelled specific antibodies to many fungi are available, and are in use in mycology laboratories for the identification of fungi on fresh and pa……….. wax sections. These antibodies have not found widespread use on fixed t…….., where identification still relies primarily on traditional staining methods.

Answer 29

Identification of Fungi

Some fungi may elicit a range of host reactions from exudative, necrotizing to granulomatous whereas other fungi produce little cellular response to indicate their presence. Fortunately, most fungi are relatively large, and their cell walls are rich in polysaccharides which can be converted by oxidation to dialdehydes and thus detected with Schiff’s reagent or hexamine-silver stains. Fungi are often weakly haematoxyphilic and can be suspected on H&E stains.

Fluorochrome-labelled specific antibodies to many fungi are available, and are in use in mycology laboratories for the identification of fungi on fresh and paraffin wax sections. These antibodies have not found widespread use on fixed tissue, where identification still relies primarily on traditional staining methods.

Question 30

Infectious organisms – fungus (candida)

An H&E stain, a Grocott methenamine (hexamine)-silver (GMS), a m………. unstai……………. section to look for pig………… and a good colour atlas when experience fails, permit most fungal infections to be identified sufficiently for diagnoses.
However, there is no sub………. for microbiological culture.

Answer 30

Infectious organisms – fungus (candida)

An H&E stain, a Grocott methenamine (hexamine)-silver (GMS), a mounted unstained section to look for pigmentation and a good colour atlas when experience fails, permit most fungal infections to be identified sufficiently for diagnoses.
However, there is no substitute for microbiological culture.

Question 31

Infectious organisms – fungus (candida)

An H&E stain, a Grocott methenamine (hexamine)-silver (GMS), a m………. unstai……………. section to look for pig………… and a good colour atlas when experience fails, permit most fungal infections to be identified sufficiently for diagnoses.
However, there is …. substitute for microbiological culture.

Answer 31

Infectious organisms – fungus (candida)

An H&E stain, a Grocott methenamine (hexamine)-silver (GMS), a mounted unstained section to look for pigmentation and a good colour atlas when experience fails, permit most fungal infections to be identified sufficiently for diagnoses.
However, there is no substitute for microbiological culture.

Question 32

Barrett’s oesophagus

Barrett’s oesophagus, or columnar lined oesophagus, is an acquired condition that results from chronic gastro-o. ……… It is characterised by the metaplastic replacement of the normal …….. epithelium of the lower oesophagus by …………. epithelium.
The diagnosis of Barrett’s oesophagus is made on en., bu……….t it has to be confirmed by the histological examination of biopsies, which show the characteristic incomplete intestinal ………….. (also called “specialised” mucosa).

Answer 32

Barrett’s oesophagus

Barrett’s oesophagus, or columnar lined oesophagus, is an acquired condition that results from chronic gastro-oesophageal reflux. It is characterised by the metaplastic replacement of the normal squamous epithelium of the lower oesophagus by columnar epithelium.
The diagnosis of Barrett’s oesophagus is made on endoscopy, but it has to be confirmed by the histological examination of biopsies, which show the characteristic incomplete intestinal metaplasia (also called “specialised” mucosa).

Question 33

Barrett’s oesophagus

Glandular m………. in the lower oesophagus presents as a red velvety mucosa over the gastro-oesophageal j……….

Serious complication of ………..
Pre-can…………. disease

Answer 33

Barrett’s oesophagus

Glandular mucosa in the lower oesophagus presents as a red velvety mucosa over the gastro-oesophageal junction.

Serious complication of GERD
Pre-cancerous disease

Question 34

Barrett’s oesophagus

Glandular mucosa in the lower oesophagus presents as a red velvety ……….. over the gastro-oesophageal junction. It can extend either circumf. ……… or as one or several tong………, and in some cases as a mixture of these two patterns.

Until recently, it was considered that this mucosa had to extend at least 30 mm over the gastro-oesophageal junction to diagnose Barrett’s oesophagus. But this definition has changed, owing to the recognition of short segment Barrett’s oesophagus measuring less than……… mm.

However, as it may be difficult to measure precisely a short segment Barrett’s oesophagus and to localise the me……… mucosa and the gastro-oesophageal ju……, it is now well admitted that the major diagnostic criteria of Barrett’s oesophagus is histological. The significance of intestinal meta……. discovered on biopsies taken from an end…….. normal junction (sometimes considered as an “ultrashort” Barrett’s oesophagus) remains controversial, and will not be discussed in this text.

Answer 34

Barrett’s oesophagus
Glandular mucosa in the lower oesophagus presents as a red velvety mucosa over the gastro-oesophageal junction. It can extend either circumferentially or as one or several tongues, and in some cases as a mixture of these two patterns.

Until recently, it was considered that this mucosa had to extend at least 30 mm over the gastro-oesophageal junction to diagnose Barrett’s oesophagus. But this definition has changed, owing to the recognition of short segment Barrett’s oesophagus measuring less than 30 mm.

However, as it may be difficult to measure precisely a short segment Barrett’s oesophagus and to localise the metaplastic mucosa and the gastro-oesophageal junction, it is now well admitted that the major diagnostic criteria of Barrett’s oesophagus is histological. The significance of intestinal metaplasia discovered on biopsies taken from an endoscopically normal junction (sometimes considered as an “ultrashort” Barrett’s oesophagus) remains controversial, and will not be discussed in this text.

Question 35

Intestinal metaplasia of gastroesophageal junction.H&E stain

Intestinal metaplasia of the oesophagus, the s……….. epithelium, is the diagnostic feature of Barrett’s oesophagus when it is located in the oesophagus and not in the upper part of the stomach. This mucosa is considered an incomplete form of int………. metaplasia, similar to type II and type III intestinal metaplasia in the stomach. Morphologically, it frequently shows a vill……… pattern. The epithelium is composed mainly of g…….. cells interspersed between intermediate m…….. cells, both in the surface and gla……. epithelium. Mature absorptive intestinal cells with a well defined brush border are rare. Pa………. .cells may be present, but they are as rare as in incomplete intestinal metaplasia of the gastric mucosa. E………….. cells can be seen on special staining in the glands. On electron microscopy, the goblet cells have characteristic apical mucin granules, and the columnar mucin cells have features intermediate between gastric m……….. cells and intestinal absorptive cells

Answer 35

Intestinal metaplasia of gastroesophageal junction.H&E stain

Intestinal metaplasia of the oesophagus, the specialised epithelium, is the diagnostic feature of Barrett’s oesophagus when it is located in the oesophagus and not in the upper part of the stomach. This mucosa is considered an incomplete form of intestinal metaplasia, similar to type II and type III intestinal metaplasia in the stomach. Morphologically, it frequently shows a villiform pattern. The epithelium is composed mainly of goblet cells interspersed between intermediate mucous cells, both in the surface and glandular epithelium. Mature absorptive intestinal cells with a well defined brush border are rare. Paneth cells may be present, but they are as rare as in incomplete intestinal metaplasia of the gastric mucosa. Endocrine cells can be seen on special staining in the glands. On electron microscopy, the goblet cells have characteristic apical mucin granules, and the columnar mucin cells have features intermediate between gastric mucous cells and intestinal absorptive cells

Question 36

Mucin stain of Barrett’s mucosa using AB / Pas methods

AB/PAS e.g. Barrett’s oesophagus

The routine alcain blue stain performed at pH ……and periodic acid-Schiff stain distinguishes between the i………… acid mucins (blue) and the ne….. mucins of gastric type.

Tissues and cells which contain both neutral and acidic mu……… will stain varying sh……… of purple due to the binding of alcian blue and the reactivity with Schiff reagent. This is seen in the go………. cells of the small intestine which contain ne………. and sialo………

Answer 36

Mucin stain of Barrett’s mucosa using AB / Pas methods

AB/PAS e.g. Barrett’s oesophagus

The routine alcain blue stain performed at pH 2.5 and periodic acid-Schiff stain distinguishes between the intestinal acid mucins (blue) and the neutral mucins of gastric type.

Tissues and cells which contain both neutral and acidic mucins will stain varying shades of purple due to the binding of alcian blue and the reactivity with Schiff reagent. This is seen in the goblet cells of the small intestine which contain neutral and sialomucins.

Question 37

AB/PAS Barrett’s Oesophagus

The goblet cells - Alcian blue (AB) positive - acid mucin. The cells between the goblet cells are Periodic Acid-Schiff (PAS) positive -neutral mucin.

Incomplete forms are more hetero…………, characterized by goblet and mu………. col………. cells but no abs…………. cells and variable patterns of mu……… Complete intestinal meta………. is characterized by the presence of ……… cells, Pa…… cells and absorptive cells with brush borders and variably developed intestinal villi.

