Histopathological Histopathological TechniquesTechniques

HISTOPATHOLOGICAL TECHNIQUES• Histopathology is the branch of pathology which

concerns with the demonstration of minute structural alterations in tissues as a result of disease.

• Most of histopathological techniques simulating to those of applied for study the normal histological structures.

• For the demonstration of minute histological changes, the tissue must be processed in such a manner that it will provide maximum information.

• Most convenient way for study of morbid tissue is to use permanent section.

• A section is prepared by cutting a thin slice from a small piece of fixed tissue.

• Thin slice is mounted upon a glass slide in a medium of suitable refractive index and covered with a glass slide.

Scope• Though the histopathological techniques are labour intensive, cumbersome and

time consuming, particularly when there are automation equipments are not available; however, their use in diagnosis of diseases is unequivocal.

• Some of the areas where histopathological diagnosis is helpful are described as follows:

• This is useful in establishing the pathogenesis and pathology of any disease caused by bacteria, virus, chlamydia, rickettsia, mycoplasma, parasite, toxin, poisons etc.

• There are certain diseases in which histopathological examination of tissues is the only alternative to diagnose the disease. e.g. Bovine spongiform encephalopathy. The agent of this disease takes a very long incubation period and very difficult to isolate and there is no immune response and inflammation in animal. Therefore, histopathology remains the only alternative for confirmatory diagnosis.

• In some cases, the tissues from dead animals are only available material for laboratory diagnosis. This may occur either due to lack of time or due to negligence for not collecting the material for serological tests or isolation studies. Sometimes the transportation of material from remote areas destroys the other material and the tissues fixed in formalin only remains for making diagnosis. In all such cases the histopathological examination has its pivotal role.

• The histopathological procedures produce permanent slides, which can be stored for a longer period and one cannot manipulate the findings; therefore, it is considered best reliable technique.

• The histopathological techniques are useful in carrying out the retrospective studies. The unstained slides and blocks can be stored for indefinite period; which can be examined even after many years for further studies.

• The presence of causative agents can also be demonstrated in tissue sections using routine histopathological techniques or special stainings. In this Gram's staining procedures are used for demonstration of bacteria while viral inclusions are demonstrated using hematoxylin and eosin or other staining techniques like Macchiavello's stain or Mann's methylene blue eosin method. The Negri bodies are demonstrated by Seller's stain in case of rabies in animals. In such cases, the isolation of causative agent or their serological examination does not require; since the presence of causal agent in infected tissues gives a confirmatory diagnosis.

• The detection of chemicals in tissues like enzymes, lipids etc. is included in histochemical examination; which not only describe the structural changes but also gives idea about the functional status of the organ.

Collection of Samples: 1. Small piece of tissue (as early as possible) 2. Piece is removed with sharp knife in a particular orientation.3. Size of piece=1cm to achieve better penetration of fixative.4. Washing the specimen with normal saline to achieve maximum penetration

of fixative. 5. At the time of tissue collection, it should be kept in mind that the

representative tissue piece should include the part of lesion and a part of normal tissue,

6. Tissue pieces for histopathological examination should be collected from all the organs.

7. Tissues should be collected directly in the fixative and not in any other pot or water.

8. The tissue pieces from hollow organs like intestines, oviduct etc should be cut transversely and placed on a hard paper.

9. The representative tissue should not be collected from such damaged portions.

10. The tissues from encapsulated organs should be collected alongwith capsule or covering.

Labelling:• Tissue is accompanied by a tag or label, bearing the lab. number given to

specimen at start, through all stages. • Thin white card with writing in soft pencil withstands all the fluids used in

tissue processing.

Sample collection and preservation

Fixation:• Hardening of the organ in such a way that its

original form is retained and its constituents do not spread out while cutting.

• Small block of tissue is immersed in Neutral Buffered formalin 10% (at least 10 times the volume of tissue)

Properties of Fixation• Preserve protoplasm with least alterations

(autolysis, bacterial decomposition)• To kill the cells suddenly, uniformly(life like

manner)• It coagulates the cell proteins and intercellular

bodies in a life position.• It prevent cell from shrinkage and deleterious

effect of cutting.• Facilitate proper staining.• Preserve tissue from drying.

Commonly used Fixatives• 10% Formalin Solution.• Buffered Neutral Formalin Solution.• Formalin Alcohol. (glyogen, good cytoplasmic fixation)• Zenker Fluid. (bone marrow aspirates)• Bouin’s Fluid. (embryological specimens, Purkinje cells)• Acetone. (Rabies)• Ethyl Alcohol. (glycogen, pigments, amyloid, hylaine,

elastic fibers and bacteria)• Glacial acetic acid. (rapid fixation, swelling of cells)• Potassium Dichromate. (cytoplasm, not nucleoproteins)• Heat. 5-10 mm thick piece of tissue, boil in 0.9 percent

Nacl solution for 2-3 min.

