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Histology2 > connective tissue > Flashcards

connective tissue Flashcards

1
Q

what are the 4 basic tissue types

A

connective, epithelial, muscular & nervous

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2
Q

what is connective tissue

A

consists of cellular portion surrounded by non cellular ground substance

provides support & connects tissues

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3
Q

cells of connective tissue

A

fibroblasts
mast cells
histocytes
adipose tissue
reticular cells
osteoblasts & osteoclasts
chrondroblasts & chrondrocytes
blood cells

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4
Q

fibroblasts

A

secrete extracellular matrix components
- usually collagen & elastin

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5
Q

macrophages

A

tissue phagocytes
aka histocytes
tissue monocytes

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6
Q

reticular fibers

A

delicate fibers
framework fro more cellular organs ( Lymph nodes, spleen, liver)
indistinct in H&E :(
weakly birefringent

use special stains
- PAS(reticulin is carbohydrate rich )
- Argyrophil silver stain ( needs extraneous reducer) q

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7
Q

cartilage vs bone

A

cartilage lacunae are much larger

cartilage typically appears blue or light purple sue to neg charge (H&E)

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8
Q

fibrous structures (3)

A

collagen ( strength )
- muscles

elastin ( flexibility )
- skin/blood vessels

reticulin ( support mesh)
- around cellular organs ( Lymph nodes, spleen etc)

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9
Q

most abundant protein in the human body and where its found

A

collagen

stength to structures

found mostly in tendons, ligaments, skin ( fibrous tissues)
in smooth muscle, blood vessels, heart & gall bladder

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10
Q

collagen

A

very eosinophilic
birefringent
strength *
may be
- dense regular ( tendons, capsules & skin )
- loose irregular ( gallbladder)

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11
Q

basement membrane

A

basal lamina

barrier between epithelium & connective tissue

stained with carbohydrate techniques(PAS) or silver stains

JMS

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12
Q

Muscle ( 3 types)

A

skeletal
- nuclei pushed to periphery
- striated, involuntary

cardiac
- central nuclei & intercalated discs
- striated involuntary

smooth
- non striated, voluntary

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13
Q

elastin

A

key protein of extracellular matrix & main component of elastic fibers

highly flexible ( Balloon )

ex. blood vessels, skin

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14
Q

fibrin

A

most commonly seen after tissue damage

part of acute inflammation
involved in clotting

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15
Q

fibrinoid

A

acellular homogenous material
similar to fibrin ( but in different disorders)

found normally in placenta

formed in connective tissue or walls of blood vessels in some diseases

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16
Q

connective tissue stains are used to asses

A

Replacement of normal tissue with
connective tissue
Tumors
Basement membranes
Elastic or reticulin fiber
Fibrin or fibrinoid
Muscle
Connective tissue cells

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17
Q

types of connective tissue stains

A

Trichrome stains
Reticulin stains
Elastin stains
Basement membrane stains
Fat stains
Metachromatic stains for mast cells
Methyl green-pyronin Y

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18
Q

trichrome stains

A

differential staining

demostrate muscle & collagen differentiation
- possibly fibrin & erythrocytes

3 dyes as a minimum ( one must be a nuclear stain )

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19
Q

in trichrome stains when is an iron mordant used

A

when using Hematoxylin because trichrome stains have acidic components

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20
Q

Weigert -van Gieson Stain

A

Trichrome stain ( not a top choice )
- different results than usual

nuclei = black ( stained with iron hematoxylin )

cytoplasm/ muscle = yellow
- stained with picric acid ( small dye molecule )

collagen = red
- stained with acid fuschion ( larg dye molecule)

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21
Q

trichrome stain - usually

A

2 or more anionic dyes in conjunction with a heteropolyacid which result in selective coloring of collagen by one of the dyes

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22
Q

Heteropolyacids

A

Phosphomolybdic acid (PMA) &/or
Phosphotungstic acid (PTA)

  • able to bind tissues from aqueous or alcoholic solutions
  • colorless anionic dyes( may be demonstrated using UV or stannous chloride ) ( molybdenum or tungesten blue )
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23
Q

how PTA or PMA work

A

bind protiens & amino acids but NOT carbohydrates

COLLAGEN binds lots
cytoplasm binds smaller amounts

nuclei has little affinity ( doesn’t interfere with nuclear staining )

small dye is more easily replaced with PTA or PMA in collagen due to large pore size of collagen
cytoplasm & muscle staining with large molecule dyes is suppressed bc the cell membrane has small pores

