UNIT I

Introduction and Definition of Histopathology

1.1 Histology, Histopathology, Biopsy, Autopsy, Autolysis, Putrefaction

1.2 Reception, recording, labeling, and preservation of histological specimen

1.3 Various Terms associated with staining: Solvents, Mordants, Metachromasia, Accelerators, Progressive and regressive staining

1.4 Theory of staining (Routine), Use of controls in staining and their significance

1.5 Principle, mechanism, and various steps of routine stain (Haematoxylin and Eosin): Deparaffinization, Hydration, Nuclear Staining, Differentiation, Blueing, Counterstaining, Dehydration, Clearing and Mounting, Results

1.6 Automation: Use of automatic stainer and cover slipper

1.1 Histology, Histopathology, Biopsy, Autopsy, Autolysis, Putrefaction

Histology: Histology is the branch of medical science and biology to study microscopic structure of the tissues.

Histopathology: Histopathology is study of abnormal conditions in tissues and cells at a microscopic level.

Biopsy: Examination of tissues sample removed from the living body to examine presence of disease, cause of disease and extent of disease is called biopsy.

Autolysis: Self-digestion and destruction of cells and tissues by their own enzyme is called autolysis.

Putrefaction: Decomposition or breakdown of the organic matter of plant or animal origin by the microorganism is called putrefaction.

1.2 Reception, recording, labelling and preservation of histological specimen

Reception, recording, labelling and preservation are the important steps to for accurate histological study of the histological specimens.

• Reception of histological sample

Histological specimen must be deliverd to medical laboratory within one hour after their collection. Pathologists shall identify the histological sample. They will cross check the patient information available on the histological sample container and the documents submitted with the sample. Its main function is to create workflow for laboratory analysis.

Specimen container shall have the following information

1. Full name, age, sex, of patients and Medical record number.

2. Type of specimen (Anatomic site).

3. Date and time of histological sample collection

4. Location of the patient.

Documents with specimen (requisition slip or requisition form) shall have following information

1. Name Age, Date of Birth, Sex. of Patient, Location of the Patient,

2. Clinical history of the patient,

3. Medical Record Number,

4. Date and Time of Collection,

5. Type of Specimen (anatomic site),

6. Physician’s name, signature, and address of physician

The histological sample shall be rejected, If these information are not available.

• Recording of histological specimen

Medical laboratory staff shall record all relevant information available on histological sample container label, requisition slip and any other relevant information.

1. Unique identification number: Medical laboratory staff shall assign a unique identification number to the specimen.

2. Laboratory information centre: Laboratory staff shall upload all information to the laboratory information system. This system will help to track the specimen in the laboratory and to access testing/analysis report of the sample.

• Labelling of the specimen container

Medical laboratory staff shall label the received container with the unique identification number, Patient’ name, medical record number other relevant information as per the SOP of the medical laboratory.

Requisition slip received with the histological specimen shall also be labelled with the all information on label of the histological sample container.

• Preservation of the histological specimen

1. Fixation: Cellular structure of histological specimen shall be preserved by using fixatives. