My Histological Techniques Notes

L1 Introduction

TERMS

TISSUES 组织

CLEARING 清除

SECTIONING 分段，切面

LOGGING 集材

INFILTRATION 浸润

FISHING 打捞

GROSSING = to examine and dissect the surgical specimen

IMPREGNATION 渗透

DRYING 烘干/风干

FIXATION 固定

EMBEDDING 嵌入

STAINING 染色

DECALCIFICATION 脱钙

ORIENTATION 

MOUNTING =preserve colored-section

DEHYDRATION 脱水

TRIMMING 修剪

LABELLING 标签

1. Logging集材 of specimens

• Laboratory receives specimens in the form of – autopsies, biopsies, whole organ.

• Small biopsies specimens are placed in fixative solution – to protect from drying.

• Large surgical specimens may arrive unfixed in plastic bags or saline moistened towels - kept in refrigerator (will slow down autolysis) - until examined by the pathologist.

• Mailing specimens MUST be fixed before mailing.

• MLT receives the specimen → record identification number in laboratory register → sketch of specimen (outline only - helpful in tracking).

• Transfer the specimens with identification tags / labels into small container with fresh fixative.

• Bone and calcified tissues need to be cut into small blocks with a saw prior to decalcification procedure.

2. Fixation固定组织，preservation

• Shortly after death or removal from the body, cells and tissue begin to undergo changes.

• Results in their breakdown and ultimate destruction. These are referred to as post- mortem changes, which may be either putrefactive or autolytic in nature.

• Basic of overall histological techniques

• The results of all subsequent procedures depend upon the correct selection and use of the fixative.

• It is therefore essential to understand the action which different fixatives have upon the cell and tissue constituents.

Aim: 

• To prevent the process of autolysis (self destruction) and putrefaction (bacterial attack).

• To be as close as possible to their living state → to allow them undergo further preparative procedures without changes.

• To retain shape or volume and preserve tissue substances / proteins.

Types: 

• Physical

 Heat fixation

 Freeze drying

• Chemical or ‘fixative’

3. Fixative

• Definition: A fixative may be described as a substance that will preserve the shape, structure, relationship and chemical constituents of tissues and cells after death.

• Do you know some of the commonly used fixative in medical labs?

Characteristics of a good fixative:

• It must kill the cell quickly and in so doing should produce minimum distortion.

• It must penetrate the tissues and cells rapidly and evenly.

• It must make the substances of cells insoluble and give good optical differentiation.

• It must inhibit bacterial decay and autolysis.

• It must harden the tissue and make it insensitive to subsequent treatment.

• It must permit, at a later date the application of numerous staining.

Types:

• Coagulant fixatives – coagulate proteins and make them insoluble.

• The most commonly used coagulant fixative is dehydrant coagulant fixative.

• E.g. alcohols such as ethanol, methanol and acetone.

• Example: Carnoy’s fluid (very rapid fixative).

• Acids such as picric acid and trichloroacetic acid are also coagulant fixative but they are less common.

• Example: Bouin’s solution (excellent for connective tissue stains)

• Cross-linking fixatives – forming cross-links whithin and between proteins and nucleic acids.

• Formaldehyde fixation

• Examples: neutral buffered formalin (NBF)(the most common fixative in diagnostic pathology), 10% formalin, Formal saline.

• The NBF is routinely preferred because it prevents the formation of formalin pigments.

• Other cross-linking fixatives

• Glutaraldehyde fixation – not widely used. Suitable for electron microscope studies.

• Osmium tetroxide fixation – toxic, soluble in water. Rarely used, usually for electron microscope studies.

• Mercuric chloride – It is toxic and no longer used routinely.

• Examples: Zenker’s solution & B5 (frequently used for bone marrow, lymph nodes, spleen).

Factors involved:

❑ pH

▪ pH suitable for fixation occurs between 6 and 8 - outside this range changes the ultrastructure of specimen - for some purposes - fixation at specific pH is chosen e.g. gastric mucosa at pH 5.5

❑ Temperature

▪ A low temperature will retard the fixation but will also reduce the autolytic action of enzymes released after death.

▪ a high temperature will decrease the time required in the fixation but will also increase autolysis.

▪ In cases where fixation is not possible until some time after death, storage at a low temperature (2-5°C) is essential.

❑ Size

▪ This process is relatively slow - blocks taken should be small or thin - large blocks of tissue such as uterus should be sliced thinly

❑ Volume ratio

▪ The fixative volume - at least 15 to 20 times greater than the tissue volume.

▪ More than recommended volume – no effect

▪ Less than recommended volume – problems occur, e.g. under fixed tissue (poor fixation)

❑ Duration of fixation

▪ Common practice allow primary fixation in buffered formalin for 4 - 8 hours during the day.

▪ There is evidence that prolonged fixation in aldehydes (e.g. formalin) can cause shrinkage and hardening of tissue and severe inhibition of enzyme activity.

Examples of several commonly used fixative solutions:

 10% formalin

Recommended for the fixation of central nervous system and general post-mortem tissue. The period required for fixation is more than 24 hrs.

 Composition:

➢ 40% formaldehyde 100ml

➢ sodium chloride 9g

➢ Dist. water 900ml

10% formalin (cont’d)

Advantages 

• Excellent for post-mortem samples

• Causes little shrinkage

• Indefinite period of sample storage

• Basis of all museum fixatives

• Restores the natural color of specimen

• Does not interfere with most staining procedures.