Answer 37

AB/PAS Barrett’s Oesophagus

The goblet cells - Alcian blue (AB) positive - acid mucin. The cells between the goblet cells are Periodic Acid-Schiff (PAS) positive -neutral mucin.

Incomplete forms are more heterogeneous, characterized by go…….. and m……… columnar cells but no absorptive cells and variable patterns of mucin. Complete intestinal metaplasia is characterized by the presence of goblet cells, Paneth cells and absorptive cells with brush borders and variably developed intestinal villi.

Question 38

Formalin

A formalin-fixed par……… section of kid……… showing the typical dep………… of acid formal…….. hematin (formalin pigment) associated with red blood cells. The pigment is brown to black in colour and is birefrin…… under pola…….. light. In this case the specimen remained in fixative for an extended period before processing.

Answer 38

Formalin

A formalin-fixed paraffin section of kidney showing the typical deposition of acid formaldehyde hematin (formalin pigment) associated with red blood cells. The pigment is brown to black in colour and is birefringent under polarized light. In this case the specimen remained in fixative for an extended period before processing.

Question 39

Lipofuscin

Section of a liver showing lipo……… pigment. It is a yellow-brown, autofluorescent, granular substance composed of pero……….. protein and lipids. It has to be distinguished from the b….. pigment, as both appear similar on H&E-stained sections, but bile pigments are not autofl………. and fail to rotate the plane of polarized light. Bile pigments can be demostrated by Fo………technique.

Answer 39

Lipofuscin

Section of a liver showing lipofuscin pigment. It is a yellow-brown, autofluorescent, granular substance composed of peroxidized protein and lipids. It has to be distinguished from the bile pigment, as both appear similar on H&E-stained sections, but bile pigments are not autofluorescent and fail to rotate the plane of polarized light. Bile pigments can be demostrated by Fouchet technique.

Question 40

Lipofuscin

Lipofuscin is believed to represent cellular waste products.

Lipofuscin is a yellow-brown, autofluorescent, granular substance composed of pero….. protein and lipids. It is seen in larger neuro….., such as the lower motor ne…….. of t he spinal . and pyra….. o. cells of the hippocampus in Alzheimer pathology.

The need to identify bile pigments arises mainly in the histological examination of the liver, where distinguishing ….. pigment from lipofuscin may be of significant importance. Both appear yellow-brown in H&E-stained p…….. wax sections, and it is worth remembering that the green colour of biliverdin is often masked by eo………. In such cases, unstained paraffin wax or frozen sections, lightly counterstained with a suitable haematoxylin (e.g. Mayer’s), will prove of value. Bile pigments are not autofluorescent and fail to rotate the plane of polarized light (monorefringent), whereas lipofuscin is autofl……….. The most commonly used routine method for the demonstration of bile pigments is the modified Fouchet technique.

Answer 40

Lipofuscin

Lipofuscin is believed to represent cellular waste products.

..Lipofuscin is a yellow-brown, autofluorescent, granular substance composed of peroxidized protein and lipids. It is seen in larger neurons, such as the lower motor neurons of the spinal cord and pyramidal cells of the hippocampus in Alzheimer pathology.

The need to identify bile pigments arises mainly in the histological examination of the liver, where distinguishing bile pigment from lipofuscin may be of significant importance. Both appear yellow-brown in H&E-stained paraffin wax sections, and it is worth remembering that the green colour of biliverdin is often masked by eosin. In such cases, unstained paraffin wax or frozen sections, lightly counterstained with a suitable haematoxylin (e.g. Mayer’s), will prove of value. Bile pigments are not autofluorescent and fail to rotate the plane of polarized light (monorefringent), whereas lipofuscin is autofluorescent. The most commonly used routine method for the demonstration of bile pigments is the modified Fouchet technique.

Question 41

Haemosiderin

Section of the spleen with haem……… pigments, which are seen as yellow to brown gra……. and normally appear intrace……… They contain iron in the form of ferric hy……. which is bound to a protein fra………. and is unmasked by various chemicals.

Answer 41

Haemosiderin

Section of the spleen with haemosiderin pigments, which are seen as yellow to brown granules and normally appear intracellularly. They contain iron in the form of ferric hydroxide which is bound to a protein framework and is unmasked by various chemicals.