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24
Q

small molecular dyes

A

PMA & PTA - there is considerable suppression of staining in all tissue components with small molecule anionic dyes ( heteropolyacids kick little molecules out of the collagen and hold that place for big molecule dyes)

ex of small dyes
Picric acid
Martius yellow
Eosin
Orange G
Biebrich scarlet

  • will stain RBCs
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25
Q

big dye molecules

A

aniline blue
light green
acid fuchsin

  • too big to be absorbed into small pores
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26
Q

factors affecting trichrome staining

A
  • different proteins form a mesh of variable pore size
  • RBCs ( small)
  • muscle/ cytoplasm ( medium )
  • Collagen/ mucin ( large )
    smaller dye can enter but will be pushed out and replaced by bigger one

pH
- 1.5- 3 proteins are basic at this pH( require low pH to stain connecive tissue fibers)

Heat
- increases the staining rate & influences penetration of large dye particles

Pore size
- or accessibility of reactive groups with dye

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27
Q

nuclear stains

A

iron mordanted

resistant to low pH
- Weigerts
- Verhoeffs
- Celestine blue

acidic component = iron mordant
will be in combo with other stains in trichrome

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28
Q

fixation of trichrome stains

A

NBF id NOT optimal but better staining can be achieved by post mordanting in bouins solution ( picric acid ) or mercuric chloride or both

the longer a tissue is fixed in formalin the more staining is suppressed bc fixative takes up binding sites so less are available for stain

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29
Q

color of each component with trichrome staining

A

1.Small anionic dye is able to pass through all the pore sizes in the tissue
- Everything is small dye color ( yellow)

2.Heterpolyacid fits through collagen pores and replaces the small anionic dye.
- Cytoplasm remains the same, collagen and mucus appear unstained ( red)

3.Large molecule dye bonds with the heterpolyacid and stains collagen
- Cytoplasm red, collagen and mucus blue (blue/green)

30
Q

Masson Trichrome stain

A
  • most popular trichrome stain
  • used to differentiate collagen & smooth muscle tumors
  • identifies increases in collahem in diseases like cirrhosis
  • stains deposits of fibrin ( seen at site of injury )
31
Q

massons trichrome control & fixation

A

No control is needed

Tissues fixed long term in NBF will have poor staining ( occupies lots of binding sites )

Bouin’s is preferred ( contains picric acid)

32
Q

Masson’s trichrome steps

A

Following hydration, mordant in Bouin’s
1 hour at 56°C

Wash yellow colour out

Stain in Weigert’s working solution

Wash, then stain in small molecule dye – biebrich scarlet- acid fuchsin
All tissue elements are now RED

Rinse & apply heteropolyacid
Binding with collagen occurs

Red dye removed from collagen (appears unstained)

Stain in larger molecule dye- aniline blue or light green
Collagen is stained blue or green

DCM

33
Q

Masson’s Trichrome Results

A

nuclei - black ( iron mordanted hematoxylin)

cytoplasm, keratin, muscle, new fibrin - red ( small molecule dye)

Collagen, mucus & old fibrin deposits- blue ( analine blue) or green ( light green)

34
Q

liver Cirrhosis

A

Masson trichrome stain will be crowded with blue staining collagen

collagen takes over in liver cirrhosis

more fat than usual may also be seen

35
Q

Gomori’s one step Trichrome steps

A

trichrome stain

Hydrate
Mordant in Bouin’s
Stain with Weigert’s ( iron mordanted )
Stain with trichrome stain which contains (small molecule dye), PTA
(heteropolyacid) and aniline blue or light green (large molecule dye)

36
Q

Gomori’s results

A

nuclei- black
cytoplasm, keratin, muscle- red
Collagen & mucus - Blue

not best choice but most used for Muscle Biopsies *

37
Q

Van Gieson stain

A

trichrome stain

Stain contains acid fuchsin and picric acid

Usually used as a counterstain **

results:
Cytoplasm – Yellow
Collagen- Red
Nuclei – usually black
due to iron hematoxylin

38
Q

MSB stain ( Lendrum )
Martius Scarlet Blue

known for stianing…
dyes used

A

Trichrome stain

Reliable FIBRIN staining **

Small dye- Martius Yellow
Nuclei – Celestine blue or iron hematoxylin
Medium dye – Crystal ponceau
Phosphotungstic acid
Large dye - aniline blue

39
Q

MSB stain results

A

Nuclei – Blue or Black
Muscle – Paler Red
Collagen – Blue
Fibrin – Red
 RBCs – Yellow

40
Q

Elastic fiber stains are used to demonstrate

A

Used to demonstrate pathological changes in elastic fibers

Thinning or loss – arteriosclerotic

Breaks or splitting – other vascular disease

Used for ID or examination of tumour invasion

41
Q

Elastic fiber stains ( non specific )

A

Elastic fibers are stained but NOT specifically

H&E
HPS (hematoxylin phloxine saffron)
Congo Red
PAS

42
Q

Elastic fiber staining ( more specific)

A

Specific or more intense stains include:
Verhoeff’s ( most widely used)
Aldehyde-fuchsin ( we use in lab)
Resorsin Fuchsin
 Orcein (oldest)

43
Q

elastin staining mechanisms ( less specific stains )

A

Eosin, Phloxine, or Congo Red stain
through ***columbic reaction between elastin protein and acid dyes

PAS stains ***glycoproteins in the fibers

44
Q

elastin stain mechanisms ( more intense or specific staining )

A

Elastin fibers are ***cross-linked by disulfide bridges

Oxidation occurs by **permanganate in Aldehyde Fuchsin & Weigert’s Resorcin
Oxidation occurs by **iodine in Verhoeff’s

Oxidation in both stains produces sulfonic acid derivatives to be formed.

These sulfonic derivatives are strongly basophilic and this may account for staining of cytoplasm and other basic tissue components

Reactions are enhanced by high electrolyte concentrations in stain solutions which inhibit uptake by chromatin, RNA etc. ( suppresses background staining)

45
Q

Verhoeff Elastic Stain colors of elastin

A

specific stain but not top choice

Some of the iron in the staining solution oxidizes the Hematoxylin, some oxidizes the elastin

Verhoeff’s imparts black color to elastin, nuclei, also myelinated nerve fibers

46
Q

Verhoeffs elastic stain 3 stock solutions

A

Lugol Iodine (iodine, potassium iodide, H2O)
- iodine is mordant & oxidizers )

10 % ferric chloride
- mordant differentiator; Regressive Stain**

Alcoholic hematoxylin

47
Q

Differentiation in Verhoeffs
- iodine remover & counterstain

A

Ferric chloride is used to remove colour until the elastic fibers are distinct (mordant differentiation)

Sodium thiosulfate is used to remove the iodine

Counterstain in van Gieson or other

48
Q

Aldehyde Fuchsin elastic stain steps

A

elastic fiber stain

Aldehyde fuchsin is made from alcoholic pararosaniline( AKA Basic fuchsin) , HCL & paraldehyde (or acetaldehyde)

Deparaffinize to 70% alcohol ( NOT TO H2O bc stain is alcoholic)
Stain, check microscopically.

Some methods are first oxidized with **permanganate &
bleached with ***oxalic acid

If not stained intensely enough return to stain, if too much, decolorize with alcohol

Counterstain with light Green or other

49
Q

Movat Pentachrome Russel mod

A

A combination of staining techniques are employed in one method
Stains mucin, fibrin, elastic fibers, muscle and collagen

50
Q

Steps of Movat pentachrome russell mod.

A

elastic fiber stain

Acid mucins are stained first
Hydrate
Stain in Alcian Blue
Place slides in alkaline alcohol
Converts alcian to monastral fast blue: insoluble

Stain nuclei and elastin
Stain in iron Hematoxylin (Verhoeff)
Differentiate with ferric chloride
Remove iodine with sodium thiosulfate

Stain muscle, fibrin and collagen
Stain in crocien scarlet- acid fuchsin
- Small molecule dye
Phosphotungstic acid
- Removes red dye from collagen, fibrin, fibrinoid
Stain with safran (saffron)
- Stains collagen yellow

51
Q

Movat pentachrome russell mod.Results

A

Nuclei & Elastin…………….Black
Collagen…………………….Yellow
Ground substance & mucin….Blue
Fibrinoid, fibrin………Intense Red
Muscle………………………….Red

52
Q

Metallic impregnation

A

No dyes are employed

Metallic ions are deposited on fibers and cells as an alternate method of providing contrast

Solutions such as **ammoniacal silver are reduced by certain tissue elements

53
Q

metal empolyed by metallic impregnantion

A

Silver *** usually silver
Gold
Osmium
Mercury chromate( toxic)
Palladium
Lead
Copper

54
Q

uses if metallic reduction

A

Reticular fibers
Bacteria (spirochetes)
Fungi
Neuropathology**
Detection of:
- Aldehydes
- Calcium
- Metal

55
Q

points to remeber with matallic impregannation

A

chemically clean glassware!
- can reduce silver = false positive

solutions shouldn’t contact metal surfaces
- use gloved hand not forceps

silver nitrate blackens skin & clothing

silver solutions must be neutralized before discard ( becomes explosive over time if not neutralized)
- NaCL can be used ( becomes cloudy)
- HCl can be used (dilute)

sections float off due to alkalinity of silver solution
- histogrip or chemical waterbath prevents this

store in a dark bottle in fridge

make solutions in fume hood

56
Q

oxidizer
sensitizer
bleach

A

Oxidizer – some methods require pre-oxidation
Example: **potassium permanganate produces aldehydes ( tuens section purple- follow with bleach)

Sensitizer – empirical enhancement of silver deposits ( sometimes used)

Bleach- Example: **oxalic acid following permanganate to remove purple colour

57
Q

stock solution
working solution
reducer

A

Stock solution- stable solution

Working solution- must be made fresh using stock solution

Reducer –reduces residual silver deposited at reaction sites
Example: formaldehyde or Hydroquinone ( extraneous reducer)

58
Q

toner
fixer

A

Toner – silver may not provide enough contrast, gold is added using gold chloride

Fixer – solution which removes unreduced silver. Sodium thiosulfate (hypo)

these two are always used

59
Q

Argyrophil & Argentaffin

A

ARGYROPHIL- metallic impregnation reactions which involve the use of an
extraneous reducer
-( Formaldehyde or hydroquininone)

ARGENTAFFIN – metallic impregnation which needs no extraneous reducer
- tissue is able to turn silver black

Tissue components have chemical characteristics which make them either
argyrophil or argentaffin. The reticulin staining method, Gordon and Sweet’s &
Gomori’s are an argyrophil techniques.

The extraneous reducer applied is
Formaldehyde.

Argyrophil methods will stain BOTH types of
tissue elements

60
Q

Gomori’s & Gordon & sweets steps

A

Hydrate to distilled (test and control)

Oxidize in potassium permanganate

Rinse
Bleach (oxalic acid) OR remove excess with potassium metabisulfite

Sensitize in ferric ammonium sulfate

Wash…wash… wash
Apply silver solution
Quick rinse*

Reduce with formaldehyde
Wash

Tone in Gold chloride
(increase contrast & permanence)

Rinse
Apply potassium metabisulfite (Gomori’s)

Fix in sodium thiosulfate

Wash
Counterstain if desired
DCM

61
Q

making the silver solution

A

This is usually the make or break part of silver impregnation
Involves silver nitrate and sodium or potassium hydroxide with ammonia
Preparation takes patience

62
Q

Mallory PTAH demonstrates

A

Used to demonstrate muscle cross striations, fibrin or glial fibers
Hematoxylin with a tungsten mordant
Zenker fixative is preferred
If Zenker is used what are the implications?

63
Q

Mallory PTAH steps

A

Oxidize in permanganate
Bleach in oxalic acid
Stain overnight
Dehydrate rapidly

64
Q

basement membrane stain

A

PAS-Methenamine Silver
Review method in book, note the use of microwave
Jones Methenamine Silver is more user
riendly

65
Q

JMS

A

Similar to Grocott methenamine silver GMS

Hexamine and methenamine refer to the same substance
HEXAMETHYLENETETRAMINE

This substance replaces the ammonia & other alkali

66
Q

lipid stain general points

A

Selective solubility stains

The dyes used are more soluble in lipid than the solvent they are in

Paraffin sections cannot be used

Alcohol & clearing agents remove lipids

Solvent is important

Isopropanol &/or propylene glycol

Dye must be strongly colored

67
Q

lipid stains

A

Oil Red O and Sudan black B are commonly used (not water soluble)
Control not required
Mashing the coverslip may remove dye from tissues

68
Q

uses of fat stains

A

Fat emboli (following bone fracture or crushing of fatty tissue)
Degenerating tissues
Identification of liposarcoma
Determine fat presence (stool, breast milk)

69
Q

osmium tetraoxide

A

Osmium the fixative, preserves fat and makes it turn black

If you see a paraffin section stained for fat this is the method

Formalin fixed tissue may be washed, then placed in Osmium

May be stained with any method ( fat is black before the staining)

Gross amounts of fat will not be fixed

Osmium has very limited penetration, face the block carefully

70
Q

toludine blue
methylene blue
methyl green-pyronin Y

A

Toluidine Blue

Methylene Blue stain
- Metachromatic staining can be used for the demonstration of mucins, cartilage, mast cells, etc..

Methyl Green- Pyronin Y for plasma cells

Histology2 (14 decks)

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