Disadvantages

• Slow fixative

• Tissue shrinkage during dehydration

• Irritant vapour injures the nasal mucosa

• Causes dermatitis with prolonged use

• Pigment is always formed when dealing with blood containing sample

• Needs periodic changes of new solution by 3 months.

Neutral Buffered Formalin (NBF):

Recommended for the preservation and storage of surgical, post-mortem and research specimens. The period of fixation is 24 hrs or longer.

 Constituents:

➢ Sodium dihydrogen phosphate  (anhydrous) 3.5g

➢ Disodium hydrogen phosphate (anhydrous) 6.5g

➢ 40% formaldehyde 100ml

➢ Dist. water 900ml

NBF (cont’d)

Advantages

• Has the same advantages as 10% formalin, but in addition it prevents formation of acid formalin pigment.

• No need of periodic changes of solution.

Disadvantages

• Disadvantages are similar to those listed in formalin, and also take longer time on the preparation of this fixatives.

Zenker’s Solution

 This is recommended for the fixation of small pieces liver and spleen. The period of fixation required is from 12-24 hours.

 Constituents:

➢ Mercuric chloride 5.0g

➢ Potassium dichromate 2.5g

➢ Sodium sulphate (Optional) 1.0g

➢ Dist. Water 100ml

 Add 5 ml of glacial acetic acid just before use.

Zenker’s Solution (cont’d)

Advantages 

• Permits excellent staining of nuclei and connective tissue fibers

• Recommended particularly for tissues which are to be stained by trichrome techniques.

Disadvantages

• Poor penetration

• Tissue pieces should not exceed 0.5 cm in thickness

• Makes brittle if the tissue kept more than 24 hrs and must be wash for several hrs in running tap water.

Bouin’s solution

 This is recommended for gastrointestinal tract biopsies / soft and delicate tissues. The period of fixation required is from 6-24 hours.

 Constituents:

➢ Saturated aqueous picric acid 75ml

➢ Formaldehyde 40% 25ml

➢ Glacial acetic acid 5ml

 Once fixed, tissue should not be washed in water, but transferred directly from fixative to 70% alcohol.

Bouin’s solution (cont’d)

Advantages 

• Produces very little micro-anatomical distortion and permits brilliant staining results.

• It is good preservatives of glycogen.

Disadvantages

• Poor penetration so not preferable for big biopsy.

4. Fixed vs Unfixed tissue

5. Decalcification 脱钙

❑ In order to obtain satisfactory paraffin section of bone and other heavily mineralized tissues - it is necessary to remove the mineral and soften the tissues

❑ It is carried out by treatment with reagents which react with calcium

❑ Before decalcification

i. Cut hard tissues into small pieces (2-6mm)

ii. Use thin blade or hack saw

iii. Minimize tearing of surrounding tissues

iv. Fix the tissue in buffered or neutral formalin

v. Tissues later must be washed

Procedures for decalcification:

1. Suspend the tissue slice in decalcifying solution - fluid 20 times the volume of tissue

2. Change the fluid daily - stirring agitation of the fluid hasten decalcification

3. The tissue may be mechanically or chemically tested

❑ bending or piercing with a sharp needle

❑ generally a day or two will be enough to decalcify

❑ twice that long for compact bone

4. Wash specimen for 24 -48 hours in running water before processing the tissue

Acid decalcifiers:

Strong acid

e.g.

Hydrochloric acid Nitric acid

-if used longer than 24-48 hour deterioration occurs

-strong acid used for urgent biopsy

Weak acid

e.g.

Formic Acetic Picric

- use in non-urgent surgical specimen decalcification should be complete 1-10 days depending on size

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L2 Tissue processing组织处理

1. Definition of tissue processing

any treatment of tissues necessary to impregnate注入/渗透 them with solid medium to facilitate the production of sections for microscope

2. Aim

 to embed嵌入 tissues in solid medium

 to support the tissues

 to enable the knife to cut the section with little damage to knife or tissues

3. Dehydration脱水

 A process by which water is removed from fixed tissues

 Also removal of aqueous and some of the lipid tissues fluids

 Tissue blocks are placed in containers or cassette with tag

 lid securely closed

 tiny fragments should be wrapped in a single layer of lens paper

 Tissues are passed through a series of increasing concentrations of alcohol

 with one change in each concentration

 duration of one hour in each

 keep covered to avoid evaporation

 Starting alcohol is usually 80%

 for soft tissues 30%, 50%,70%

 it is important that the final bath of alcohol is pure and change 3 times

 free from water

4. Dehydrating agents

 Ethyl alcohol (ethanol) C2H5OH

 clear colourless inflammable liquid with pleasant odour

 hydrophillic-miscible with water in all volumes

 the most common

 Methylated spirit (denatured alcohol)

 same physical characteristics as ethanol

 stronger smell

 consists of ethanol to which has been added a proportion of methanol

 Methyl alcohol (methanol) CH3OH

 clear,colourless inflammable and poisonous fluid

 unpleasant odour, miscible with water, ethanol

 Isopropyl alcohol (2 propanol) CH3CHOHCH3

 miscible with water, ethanol

 does not harden the tissue

 99% is the best substitute for ethyl alcohol

 Acetone CH3COCH3

 clear,colourless,inflammable fluid

 pungent辛辣 odours

 miscible可混溶 with water, ethanol

 more volatile易挥发/气化

 causes brittleness易脆 of tissue if treatment is prolonged

 when speed is essential -acetone is preferred

5. Clearing清除

 The removal of dehydrating agents from tissue

 To make tissue transparent透明

 Selection of a suitable clearing agent must be based on:

 Speed of removal of alcohol

 Ease of removal

 Gentleness towards tissue

 Flammability

 Toxicity

 Cost

6.  Clearing agents

 Xylene

 for regular size

 not thicker than 3-4mm

 immersion time must not be prolonged

 tissues become brittle

 Toulene甲苯C7H8

 less damaging on prolonged immersion of tissues

 suitable for automated tissue processing

 Chloroform

 slower in action causes brittleness

 thicker tissue up to 1cm thickness can be processed

 dangerous -releases toxic gas

 Benzene

 carcinogenic properties

 similar to xylene

 Carbon tetrachloride

 similar properties to chloroform

 Toxic

 Paraffin

 variable mixture of hydrocarbon

 less flammable

 less dangerous

 Petrol

 similar properties to xylene

 various additives

 not recommended

 Amyl acetate

 remove alcohol fairly rapidly

 Costly

 Methyl benzoate and methyl salicylate

 fluids moderate in speed action

 cause minimal distortion of tissue

 Cedar wood oil

 slow in action

 causes little hardening or shrinkage of tissues

 low volatile properties

7. Infiltration浸润 and impregnation渗透

 Tissues are transferred to a bath of molten paraffin wax

 During this process xylene is eliminated from tissues by diffusion –called infiltration

 Wax diffuses into the tissue to replace the clearing agent -called impregnation

 To completely remove the clearing agent

 Inadequate impregnation lead to drying and shrinking of tissues-crack and crumble develop

 High temperature will over harden the tissues

8. Embedding嵌入

 Placing the infiltrated impregnated tissue in warm liquid paraffin wax that solidifies into a firm block when cool to room temperature

 Precaution should be taken

 no dust particles must be present

 after embedding the wax must be rapidly cooled to reduce the wax crystal

 Orientation of tissues

 tissues of tubular nature cut transversely

 skin-cut in a plane at right angles

 muscle biopsies are sectioned both transverse and longitudinal planes

 Little volume of wax is dispensed into the mould first

 When a thin film of semi-solid wax has formed on the base of the mould

 tissue is introduced with warm forceps gently pressing

the tissue into the semi-solid wax correctly orientated plane

 Then top up the mould with wax again

 make sure that there is no air bubbles

 Transfer blocks to refrigerator to complete hardening

 Identification label must accompany the specimen through all types of tissues processing

9. Embedding media

 Paraffin wax

 most popular

 large number of tissue blocks may be processed in a short time

 sectioning and staining - fewer difficulties

 Cheaper

 melting point in range 40-70°C

 for satisfactory sections melting point 54-58°C

 Other waxes

 Ester wax

 microcrystalline wax

 Resins

 Acrylic

 Epoxy

 urea-formaldehyde

 Other media

 Agar

 Gelatin

 celloidin

 Different medium is required if

 medium not sufficiently hard and fail to provide adequate support

 affected by heat

 distort the tissue

 tissue breaking away from the wax during sectioning

 sections cannot be cut thin enough

 Manual tissue processing

 Necessary in the following

 speed processing

 electrical power failure / machine breakdown

 small tissue slices -need optimum time

 Advantage

 flexibility tissues are treated for the optimum duration in each fluid

 Rapid technique for thin slices of tissue

1.10% formalin 60°C ……….20 min

2. fresh acetone………………..20 min

3. fresh acetone ……………….20 min

4.fresh acetone ……………….20 min 5.

xylene ………………………….10 min 6.

xylene ………………………….15 min 7.

wax ………………………………30 min

8. wax ………………………………60 min (3 hours 5 min)

 agitate搅动 frequently to assist in the transfer of fluids

 Automated tissue processing

 Most automated tissues processor have 12 containers processing cycle

 Some machines apply -heat and vacuum to increase the rate of processing

 Care must be taken for type of tissue e.g. spleen, muscle, skin, decalcified tissue -become hardened if heat and vacuum is applied

 Vacuum..

 the degree of vacuum should not exceed 40-50mmHg

 normally in a sealed container of molten paraffin wax- applying suction to the container

 Aims:

 to remove air bubbles in the tissue

 to remove clearing agents

 For increasing speed of processing

 use warm (40-50°C) fixative to ensure fixation is complete

 use a fast -acting clearing agent (e.g. xylene)

 use of vacuum infiltration at all wax stages

 agitation at all stages, even during fixation

Tissue Processor

 Routine overnight processing (automated)

1. 4% formalin (1.5 lit.) ……………………………………………………2 hrs

 (40 ml formaldehyde 960 ml water add 9 g NaCl)

2. 4 % formalin (1.5 lit.)…………………………………………………….2 hrs

3. 70 % alcohol (1.5 lit.)…………………………………………………………….1 hr

 (700 ml alcohol 300 ml water)

4. 90 % alcohol (1.5 lit.).................................................................1 hr

 (900 ml alcohol 100 ml water)

5. absolute alcohol (100% alcohol) (1.5 lit.)…………………………….1 hr

6. absolute alcohol (1.5 lit.)……………………………………………………2 hrs

7. absolute alcohol (1.5 lit.)…………………………………………………….2 hrs

8. xylene (1.5 lit.).......................................................1 hr

9. xylene (1.5 lit.)…………………………………………………..1.5 hrs

10. xylene (1.5 lit.).......................................................1.5 hrs

