7.Histology of MSK system Flashcards
what is HISTOLOGY
EXAMINING TISSUES/CELLS using a MICROSCOPE
HISTOLOGY needs PREPARATION of the TISSUE. 4 steps:
- FIXATION
- EMBEDDING
- SECTIONING
- STAINING
Why do we need FIXATION (4)
- To PRESERVE tissue
- To PROTECT and FIX STRUCTURES
- To make INTRACELLULAR STRUCTURES ACCESSIBLE to reagents
(Immuno/histochemistry) - To STOP a PROCESS at a CERTAIN POINT in time
2 COMMON FIXATION METHODS
- CHEMICAL FIXATIVES such as formaldehyde, EtOH
(chemicals may alter tissue) - FREEZING
(structures less well-defined)
why do we need EMBEDDING of tissues (2)
- to SUPPORT the sample DURING SECTIONING
- to PRESERVE FRAGILE 3D STRUCTURES
3 COMMON EMBEDDING MATERIALS
- WAX (most common)
- SUCROSE solutions for cry-sectioning (frozen samples)
- Hard PLASTICS for HARD TISSUES eg bone, teeth
WHY do we SECTION tissues
to produce samples that are THIN enough
for the Microscope LIGHT to SHINE THROUGH
most common INSTRUMENT for SECTIONING is called
ROTARY MICROTOME
(can change thickness)
TYPES of KNIVES used:
steel (MOST)
diamaond,
glass
why is STAINING important
- normal tissues show very little contrast
staining needed to INCREASE CONTRAST
& DIFFERENTIATE between STRUCTURES with SIMILAR CONTRASTS
example of a general STAIN used
HAEMATOXYLIN/EOSIN STAIN (H&E)
H&E STAINS NUCLEI which colour
PURPLE
2 STAINS used for ELASTIC FIBRES (eg Dermis)
- MASSON’S TRICHROME (PURPLE)
- VERHOEFF’S STAIN
(PURPLE elastic fibres but other structures RED)
stain for Reticular Fibres (in bone marrow)
Collagen Type III STAIN
HISTOLOGY of SKELETAL MUSCLE
(nucleus, striations, intercalated borders, branching, size of fibre)
MULTI-NUCLEATED
nucleus is PERIPHERAL not central
STRIATED
LARGE Fibre size
no intercalated borders
no branching
HISTOLOGY of CARDIAC MUSCLE
(nucleus, striations, intercalated borders, branching, size of fibre)
ONE NUCLEUS
CENTRAL
STRIATED
INTERCALATED BORDERS (intercalated DISCS where attach to each other)
BRANCHING
LARGE SIZE
HISTOLOGY of SMOOTH MUSCLE (eg small intestine)
(nucleus, striations, intercalated borders, branching, size of fibre)
ONE NUCLEUS,
CENTRAL
SMALL SIZE
2 LAYERS - PERPENDICULAR
not striated,
no borders,
no branching
how are SMOOTH MUSCLE LAYERS in SMALL INTESTINE
(stain H&E)
2 LAYERS
fibres in each layer are PERPENDICULAR to each other
INNER LAYER: LONGITUDINAL
OUTER LAYER: CROSS SECTIONAL
STAINS used for SKELETAL MUSCLE (3 examples)
H&E (RED, PURPLE)
STAIN FOR ATPase ACTIVITY (shows type 1 and type 2) (brown)
SIRIUS RED STAIN - STAINS COLLAGEN in the ECM
- RED (rest is YELLOW)
what does the ATPase STAIN show in skeletal muscle
TYPE 1 - SLOW TWITCH & TYPE 2 - FAST TWITCH muscle
ATPase pH 9.4
(Colours switched at pH 4.3)
how is the NUCLEUS in SKELETAL MUSCLE
MULTI-NUCLEATED
PERIPHERAL
how is the NUCLEUS in SKELETAL MUSCLE
MULTI-NUCLEATED
PERIPHERAL
Which types of MUSCLE has 1 NUCLEUS per cell/fibre
CARDIAC
& SMOOTH
which muscle type has BRANCHING
CARDIAC
which muscle types have STRIATIONS
SKELETAL
CARDIAC
2 LAYERS OF SMOOTH MUSCLE:
(small intestine)
Inner layer: LONGITUDINAL
Outer layer: CROSS SECTIONAL
which muscle type has INTERCALATED BORDERS
CARDIAC
which muscle type has SMALL fibre size
SMOOTH
how are COLLAGEN FIBRES arranged in TENDONS & LIGAMENTS
LONGITUDINALLY
(tendon: parallel, ligament: cross-connecting)
COLLAGEN types in TENDONS & LIGAMENTS
TYPE 1
some TYPE 3 INCREASES AFTER INJURY
composition of ELASTIC CARTILAGE
HIGH CELL DENSITY
CHONDROCYTES (rounded appearance) in lacunae
collagaen fibres
elastic fibres
which STAIN allows ELASTIC FIBRES to be VISIBLE in ELASTIC CARTILAGE (opposed to H&E)
MASSON’S TRICHROME
what colour do ELASTIC FIBRES appear in elastic cartilage using MASSON’S TRICHROME STAIN
DARK PURPLE
CHONDROCYTES APPEARANCE:
ROUND
how is CELL DENSITY in FIBROCARTILAGE as opposed to in elastic cartilage
INTERMEDIATE CELL DENSITY
- NOT as closely packed as in elastic cartilage
how is CELL DENSITY in HYALINE CARTILAGE as opposed to elastic cartilage and fibrocartilage
LOWER CELL DENSITY
- space around cells
COLLAGEN in FIBROCARTILAGE
TYPE 1 & TYPE 2
what ‘mark’ do you find in ARTICULAR CARTILAGE
TIDE MARK
- BOUNDARY for CALCIFIED CARTILAGE
besides H&E, what other STAINS can be used for ARTICULAR CARTILAGE
(what colours)
SAFRANIN O
& FAST GREEN
(with Haematoxylin counterstain)
- stains GLYCOPROTEINS: ORANGE
- TYPE 1 COLLAGEN: GREEN
- cells: PURPLE
why is HISTOLOGY of bone DIFFICULT
bone is MINERALISED
2 options for HISTOLOGY of bone
- DEMINERALISE -> STANDARD WAX HISTOLOGY
- DON’T demineralise -> EMBED in HARD RESIN
ADVANTAGES and DISADVANTAGES in DEMINERALISING bone for HISTOLOGY (& using standard wax histology)
adv: ALL NORMAL STAINS AVAILABLE
disadv: INFORMATION on the bone MINERALISATION is LOST
ADVANTAGES and DISADVANTAGES in NOT DEMINERALISING & EMBEDDING IN HARD RESIN for HISTOLOGY
adv: MINERALISATION INFORMATION RETAINED
disadv:
-SPECIAL SECTIONING EQUIPMENT required
- NOT ALL stains work
how is CORTICAL BONE
DENSE,
MOSTLY mineralised bone matrix
can see OSTEOCYTES (mechanoreceptors)
OSTEONS (contain blood vessels)
Osteoblasts on edge
how is TRABECULAR BONE
OPEN STRUCTURE,
ABUNDANT BONE MARROW SPACE
Adipocytes, Osteoblasts on edge,
which bone cells are RARE to find and what are they like
OSTEOCLASTS
- LARGE
- MULTI-NUCLEATED
what can you EMBED UNDEMINERALISED (mineralised) bone in
Methyl-Methacrylate
STAIN you can use for MINERALISED / UNDEMINERALISED bone and what does it allow you to see
GOLDNERS TRICHROME STAIN
- can identify mineralised bone (GREEN)
- can see NEW BONE TISSUE that has NOT YET BEEN MINERALISED (ORANGE)
VAN KOSSA STAIN
stains mineralised bone:
stains osteoid (new bone, not yet mineralised):
mineralised: BLACK
new bone: PINK
Calcein Double Labelling allows you to see..
(in mineralised)
PROGESS of BONE GROWTH
(rate)
STAIN for OSTEOCLASTS
TRAP stain
(RED)
stain TRAP enzymes in osteoclasts
(mineralised bone BLUE)
