1

HISTOTECHNIQUES – PART 1

Dr Swati Patil

2

INTRODUCTION • Histological technique deals with the preparation of

tissue for microscopic examination.• Complete tissue or a selected part undergoes series of

processes, viz.- Fixation- Dehydration- Clearing- Embedding- Cutting - Staining

3

• Section :• Study of cell microanatomy:

– Living cells– Dead cells

• Preparation depends upon – Liquid– Solid:

• Scraping• Teasing• Maceration• Sectioning

• Cinematography

4

• Minimum handling-• Cut with very sharp knife-• Osmotic injury-• Drying-• Thickness of tissue slice-• Excess blood or mucous in tissues-• Refrigeration-

TISSUE PROCUREMENT

5

FIXATION • Def : Process by which constituents of cell/ tissues are fixed

in physical & partly chemical state, so that they will withstand subsequent treatment with various reagents with minimum of loss/ distortion/ decomposition.

• Most fixatives act by denaturing or precipitating proteins – specifically of cell membrane & cytoplasm – thus forming sponge or meshwork holding other cell constituents.

• Fixative: - chemical substance - Dilution of chemical substance with

some other chemicals that can be used to fix tissue.

6

AIM OF FIXATION Preservation of cells and tissue constituents in a condition

identical to that existing during life So purpose of fixations is:1. To prevent or arrest autolysis & bacterial decomposition &

putrefaction.2. To coagulate tissue proteins as to prevent loss of diffusible

substances3. To fortify tissue against deleterious effect of various stages of

preparation of sections.4. To leave tissue in a condition which facilitates differential

staining with dyes and other reagents.

7

QUALITIES OF A GOOD FIXATIVE

• Good tissue penetration• Stabilizes the tissue, preserving the character and distribution

of cellular components• Prevents fixation artifacts• Prevents structure deformation – maintaining shape and

volume• Preserves cellular constituents

8

QUALITIES OF A GOOD FIXATIVE

• Destroys microorganisms• Extracts inactivated autolytic enzymes• Increases tissue consistency• Raises optical differentiation for better visualization• Maintains its chemical composition• Cheap, nontoxic, noninflammable, nonirritant and easy to

prepare

9

• THE IDEAL FIXATIVE:

So ideal fixative is one which:1. Prevents bacterial decomposition & autolysis2. Preserves tissue in their natural state & fixes all components

(proteins, carbohydrates, lipids)3. Makes cellular components insoluble to liquids encountered in

tissue processing4. Preserves tissue volume5. Avoids excessive hardening of fixed tissue6. Allows enhanced staining of tissues7. Nontoxic & non allergic

10

METHODS OF FIXATION

• Fixation by immersion:

• Fixation by perfusion:-high quality research work

11

CLASSIFICATION OF FIXATIVE

1. General classification: Routine fixative Special fixative

2. Depending upon mechanism of action: Precipitant fixative Non-precipitant fixative

3. Depending upon use of fixative: Micro anatomical fixative Cytological fixative

12

GENERAL PRINCIPLES IN HANDLING & FIXATION OF SPECIMENS

• Amount of fixing fluid:– Should be 10-20 times volume of specimen

• Specimens:– Should be placed in fixative as soon as possible after removal.– For bulky specimens like specimens of hysterectomy,

mastectomy:• make appropriate incisions to allow penetration of fixative.

13

• EFFECTS OF FIXATIVE ON TISSUE:1. Tissue hardening: helps in cutting tissue2. Acts as mordants for certain stains: e.g. mercuric fixatives,

iodine (fixative+ dye) + tissue3. Changes optical resolution , so it increases optical

differentiation of cell & tissue constituents.4. Renders cell insensitive to hypo / hypertonic solutions. 5. Micro organisms are composed of proteins , hence they also

get fixed:• prevents putrefactive changes in tissue • minimizing risk of infection among tissue handlers.

14

FIXATION MECHANISM• Forms cross-links between proteins, thereby forming a gel –

keeps structures in their in vivo relations to one another• Soluble proteins are fixed to structural proteins – insoluble →

gives mechanical strength for next steps• Protein denaturation- loss of solubility to communal aggregation

CHAIN FORMATION DENATURATION

15

16

COMMON FIXING AGENTS• Numerous substances or chemicals are used singly or in

combination:– have ability to preserve tissue – allow demonstration of every tissue component.

Coagulant fixative Non coagulant fixative

Alcohol Formalin

Acetone Osmium tetroxide

Acetic acid Potassium dichromate

Mercuric Chloride(corrosive sublimate)

Gluteraldehyde

Picric acid

Trichloracetic acid

17

FORMALDEHYDE

Formalin: Formaldehyde gas which is soluble in water to maximum of 40% by weight

Diluents : used in 1:9 proportion 1. Tap water2. Physiological saline3. Buffered salt solution

100% formalin = saturated solution = 40% solution of formaldehyde gas in

water

18

MECHANISM OF ACTION:Aldehyde group (H-CHO) of formaldehyderesponsible for polymerization. POLYMERIZATION : formation of additive compounds or

complexes by development of various links e.g. methylene bridges between protein mole.These reactions naturally alters cell physiochemistry &

reactivity of tissue to certain histochemical stains.

Many of these changes are reversible with water.

19

• FORMALIN FIXATION & EFFECT OF TEMPERATURE:

- At room temp. block of upto 4mm thickness, is adequately fixed when immersed in buffered formalin solution 10-20 times it’s vol. for 8 hrs.