11. wax …………………………………………………………………..2 hrs

12. Wax…………………………………………………………………..3 hrs

Total 20 hrs

 Day time processing -for small specimen

1. formalin ……………………………………………………………….…..15 min

2. formalin …….……………………………………………………………..15 min

3. 70% alcohol ………………………………………………………………15 min

4. 90%alcohol ……………………………………………………………….15 min

5. 100%alcohol …………………………………………………………… 15 min

6. 100%alcohol ………………………………………………………………15 min

7. 100%alcohol ………………………………………………………………15 min

8. toluene ………………………………………………………………………15 min

9. toluene ………………………………………………………………………15 min

10. toluene ……………………………………………………………………...15 min

11. wax ……………………………………………………………………………30 min

12. wax ……………………………………………………………… ……………30min

3hrs 30min

Automated Tissue Processor (latest)

10.  Factors influencing the rate of impregnation渗透

 Agitation搅动

 allow the tissue to sink to the base of container-to make sure interchange of fluids occur

 mechanical device -vertical agitation

 average speed of tissue movement is 10-12 inches per minute

 improved impregnation by 25-35%

 Heat

 increases the rate of penetration

 care must be taken not to overheat tissues and cause shrinkage, brittleness and

 difficulties in sectioning

 Viscosity黏度

 the larger the molecule the higher the viscosity the slower the rate of penetration

 Vacuum

 serve to remove air bubbles trapped within the tissue –this will increase the contact with fluid to tissue

 Ultrasonic -not widely used

 Points to note

 beakers and wax bath must be filled to the correct fluid level

 any spillage of fluid should be wiped

 accumulations of wax must be removed

 wax bath thermostats set at satisfactory level

 checked -timing

 electrical plugs

 changing of solution after using them for 2-3 days

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L3 Sectioning分段，切面

TERMS

TRIMMING修剪 10micron

SECTIONING into ribbons 2.5micron

FISHING打捞 waterbath 

DRYING 37°C

1. INTRODUCTION

EQUIPMENT

➢ROTARY MICROTOME

▪ For production of large number of sections

▪ Serial section

➢FLOATATION BATH become straight

▪ Thermostically controlled

▪ For paraffin wax 60oC melting point water bath temperature is about 45oC

▪ A variety of fluids are recommended from distilled water to quite high concentration of alcohol

➢ANTIQUE MICROTOME

➢HOT PLATE OR DRYING OVEN

▪ Recommended drying of section onto slides should take place at temperature as low as 37°C

▪ Section dried at low temperature drying is prolonged - over night

▪ Drying section on a hot plate is necessary if staining is to be carried out urgently

➢BRUSH, FORCEPS夹子

▪ Necessary during sectioning for removal of folds

➢SLIDES, GLASS MARKER

▪ Size 76mm x 25mm, 1 to 2mm thick

▪ Mark slides with a glass marker

➢SECTION ADHESIVES 

Used when fishing, the tissue cannot paste on it

▪ Most tissue section do not requires any adhesive provided sections are initially dried adequately on to slides

▪ If staining solution tend to remove section from slides so adhesive should be use

▪ Types of adhesive:

• Albumin

•Gelatin

• Starch

• Cellulose

• sodium silicate

• Resin

▪ Tissues impregnated with ester wax or polyester wax will usually require the use of section adhesive to minimize loss e.g. Mayer’s glycerol albumen

• Mix and agitate ingredient

• Filter through coarse filter paper

• Thin smear is needed on the slide

2. SECTIONING

SETTING UP THE MICROTOME

Tighten knife clamp (blade) screws securely

Block clamp screw as well

Exposed ends of knife must be protected

TRIMMING OF TISSUE BLOCK

For smooth and straight 

To trim any surplus wax

To expose suitable area of tissue for sectioning - thickness at 20um

On exposing - section thickness set to 0.5-4um

Top and bottom of block parallel and horizontal to the edge of knife at the moment of impact

CUTTING SECTIONS

all tissues desired on the slide should be exposed on the surface

no scratch marks on the surface

if scratch marks are visible the knife must be moved laterally

the face trimmed again

speed of block is important

softer tissues - at a slower rate

Optimal speed is obtained through experience

Maintain a regular cutting rhythm

A ribbon section is produced

Use fine forceps to hold the free end of the ribbon-left hand (right hand hold hand wheel for rotary microtome movement)

Use soft paint brush to brush away the last section from the knife

Transfer ribbon to water bath-left hand with forceps

To obtain flat section its is necessary to spend time in the cutting and gentle

Stretching of the ribbon - before floating on the water surface

3. RIBBONS

4. FLOATING OUT SECTIONS

action in floating out must be smooth with trailing end of the ribbon making contact with the water

slight drag when ribbon touches the water will produce tension in ribbon

remove folds from section-making it straight

when ribbon has come to rest on the water - remaining wrinkles and folds are removed by teasing a part using forceps or seeker

Prolonged floating out of section on the water bath must be avoided as tissues may expand and become distorted

Advantage if water bath has a black base - section are more easily seen

Some tissues e.g. cartilage are difficult to flatten on the water surface

5. FROM RIBBONS TO WATERBATH

Before fishing用slide钓, ensure the tissue is straight and smooth with no wrinkles or folds

6. DRYING SECTIONS

if staining is required urgently drying section on a hot plate or oven (at 45°C) is necessary

drying time is 10 to 45 minutes

advantage of oven drying - dust is less likely to settle on sections

after removal from hot plate or oven - cooled sections are stored in dust free containers

if overnight the temperature is 37o C

7. SLIDES DRIED IN THE OVEN

8. SUCCESSFUL SECTIONING

Properly prepared material

 Tissue blocks must be well fixed, adequately processed and embedded in an

 appropriate embedding material.