Question 42

Hemosiderin

These pigments are seen as yellow to brown granules and normally appear intracellularly.

They contain iron in the form of ferric hydr…….. which is bound to a protein framework and is unmasked by various chemicals. Iron is a vital component of the human body as it is an essential constituent of the oxygen-carrying h………………. found in red blood cells, where 60% of the body’s total iron content resides. It also occurs in m……… and certain enzymes, such as cyto……….. oxid….. and the peroxidases.

Answer 42

These pigments are seen as yellow to brown granules and normally appear intracellularly.
They contain iron in the form of ferric hydroxide which is bound to a protein framework and is unmasked by various chemicals. Iron is a vital component of the human body as it is an essential constituent of the oxygen-carrying haemoglobin found in red blood cells, where 60% of the body’s total iron content resides. It also occurs in myoglobin and certain enzymes, such as cytochrome oxidase and the peroxidases.

Question 43

Normal and abnormal iron metabolism

Bone Marrow normally contains ~30% stored within the reticul………….. system.

Bone marrow is the main site of i……. utilization.
Under normal conditions system of recycling of iron is efficient.

The small intestine normally only absorbs suff……… iron from the excess iron in the diet to counteract any losses.
In excessive blood loss the dietary content may be relatively inad…… for the need.

In iron defic………. will be observed by the ab……… of stainable i…. in the bone marrow.

Answer 43

Normal and abnormal iron metabolism

Bone Marrow normally contains ~30% stored within the reticuloendothelial system.
Bone marrow is the main site of iron utilization.
Under normal conditions system of recycling of iron is efficient.
The small intestine normally only absorbs sufficient iron from the excess iron in the diet to counteract any losses.
In excessive blood loss the dietary content may be relatively inadequate for the need.
In iron deficiency will be observed by the absence of stainable iron in the bone marrow.

Question 44

Normal and abnormal iron metabolism

The main reason for loss of iron from the body is haem…….e in the form of either ch……… bleeding (e.g. a peptic ulcer or bowel neoplasia), or in the female by men………., with approximately 25% of females being iron deficient.

The small intestine normally only absorbs sufficient iron from the excess iron in the diet to counteract any losses, but in ex……….. blood loss the dietary content may be relatively inadequate for the need, and even though the absorption mechanism is working at full efficiency, a state of clinical iron deficiency occurs.

In iron defic…….., the …….. .stores in the bone marrow become depleted, insufficient haemoglobin is produced because of the lack of iron, and a………. develops in which the r…….. cells contain diminished amounts of haem………… The iron deficiency is charact………. demonstrated by the absence of stai.. iron in the bone marrow.

Answer 44

Normal and abnormal iron metabolism

The main reason for loss of iron from the body is haemorrhage in the form of either chronic bleeding (e.g. a peptic ulcer or bowel neoplasia), or in the female by menstruation, with approximately 25% of females being iron deficient.

The small intestine normally only absorbs sufficient iron from the excess iron in the diet to counteract any losses, but in excessive blood loss the dietary content may be relatively inadequate for the need, and even though the absorption mechanism is working at full efficiency, a state of clinical iron deficiency occurs.

In iron deficiency, the iron stores in the bone marrow become depleted, insufficient haemoglobin is produced because of the lack of iron, and anaemia develops in which the red cells contain diminished amounts of haemoglobin. The iron deficiency is characteristically demonstrated by the absence of stainable iron in the bone marrow.

Question 45

Normal and abnormal iron metabolism

There is …… active method of iron excretion from the body.

Iron excess is a much……. common condition.

The intestine will not absorb ……. from the diet when there is already a . ……. within the body.

Liver is commonly seen first to accumulate an ………amount of he………… with increase iron absorption and deposition.

Hemosiderosis – common term to describe accumulation of hemosiderin, which can be a result of disorder or event.

Hemochromatosis - genetic disease.

Answer 45

Normal and abnormal iron metabolism

There is no active method of iron excretion from the body.

Iron excess is a much less common condition.

The intestine will not absorb iron from the diet when there is already a surplus within the body.

Liver is commonly seen first to accumulate an excessive amount of hemosiderin with increase iron absorption and deposition.

Hemosiderosis – common term to describe accumulation of hemosiderin, which can be a result of disorder or event.
Hemochromatosis - genetic disease.

Question 46

Normal and abnormal iron metabolism

There is no active method of iron excretion from the body.