10% Formalin Solution37%-40% Formaldehyde 100 mlDistilled water 900 ml

Neutral buffered formalin37%-40% Formaldehyde 100 mlDistilled water 900 mlSodium Phosphate monobasic 4 gmSodium Phosphate dibasic (anhydrous) 6.5 gm

Trimming of tissue • Tissue, enclosed in tissue cassette

and placed in basket of automatic tissue processor.

Washing of the Tissue• Washing of tissue under running

tape water overnight (12 hours) remove excessive Fixative Solution.

PROCESSINGDehydration•The purpose of dehydration to remove fixative and water from the tissue and replace them with dehydrating fluid.•There are a variety of compounds many of which are alcohols. Several are hydrophilic so attract water from tissue.Types of dehydrating agents:•Ethanol•Methanol•Acetone

• To minimize tissue distortion from diffusion currents, delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol.Ethyl alcohol 70% 2 hourEthyl alcohol 80% 1 hourEthyl alcohol 95% 1 hourEthyl alcohol (Absolute 1) .5 hourEthyl alcohol (Absolute 2) .5 hour

• To increase the process of dehydration, the tissue blocks should be agitated either mechanically in an automatic tissue processor or by shaking the container periodically.

• The volume of alcohol should be at least 50 times more than the tissue placed for dehydration.

Clearing:• It involves removal of dehydrating agent and replacement by

some fluid which is miscible with dehydrating agent and embedding medium.

Choice of a clearing agent depends upon the following: Choice of a clearing agent depends upon the following: • The type of tissues to be processed, and the type

of processing to be undertaken.• The processor system to be used.• Intended processing conditions such as temperature,

vacuum and pressure.• Safety factors.• Cost and convenience.• Speedy removal of dehydrating agent .• Ease of removal by molten paraffin wax .• Minimal tissue damage .

• Like ethanol, xylene should also be kept in tightly stoppered bottle to prevent the evaporation.Equal parts of Absolute alcohol +Xylene .5 hour

Xylene I 1 hourXylene II 1 hour

• If xylene is not available then benzene may be used for 3 hr as its action of clearing is slower than xylene.

• On complete clearing, the tissue becomes transparent, then they should be transferred in paraffin wax for impregnation.

Wax Impregnation/Infiltration•It is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning.•Tissue is impregnated with melted wax.•Xylene +Paraffin 1 hour•Paraffin wax I 2 hours•Paraffin wax II 3 hours

BLOCKING/ EMBEDDING• At the time of transfer of tissue blocks

from xylene II, the paraffin wax must be kept at (60-62°C) in liquid form at a temp. 2 or 3 above the melting point .

• For impregnation/dispension into mould to depth more than adequate to cover the tissue block.

• The tissue is introduced with warmed forceps.

• As soon as a film of solid has formed on the surface

• Submerged beneath cold water to

hasten solidification of wax.

MICROTOMY

• Paraffin blocks are cooled before sectioning.

• The section thickness is adjusted at 15µm to trim away any surplus wax and to expose a suitable area of tissue.

• On exposing the suitable area, thickness is set at 2-5um.

• Sections adhere to each other to produce a ribbon.

• First section is held with fingers or forceps and the last being detached by means of a small camel hair brush.

FLOATING &PICKING UP SECTIONFLOATING &PICKING UP SECTION• The ribbon is floated on the water

surface heated at 45 C, with the trailing end of the ribbon making contact with the water first.

• The ribbon may be split into individual or group of sections by the use of forceps

• Picking up a section on a slide is

achieved by immersing the slide vertically in the water to three quarter of it’s length

DRYING SECTIONSDRYING SECTIONS• The slides are placed on the hot

plate adjusted at melting point of wax for ten minutes.

• Drying in oven at the same temperature should be done for 45 minutes.

PRE STAINING PREPARATIONPRE STAINING PREPARATIONDeparaffinization with XyleneDeparaffinization with Xylene

• Xylene I Xylene I 3 minutes3 minutes

• Xylene IIXylene II 3 minutes3 minutes

RehydrationRehydration• Absolute ethyl alcoholAbsolute ethyl alcohol II 3 minutes3 minutes

• Absolute ethyl alcoholAbsolute ethyl alcohol IIII 3 minutes3 minutes

• Ethyl alcohol 90%Ethyl alcohol 90% 2 minutes2 minutes

• Ethyl alcohol 70%Ethyl alcohol 70% 2 minutes2 minutes

• Distilled waterDistilled water 3 minutes3 minutes

• Wash in tap water and rinse in Distilled waterWash in tap water and rinse in Distilled water

STAININGSTAINING

• Harris haematoxylinHarris haematoxylin 15 min15 min• Distilled waterDistilled water 5 min5 min• Acid alcohol 70%Acid alcohol 70% 4-10 dips 4-10 dips• Ammonia Alcohol Ammonia Alcohol 5 min5 min• Wash in running tap waterWash in running tap water 5 min or less5 min or less• EosinEosin 2 min 2 min• Wash in running tap waterWash in running tap water 1-5 minutes1-5 minutes