A well-maintained microtome.

A sharp microtome knife set at the correct clearance angle. This is the angle

 between the knife edge bevel and the block. For paraffin blocks this angle should be at 5 on the setting on the knife holder (settings between 0-10). If the angle is too great, it can cause compression of the cut sections, if too fine the edge of the knife can vibrate and cause ‘chatter’ in the sections.

 The tissue block must be positioned so that the vertical and horizontal planes are set to cut the whole face of the block.

 Block edges must also be parallel with the knife edge. All clamps and screws should be firmly tightened so that the vertical and horizontal

9. FAULTS & REMEDIES IN PARAFFIN WAX SECTIONING

Sections do not join to form a ribbon:

Causes

 wax too hard

 dust on knife edge

 knife angle too wide or narrow

Remedies

 warm or re-embed

 clean with xylene

 adjust angle

Sections roll into a coil:

Causes

Blunt knife

knife angle too wide

section thickness too great

Remedies

replace knife

reduce knife tilt

reduce section thickness

When sections are curved:

Causes

 knife blunt in one area

 excess wax in one side

Remedies

 use different part of knife or replace with a new one

 trim away excess wax-important

Splitting of sections:

Causes

damage in knife edge

hard particles in tissue

hard particles in wax

Remedies

use different part of knife or replace with a new one

remove particles from tissue

re-embed in fresh wax-embedding important too

Compression of sections:

Section should be hard enough

Causes

 blunt knife

 angle knife too wide

 wax too soft

Remedies

 replace with a new knife

 reduce knife tilt

 cool block in refrigerator

Sections expand and disintegrate on water surface:

Causes

poor impregnation of tissues

water temperature too high

floatation bath dirty

Remedies

return tissue to vacuum impregnation container for a few hours

cool water

clean floatation bath

https://drive.google.com/file/d/1ArgQ4oEVNA5IBu--Jxtpjm_J7cQzpneD/view?usp=drivesdk

https://www.youtube.com/watch?v=ml4fBEmH8Sg

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L4 Haematoxylin & Eosin (H&E) Staining

1. Theory of staining

 Entry of dyes or reagents into tissues may be passive – involving diffusion

 Or active -this is due to dye - tissue / reagent – tissue affinities

 Affinity – attractive force that bind dye to tissue or measure of the tendency of dye to transfer to tissues

 High affinity means –intensely stained

2. Staining mechanisms

 Both chemical and physical reactions occur such as:

 Simple absorption such as the Oil Red O stain for lipids.

 Adsorption such as in colloid dyes

 Coloumbic attraction as seen in acidic and basic dyes.

 Van der Waal Forces such as hydrogen bonding, covalent bonding and hydrophobic bonding may all be involved.

3. Other factors affecting staining reactions include the following:

a) pH of the dye e.g. Alcian Blue at different pH stain different mucins.

b) Concentration of the dye, can stain quicker up to a point at which all dye uptake sites have been utilised.

c) Type of fixative used. Formalin fixation enhances basic dye uptake. Mercuric chloride enhances acid dye uptake.

d) Temperature, increase in temperature usually increases the rate of staining.

e) Mordants=perantara

 They affect dye uptake and retention and their function is to help in the attachment of the dye to the tissue. The compound formed between a dye radical and the mordant is called a dye lake.

 When attached to the tissue the dye lake is relatively permanent ,it is insoluble in neutral solutions and does not decolourise during dehydration and subsequent staining.

 Mordants can be used before applying the dye, at the same time as the dye or after the dye.

4. Classification of dyes

a) By the origin of dye

 Natural: Haematoxylin

 Synthetic: Aniline

b) By physico - chemical properties of a dye

 Fluorescent: Acridine Orange

 Acid: Eosin

 Basic: Safranin

c) By Dye structure

 Iron complex: Haematein

f) By usage in biological staining

 Fat/lipids: Oil Red O

g)  By mode of action

 Mordant: Gallocyanin

 Reactive: Mercury Orange

5. Standardization of dyes

 Commonly used dyes are identified by the name but confusion may be occur, e.g. sudan IV a stain for lipid may also be called scarlet red or oil red IV

 To avoid the confusion Colour Index which is a system of dye indexing is used

 E.g. C.I 42685 for acid fuchsin or acid magenta

6. Haematoxylin 

a) Alum haematoxylin-nucleus-purple color

 produce good nuclear staining

 the mordant is aluminium

 Harris's haematoxylin:

Haematoxylin 2.5g

Absolute alcohol 25cm3

Potassium alum 50g

Distilled water 455cm3

Mercuric oxide 1.25g

Glacial acetic acid 20cm3

#REU=ready to use

b) Iron haematoxylin

 iron salts - used as oxidizing agent and as mordant

 common iron salts:

ferric chloride

ferric ammonium sulphate

– Weigert`s haematoxylin

✓ Haematoxylin solution:

Haematoxylin　1 g

Absolute alcohol　100 cm3

Allowed to ripen for 4 weeks

✓ Iron solution

30% aqueous ferric chloride 4 cm3

HCl (conc)　1 cm3

Distilled water　95 cm3

this solution is filtered- add equal volume of Haematoxylin solution before use

violet black colour- if brown - discard

c) Tungsten haematoxylin

 Phosphotungstic acid as mordant

 Tungsten acid haematoxylin: 