Iron excess is a much less common condition; under normal conditions the intestine will not absorb iron from the diet when there is already a surplus within the body.

However, this controlling mechanism may be bypassed when iron is given thera………, such as in the form of either iron injections or a blood transfusion. If excess iron is given this way, the iron stores may become overloaded, and excessive amounts of hem………. may be deposited in the organs with a ………..prominent reticulo. component e.g. spleen, bone marrow or liver.

This condition is called hemosi……….. A rarer cause of iron overload is the genetic disease hemochromatosis.

Answer 46

Normal and abnormal iron metabolism

There is no active method of iron excretion from the body.

Iron excess is a much less common condition; under normal conditions the intestine will not absorb iron from the diet when there is already a surplus within the body.

However, this controlling mechanism may be bypassed when iron is given therapeutically, such as in the form of either iron injections or a blood transfusion. If excess iron is given this way, the iron stores may become overloaded, and excessive amounts of hemosiderin may be deposited in the organs with a prominent reticuloendothelial component e.g. spleen, bone marrow or liver.

This condition is called hemosiderosis. A rarer cause of iron overload is the genetic disease hemochromatosis.

Question 47

hemochromatosis

Iron disorder in which the body simply loads too much iron; if left untreated, can damage joints, organs, and eventually be fatal

There are several types of hemochromatosis: type….., also called Classic
Hemochromatosis (HHC), is more common

Genetic disease: mutations in the HFE gene and the excess iron

Affects he……, liver, pancreas, joints, pituitary gland, testis, etc

Most common symptoms: chronic fatigue and joint pain

Can cause: diabetes mellitus, irregular heart beat or heart attack, arthritis, cirrhosis or liver cancer, gall bladder disease, depression, impotence and infertility, skin disorder (bronze, reddish or ashen-gray colour), hypothyroidism, enlarged spleen

Answer 47

hemochromatosis

Iron disorder in which the body simply loads too much iron; if left untreated, can damage joints, organs, and eventually be fatal
There are several types of hemochromatosis: type 1, also called Classic Hemochromatosis (HHC), is more common
Genetic disease: mutations in the HFE gene and the excess iron
Affects heart, liver, pancreas, joints, pituitary gland, testis, etc
Most common symptoms: chronic fatigue and joint pain
Can cause: diabetes mellitus, irregular heart beat or heart attack, arthritis, cirrhosis or liver cancer, gall bladder disease, depression, impotence and infertility, skin disorder (bronze, reddish or ashen-gray colour), hypothyroidism, enlarged spleen

Question 48

Br……… pigmentation is markedly seen in the white skin in hemochr…………. compared to normal white skin (outer two hands).

Answer 48

Brown pigmentation is markedly seen in the white skin in hemochromatosis compared to normal white skin (outer two hands).

Question 49

Demonstration of haemosiderin and iron

In unfixed tissue, hemosiderin is ins…….. in al…….. but freely soluble in strong a……….solutions.

After fixation in formalin, it is slowly soluble in ……… acids, especially oxalic acid.

In tissues certain type of iron is tightly bound within a protein complex. Both hae. and……… myo……….. are examples of such protein complexes and, if treated with hydrogen peroxide, the iron is released and can then be demonstrated using P………. Prussian blue reaction.

Metallic iron d………, or inert iron oxide seen in tissues because of in…….. .exposure, are not positive when treated with …….. ferro…….. solutions.

In almost all the instances where demonstrable iron appears in tissues, it does so in the form of a ferric salt.

Answer 49

Demonstration of haemosiderin and iron
In unfixed tissue, hemosiderin is insoluble in alkalis but freely soluble in strong acid solutions.

After fixation in formalin, it is slowly soluble in dilute acids, especially oxalic acid.

In tissues certain type of iron is tightly bound within a protein complex. Both haemoglobin and myoglobin are examples of such protein complexes and, if treated with hydrogen peroxide, the iron is released and can then be demonstrated using Perls’ Prussian blue reaction.

Metallic iron deposits, or inert iron oxide seen in tissues because of industrial exposure, are not positive when treated with acid ferrocyanide solutions.

In almost all the instances where demonstrable iron appears in tissues, it does so in the form of a ferric salt.

Question 50

Demonstration of haemosiderin and iron

In unfixed tissue, hemosiderin is insoluble in alkalis but freely soluble in strong acid solutions.