- If agitated, 4hrs immersion is adequate

- If temp is raised up to 450c, immersion time shortened by 25-40% (there may be some loss of quality)

20

• FORMALIN IMPURITIES: – Formic acid– Paraformaldehyde– Formalin pigment:

• Acid formaldehyde hematine • granular, black, extracellular material, bifringent

21

ROUTINE FORMALIN FIXATIVES:1. 10% formal saline: Most commonly used fixativeWater (distilled) 900mlSodium chloride 8.5gmFormalin 100ml

2. 10% buffered neutral formalin: (better than formal saline)Water (distilled) 900mlNaH2PO4 3.5gm

Na2HPO4 6.5gm

22

3. Formaline mercuric chloride: (formal-sublimate)Mercuric chloride 30gmD/W 900mlFormalin 100mlFormaldehyde is added just before use as added solution is

unstable

Adv -useful for secondary fixative -excellent micro-anat fixativeDisadv - produces mercuric pigments -corrosive action on metals

23

4. Zenker’s fluid:Mercuric chloride 5gmPotassium dichromate 2.5gmSodium sulphate 1gmd/water 100mlAcetic acid 5ml(added immediately before use)

-Efficient micro anatomical fixative.-Beneficial effect on staining particularly with cytoplasmic & fiber stains

24

5. Bouin’s fluid (formalin-picric-acetic)Saturated aqueous picric acid 75mlFormalin 25mlAcetic acid 5ml

-Micro anatomical as well as cytological fixative

-Used for demonstration of chromosomes

-Glycogen is well preserved.

25

6. Helly’s fluid (zenker-formal):Acetic acid omitted & formalin 5 ml is added inzenker’s fluid

-Micro anatomical as well as cytological fixative

-Like formal sublimate used as secondary fixative

-Particularly suitable for use with bone marrow, spleen, lymph node, pituitary, pancreas where accurate preservation of cytoplasm as well as nuclei is desired.

26

7. Carnoy’s fluid:Absolute alcohol 60mlChloroform 30mlAcetic acid 10ml

-Rapidly penetrating & acting fixative -Due to quick fixation, it can be used for urgentdiagnosis.

-Glycogen is preserved.

27

8. Sanfelice’s fluid: Solution A formalin 128ml acetic acid 16mlSolution B 1% chromic acid 100ml mix of 9ml of solu. A & 16ml of solu. B -Cytological fixative

-Excellent fixative for mitotic figures & chromosomes

28

OTHER FIXATING AGENTS• Coagulants1. Acetic acid : Glacial acetic acidAdv : -best fixative for nuclei. -counteracts the shrinking effect of others.Disadv : -pronounced swelling of collagen fibers -distorts mitochondria & Golgi body2. Acetone :Adv : -best fixative for certain enzymes (acid phosphatase &

lipase).

29

3. Ethyl alcohol :Adv : -best for alkaline phosphatase & lipase -coagulates proteinDisadv : -powerful dehydrating agent- hardening & shrinkage -Improper fixation of chromatin -Distorts mitochondria & Golgi

4. Chromic acid:Adv : good fixative for carbohydrateDisadv : -powerful oxidising & rapid hardening with brittleness -washing with tap water for several hours required after

dehydration to avoid insoluble precipitate formation.

30

5. Picric acid: Adv : - good fixative for proteins, carbohydrates & glycogen - staining becomes better - enhances results with cytoplasmic stainsDisadv : much shrinkage & less hardening 6. Mercuric chloride:Adv : -good fixative for proteins -it shrinks but doesn’t distort tissue -fixes both nucleus & cytoplasm -act as secondary fixative Disadv :-mercury pigments

31

• NON COAGULANTS FIXATIVES:1. Potassium dichromate :Adv : -excellent fixation of mitochondria, phospholipids & myelin -acts as a mordant for mitochondria -iron staining of mitochondria -iron-containing pigments better fixed at higher pH Disadv : -chromatin gets dissolved -mitochondria gets thickened -formation of insoluble precipitants -brittleness of tissues

32

2. Glutaraldehyde:Adv: - cytological fixative - requires short time to fix small fragmentsDisadv:-poor penetration in larger pieces -gives generalized PAS +ve reaction

- much more costly.3. Osmium tetroxide :Adv : -valuable for fixation of cytoplasmic organelles.Disadv: -tissue becomes very brittle & nuclei not fixed well - cause over-blackening of tissues eg myelin -cost of fixative very high

33

SECONDARY FIXATION:- Tissue fixed by 10% formal saline is still susceptible to the effects of other more vigorous precipitant fixatives.- Tissues will show improvement in preservation and staining - Secondary fixatives: Mercuric chloride-formalin & Helly’s fluid

Adv: -Sections are easily cut & flattened better -Sections get stained more brilliantly.

34

POST MORDANTING & POST CHROMING

• Methods designed to facilitate preservation and subsequent staining of particular tissue component like lipid & lipoproteins with chrome salts.

• Tissues fixed in primary fixatives like formalin, treated with solution of potassium dichromate for extended period, usually days or weeks.

• It does not improve preservation of materials such as mitochondria & myelin so they can be demonstrated in paraffin sections

35

FACTORS AFFECTING THE FIXATION:1. Hydrogen ion concentration2. Temperature3. Penetration4. Osmolarity5. Concentration6. Duration

36

THANK YOU