Haematoxylin 0.5 g

Phosphotungstic acid 10 g

Distilled water 475 cm3

Aqueous potassium permanganate 25 cm3

d) Molybdenum haematoxylin

 Molybdic acid as mordant

 Molybdenum haematoxylin

 Haematoxylin solution:

Haematoxylin 2.5g

Dioxane 49cm3

Hydrogen peroxide 1cm3

 Phosphomolybdic acid solution:

Phosphomolybdic acid 16.5g

Distilled water 44cm3

Diethylene glycol 6cm3

 Phosphomolybdic acid solution is filtered

 50cm3 of filtrate added to Haematoxylin solution

 Dark violet in colour

 Stand 24hrs before use

e) Lead haematoxylin

 Lead salts as mordant

 Lead haematoxylin

 Lead solution (stabilized): 

5% lead nitrate (aq) 50cm3

Ammonium acetate (aq) 50cm3

filter then add 2cm3 of 40% formaldehyde

 Lead staining solution:

Stabilized lead solution 10cm3

0.2g haematoxylin in 1.5ml 95% ethanol 1.5cm3

Distilled water 10cm3

 Mix and stir

 Stand 30min , filter

 Filtrate made up to 75 cm3 with distilled water

f) Haematoxylin without mordant

 freshly prepared haematoxylin without mordant

 stain various minerals

7. Eosin-cytoplasm

 Most suitable stain to combine with an Alum Haematoxylin

 Used to distinguish the cytoplasm of different types of cells and different types of connective tissues fibers

 3 types of eosin

 Eosin Y:

yellowish

water soluble

the most widely used-common

 Ethyl eosin: 

Alcohol

 Eosin B: 

bluish

8. Outlines of procedure for staining paraffin sections

a) Removal of paraffin wax with xylene

 paraffin wax poorly permeable to stains

 removal with solvent is necessary by using xylene

 1-5 minutes in each of 2 changes of xylene is sufficient

b) Removal of xylene with absolute alcohol 100%

 xylene is not miscible with aqueous solutions and low grade alcohols

 necessary to remove with absolute alcohol 1 minute in each of 2 changes

c) Treatment with descending grades of alcohol 95%→70%

 to avoid damage and detachment of the section

 to introduce water in the tissue

d) Water

 Must be the same as the solvent of the stain to be used in next step

 commonly distilled water

e) Staining

 may involve treatment with a single stain solution or 2 or more separate stains (H&E)

 may take several minutes or hours

f) Dehydration

 paraffin sections are mounted in media miscible with xylene

 necessary to dehydrate tissues in alcohol before passing to mounting

g) Clearing

 1 minute of xylene is sufficient to achieve transparency of the section

 also removal of alcohol

8. Standard haematoxylin and eosin stain for paraffin sections (H&E) SOP

Progressive

a) Soak the section in 200 ml of xylene for 5 minutes and agitate

b) Place the section in another 200 ml of xylene for 5 minutes

c) Transfer to 200 ml of 100% alcohol for 1 minute

d) Transfer to 200 ml of 100% alcohol for 1 minute→90%→80%

e) Transfer to 200 ml of 70% alcohol for 1 minute

f) Wash in running tap water, rinse in distilled water, drain well

g) Stain with Harris’s haematoxylin for 10 minutes

h) Wash in running tap water

i) Dip several times in 200 ml of 1% acid alcohol (2 ml of HCl into 198 ml of 70% alcohol)

j) Wash in running tap water

k) Dip several times in 200 ml of dilute ammonia water-mordant (1 ml of ammonia into 199 ml of distilled water) - the section will change to a blue colour

l) Wash in running tap water

m) Counterstain in 200 ml of eosin for 1 to 2 minutes

n) Wash quickly in running tap water

o) Transfer to 200 ml of 70% alcohol for 1 min

p) Transfer to 200 ml of 95% alcohol for 1 min

q) Transfer to 200 ml of 100% alcohol 1 min

r) Transfer to another 200 ml of 100% alcohol 1 min

s) Transfer to 200 ml of xylene (5 mins)

t) Transfer to another 200 ml of xylene (5 mins)

u) Drain, mount in DPX (mixture of distyrene, a plasticizer, and xylene used as a synthetic resin mounting media) and label the slide

 Results:

nuclei - blue black

cytoplasm - pink

muscle fiber- pinky red

collagen - pale pinky red

RBC - orange/red

9. Common terms in staining

 Progressive staining technique:

is one in which the different elements in the tissues are coloured in sequence

satisfactory differential colouration of the tissues is achieved

 Regressive staining technique:

is one in which the tissue is first over-stained and then destained or differentiated

by removing excess stains from unwanted parts of the tissue

 Direct staining:

does not require an additional intermediate substance known as mordant before satisfactory combination with tissues takes place

E.g. aniline dyes

 Indirect staining:

requires mordant for satisfactory staining

mordanted dye combines with the tissue to form tissue-mordant-dye complex

insoluble in aqueous or alcoholic solvents allowing counterstaining and dehydration to be easily carried out

e.g. haematoxylin with potassium alum

 Differentiation:

Destaining or differentiation of an over-stained tissue in a regressive technique

Can be by washing in simple solutions or by acids and oxidising agents

 Counterstain (eosin):

Application of a different colour to provide contrast and background to the tissue

Staining component

The purpose of counterstains is supplementary e.g.eosin

Summary for staining

Staining→xylene→absolute alcohol→90% alcohol→80% alcohol→70% alcohol→haematoxylin-color nucleus→acid alcohol→ammonia-mordant→eosin-color cytoplasm→DPX

SUMMARY:

https://www.researchgate.net/publication/47535516_Histopathology_Procedures_From_Tissue_Sampling_to_Histopathological_Evaluation

_____

L5 Frozen Section

1. Frozen section

• The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen. It is used most often in oncological surgery.