After fixation in formalin, it is slowly soluble in dilute acids, especially oxalic acid. Fixatives which contain acids but no formalin can remove hemosiderin or alter it in such a way that reactions for iron are negative.

Certain types of iron found in tissues are not demonstrable using traditional techniques. This is because the ir….. is tightly bound within a protein comp…….lex. Both haem. and myoglobin are examples of such protein complexes and, if…….. treated with hydr……. pe., the iron is released and can then be demonstrated using Perls’ Prussian blue reaction.

Answer 50

Demonstration of haemosiderin and iron

In unfixed tissue, hemosiderin is insoluble in alkalis but freely soluble in strong acid solutions.
After fixation in formalin, it is slowly soluble in dilute acids, especially oxalic acid. Fixatives which contain acids but no formalin can remove hemosiderin or alter it in such a way that reactions for iron are negative.
Certain types of iron found in tissues are not demonstrable using traditional techniques. This is because the iron is tightly bound within a protein complex. Both haemoglobin and myoglobin are examples of such protein complexes and, if treated with hydrogen peroxide, the iron is released and can then be demonstrated using Perls’ Prussian blue reaction.

Question 51

Demonstration of hemosiderin and iron

Metallic iron deposits, or inert iron oxide seen in tissues because of industrial exposure, are not po……. when treated with acid ferro……….. solutions.

As a consequence of the tissue response, various mechanisms release some of the iron in a demonstrable form, and such deposits are almost invariably surrounded by he……………

In almost all the instances where demonstrable iron appears in tissues, it does so in the form of a ……….. salt. On those rare occasions when iron appears in its reduced state as the f……… salt, then Lillie’s method may be used to achieve the Turnbull’s blue reaction to v………. its presence in tissue sections.

Answer 51

Demonstration of hemosiderin and iron

Metallic iron deposits, or inert iron oxide seen in tissues because of industrial exposure, are not positive when treated with acid ferrocyanide solutions.

As a consequence of the tissue response, various mechanisms release some of the iron in a demonstrable form, and such deposits are almost invariably surrounded by hemosiderin.

In almost all the instances where demonstrable iron appears in tissues, it does so in the form of a ferric salt. On those rare occasions when iron appears in its reduced state as the ferrous salt, then Lillie’s method may be used to achieve the Turnbull’s blue reaction to visualize its presence in tissue sections.

Question 52

Pigment: Perls’ Prussian Blue Iron

Perls’ for Fe3+ (liver)
Free and in ma………..

Perls’ Prussian Blue Iron (liver).
This stain is used to detect and identify ferric (Fe3+) iron in t……. p…………, blood smears,or bone marrow smears. Minute amounts of ferric ir…… (haemosiderin) are commonly found in ……….. ………. and in the spleen. Abnormal amounts of iron can indica………… hemochro……….. and hemo..
……..

Answer 52

Pigment: Perls’ Prussian Blue Iron

Perls’ for Fe3+ (liver)
Free and in macrophages

Perls’ Prussian Blue Iron (liver).
This stain is used to detect and identify ferric (Fe3+) iron in tissue preparations, blood smears,or bone marrow smears. Minute amounts of ferric iron (haemosiderin) are commonly found in bone marrow and in the spleen. Abnormal amounts of iron can indicate hemochromatosis and hemosiderosis.

Question 53

Haemochromatosis - liver

Long-standing hemochromatosis often results in significant hem……….. deposition in hepatocytes and increased risk of hepatocarc…………. Hemosiderin can be distinguished from ….. by po………. iron stains.

In hemochromatosis, non-neoplastic hepatocytes may show marked hemosiderin deposition (granular golden brown cytoplasmic pigment), which can be distin…….. from bile by po…….. ir…….staining. Note the reactive nuclear atypia (enlargement, prominent nucleoli) in these non-neoplastic hep…………

Answer 53

Haemochromatosis - liver

Long-standing hemochromatosis often results in significant hemosiderin deposition in hepatocytes and increased risk of hepatocarcinogensis. Hemosiderin can be distinguished from bile by positive iron stains.

In hemochromatosis, non-neoplastic hepatocytes may show marked hemosiderin deposition (granular golden brown cytoplasmic pigment), which can be distinguished from bile by positive iron staining. Note the reactive nuclear atypia (enlargement, prominent nucleoli) in these non-neoplastic hepatocytes.