POST STAINING PROCEDURESPOST STAINING PROCEDURES

• Ethyl alcohol 95%Ethyl alcohol 95% 2 min2 min

• Ethyl alcohol 95%Ethyl alcohol 95% 2 min2 min

• Absolute Ethyl alcoholAbsolute Ethyl alcohol 2 min2 min

• Absolute Ethyl alcoholAbsolute Ethyl alcohol 3 min3 min

• Xylene I Xylene I 3 min 3 min

• Xylene IIXylene II 3 min3 min

Harris HaematoxylinHarris Haematoxylin

Haematoxylin crystalsHaematoxylin crystals 1 gm1 gm

Alcohol 95%Alcohol 95% 10 ml 10 ml

Potassium alumPotassium alum(Aluminum potassium sulphate)(Aluminum potassium sulphate) 20 gm 20 gm

Distilled waterDistilled water 200 ml200 ml

Mercuric oxideMercuric oxide 0.5 gm0.5 gm

Glacial acetic acidGlacial acetic acid 8.0 ml8.0 ml

Eosin solutionEosin solution• EosinEosin 1 gm 1 gm• Distilled waterDistilled water 100 ml 100 ml• Acetic acidAcetic acid 0.05 ml 0.05 mlAcid alcohol:Acid alcohol:• Ethyl alcohol 70%Ethyl alcohol 70% 990 ml 990 ml• HCl (concentrated)HCl (concentrated) 10 ml10 ml

Dilute ammonia solutionDilute ammonia solution• Distilled waterDistilled water 250 ml 250 ml• Ammonia (concentrated) Ammonia (concentrated) 2 drops 2 drops

• Acid dyeAcid dye: When the staining property is in : When the staining property is in the acid radical of the neutral salt, the the acid radical of the neutral salt, the stain is called an acid dye e.g., stain is called an acid dye e.g., eosineosin, , picric acid, azo dyes, tryan red, orange G.picric acid, azo dyes, tryan red, orange G.

• Basic dyeBasic dye: The coloring property is in the : The coloring property is in the basic radical of the neutral salt, the stain is basic radical of the neutral salt, the stain is called a basic dye e.g., called a basic dye e.g., hematoxylinhematoxylin, , toluidine blue, methylene blue.toluidine blue, methylene blue.

PRECAUTIONS PRECAUTIONS • Rapid fixation of fresh specimen is essential as post-mortem

degeneration markedly alters the appearance of the various cell types in tissue making interpretation unrewarding.

• Quality of nuclear staining begins to deteriorate and precipitate is formed in stored stain after a few months. At this stage, stain should be filtered and staining time should be increased.

• It is better to prepare a fresh batch of stain every month Preparation of Haematoxylin: • Haematoxylin is dissolved in the absolute alcohol• Alum is dissolved in the warm distilled water • Haematoxylin solution is added to the alum solution and

rapidly brought to boil• Mercuric oxide is then added• Stain is rapidly cooled by plunging the flask into cold water

and acetic acid is added when cold

MOUNTINGMOUNTING

• Mounting with canada balsam, Mounting with canada balsam, apply coverslip and examine under apply coverslip and examine under the microscope.the microscope.

HELPFUL HINTSHELPFUL HINTS• Lenses of your microscope should be cleanLenses of your microscope should be clean

• Make sure that fine adjustment knob is near the middle of its range of Make sure that fine adjustment knob is near the middle of its range of rotationrotation

• To start your observation using the lowest power objectiveTo start your observation using the lowest power objective

• By knowing the app. dia. Of RBC in the section, you can estimate the By knowing the app. dia. Of RBC in the section, you can estimate the size of other tissue components.size of other tissue components.

• Goat: 2.4 umGoat: 2.4 um• Dog: 4.9umDog: 4.9um• Chichen: 9.4um Chichen: 9.4um

• Average value based on total of 20-30 cell measured from different Average value based on total of 20-30 cell measured from different slide preparations slide preparations

ARTIFACTSARTIFACTS

FoldFoldKnife marksKnife marksSpacesSpacesStain precipitatesStain precipitatesShrinkage Shrinkage Air bubbles Air bubbles

examples of commonly occurring imperfections seen in examples of commonly occurring imperfections seen in

slide preparationsslide preparations

STAIN PRECIPITATESSTAIN PRECIPITATES

• Occasionally , solution Occasionally , solution accumulate precipitate that accumulate precipitate that may stick to the surface may stick to the surface tissue section during tissue section during staining procedurestaining procedure

SEPARATION (SPACES)SEPARATION (SPACES)

• Tissue may be subjected to Tissue may be subjected to excessive pressure, tension excessive pressure, tension or shrinkage during or shrinkage during processing resulting in processing resulting in separations of tissue separations of tissue

KNIFE MARKSKNIFE MARKS

• Caused by defects in the Caused by defects in the microtome knife or by microtome knife or by accumulation of debris on accumulation of debris on the knife edgethe knife edge

FOLDSFOLDS

• Folds occurs when the Folds occurs when the tissue sections fail to spread tissue sections fail to spread properlyproperly