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

• While diagnosis can be rendered in many cases, fixed tissue processing is preferred in many conditions for more accurate diagnosis.

2. Uses of frozen section

• If a tumor appears to have metastasized (merebak), 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.

• If a tumor has been resected but it is unclear whether the surgical margin is free of tumor, an intraoperative consultation is requested to assess the need to make a further resection for clear margins.

 E.g. in a sentinel node procedure, a sentinel node containing tumor tissue prompts a further lymph node dissection, while a benign node will avoid such a procedure.

 Rarely, cryosections (frozen sections) 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.

3. Method for preparation of frozen sections

 Start with a sharp blade

 Practice one patient one blade

 Sitting or standing

 Depends on you, but you need to be relax and comfortable

 The Brush

 You must learn to be good with a brush

 The purpose of the brush is to grab and maneuver the section across the stage. unless you have perfect temperature, a cold section will by nature trying to curl up and pull away from the brush.

 use a brush with stiff bristles and a fairly wide gripping surface.

 Holding the brush

 Hold the brush like a pen in the left hand and stabilize the hand (e.g. by gently resting the side of the fifth finger on the stage).

 Turning the wheel

 Turn the wheel in a continuous uniform motion without hesitation.

 many MLT using a brush stop at the beginning of the section, slowly grab the tissue and then start to turn the wheel. This is not good and adds to potential artifacts at the beginning of the section, variations in thickness or difficulties when approaching tissues containing fat.

 Movement of the brush

 As the block begins to move toward the knife the brush moves downward in pace with the block.The brush can gently rest on the bottom 2mm of the block and "ride the block" pulling away just as the block meets the knife. It is the downward movement of the brush that allows you to keep a continuous motion as you grab the section.

 Retrieving the section

 Tissue can be picked up from the cryostat stage or from the block.

 When the section is complete the tissue can be picked up by holding the slide just above the section and angle the slide down to touch a portion of the tissue.

 Static attraction will draw the section to adhere to and quickly melt on to the warm slide.

 Retrieving from stage

 Slide levers down to gently touch the section which will float onto the slide with static or cohesive attraction.Try avoid stretching or folding the section during this process by keeping the a steady hand and the transverse axis of the slide parallel to thee section.

Retrieving from the block

1. section is cut leaving an attachment of medium at the top.

2. The wheel is turned in opposite direction bring the section back to the face of the block.

3. Section is retrieved by placing the slide over the tissue on the face of the block.

Rapid fixation

 Have your fixative (warm formalin) opened in an immediately reachable location.

 If there is delay in fixing the tissue there will be significant drying artifact (loss of nuclear detail and leakage of fluids from the cytoplasm).

4. Rapid H&E for frozen section

1. Warm formalin 1 dip

2. Running tap water - dip until clear

3. Hematoxylin – 1 minute

4. Running tap water - until clear

5. Bluing Reagent (1% ammonia) - dip 1 time

6. Running tap water - 30 seconds

7. Eosin Y - 1 - 2 dips

8. Running tap water - 30 seconds

9. 95% Ethanol (2 changes) 5 - 10 dips

10. 100% Ethanol (3 changes) - 5 - 10 dips

11. Xylene (2 changes) 5 - 10 dips

12. Mounting

5. Limitations of Frozen Section

 True limitations:

 Time

 Limited special stains and studies

 Freezing artifacts

 Avoidable limitations:

 Drying artifacts

 Sampling error

 Fat

 Etc.

SUMMARY:

https://www.youtube.com/watch?v=UnCLhowHucU

Basic Immunohistochemistry

1. Immunohistochemistry

• Histochemistry is a science that combines the techniques of biochemistry and histology in the study of the chemical constitution of tissues and cells.

• Immunology is a science that deals with the immune system, cell-mediated and humoral aspects of immunity and immune responses.

• Immunohistochemistry (IHC) is the integration of the above mentioned disciplines.

• The basic principle of any IHC procedures is that an antibody will specifically bind with an antigen to produce an exclusive antibody-antigen complex.

• This bonding is used to visualize both normal and diseased states of tissues, infectious agents and other components that may not be demonstrated by histochemical or special stains.

2. Antibody-Antigen Reaction

• The rate of antigen-antibody reaction is affected by temperature and pH of buffers and diluents used in IHC procedures.

• Higher incubation temperature permits rapid antigen-antibody binding.

• The buffer‟s pH and ionic content can affect the charge of amino acids in both the antibody and antigen.

• Polyclonal vs Monoclonal antibody – Which is better?

3. Primary Antibodies

• Primary antibody refers to the antibody that is directed against the antigen of interest. For example, CD20 can be used as the primary antibody to demonstrate B-cells on a tissue section.

• Both polyclonal and monoclonal antibodies can be used as primaries.

4. Antibody Titer and Dilution

• Titer is the highest dilution of the antibody resulting in strong specific staining with the least amount of background. Background includes all nonspecific staining as a result of procedural artifacts.

• Dilution is the ratio of the concentrated antibody to the total volume of the desired dilution. For example, a 1:5 dilution means one part concentrated antibody and four parts diluent.

• Optimal working dilution is typically determined by titration or dilution series.

• The next slide shows IHC staining results of serially diluted Chromogranin A antibody on pancreas.

• At 1:50, the Islet cells stain strongly but there is also a strong background staining.

• At 1:800, there is no background staining but the Islet cells stain very weak.

• At 1:200, there is good contrast and no background staining, it is therefore the optimal working dilution.

5. Antibody Incubation Time

• Incubation time is inversely proportional to antibody concentration. Higher concentration of antibody allows shorter incubation time.

• It can be from minutes to hours, with 30-60 minutes the most common practice.

6. Antibody Incubation Temperature

• Antibody-antigen reaction is hastened at 37°C as compared to room temperature. An increase in temperature also allows for a higher dilution of the antibody.

• Humidity chambers must be used when incubating at higher temperature to prevent drying of tissue sections.

7. Antigen Retrieval

• During the process of formalin fixation, many antigenic sites are "masked" and are therefore sometimes difficult or impossible to stain without antigen retrieval.

• Antigen retrieval is a process of treating formalin fixed-paraffin embedded tissue sections with proteolytic enzymes or heating them in various buffer solutions in order to expose the antigen.

• Commonly used proteolytic enzymes include trypsin, pepsin and protease.

• Heat induced epitope retrieval (HIER) includes microwaving, pressure cooking, steaming, autoclaving or using the PreTreatment Module™.

• Requires buffer of different concentrations and pH. Commonly used buffers include:

• citrate at pH 6.0

• EDTA at pH 8.0

• Tris-HCL at pH 10.0

• These photos show the staining results of CD3 antibody on tonsil, with and without antigen retrieval.

6. Enzymes and Chromogens

• Detection systems attach enzyme labels to primary or secondary antibodies to visualize the localized antibody-antigen binding in tissue section.

• Enzymes are proteins that act as catalysts to increase the rate of chemical reaction. They are used in IHC to convert a colorless reagent into a stable colored product (chromogen) that marks the site of antibody-antigen complex.

• A chromogen is a substance that absorbs light, *producing color.

• Commonly used enzyme labels for IHC procedures include:

• horseradish peroxidase (HRP)

• alkaline phophatase (AP)

• Commonly used chromogens for HRP include:

• 3-amino-9-ethylcarbazole (AEC)

• AEC is oxidized by HRP producing a bright red reaction product. This  reaction product is not stable and may fade over time.

• AEC is soluble in alcohol, stained slides should therefore be counterstained with non-alcoholic hematoxylins (Mayer‟s or Gills).

• 3,3‟-diaminobenzidine (DAB)

• DAB is oxidized by HRP producing a dark brown reaction product. This reaction product is stable and does not fade over time.

• In some IHC procedures, the dark brown reaction product can be modified and intensified by adding metals (copper or cobalt) to DAB solution.

• DAB is not suited for staining melanoma cases because the endogenous dark brown pigments in tumor cells can be confused with DAB reaction product.

• AEC & DAB examples

7. IHC Staining Methods

• This following slides will discuss the various IHC staining methods which can be used to localize tissue antigens for light microscopy.

• Direct Method

• Two-Step Indirect Method

• Three-Step Indirect Method

• Peroxidase-Antiperoxidase (PAP) Method

• Alkaline-Antialkaline Phosphatase (APAAP) Method

• Avidin-Biotin Complex (ABC) Method

• Labeled Streptavidin-Biotin (LSAB) Method

8. LSAB Method

• Uses enzyme-conjugated streptavidin. Streptavidin is conjugated to several molecules of enzyme horseradish peroxidase (HRP) or alkaline phosphatase (AP).

• The secondary antibody is conjugated to numerous biotin molecules, each of which can potentially bind to an enzyme-conjugated streptavidin.

• Procedure:

An unlabeled primary antibody binds to tissue antigen.

A biotinylated secondary antibody binds to the primary antibody.

Each secondary antibody contains multiple biotin molecules; several secondary antibodies can bind to the primary antibody.

An enzyme-labeled streptavidin is added and binds to the secondary antibody.

A substrate-chromogen solution is added producing a colored end-product.

10. LSAB Staining Procedure

• This section shows the step by step LSAB staining procedure using carcinoembryonic antigen (CEA) antibody on colon carcinoma.

• Procedure:

Remove paraffin wax and hydrate tissue section.

3% hydrogen peroxide solution for 10 minutes to inactivate endogenous peroxidase activity.

Rinse in distilled water and wash 2 times in PBS buffer.

Microwave for 20 minutes using citrate buffer solution pH 6.0.

Thorough rinse in distilled water and wash 2 times in PBS buffer.

3% bovine serum albumin (BSA) to block nonspecific staining. Drain excess BSA after incubation.

Optimally diluted CEA antibody for 30 minutes.

Biotinylated link antibody for 10 minutes.

Wash 2 times in PBS buffer.

HRP conjugated streptavidin for 10 minutes.

Wash 2 times in PBS buffer.

AEC chromogen for 10 minutes.

Wash 2 times in PBS buffer and rinse in distilled water.

Counterstain in Mayer's Hematoxylin for 1 minute. SPECIMEN Lab Vision Corp. NM-123 Colon carcinoma MS-1375 CEA 

Thorough wash in tap water to “blue” the nuclei.

Coverslip using an aqueous mount.

10. IHC Staining Pattern

• The following slides show photos of various staining patterns that can be achieved after performing an IHC stain.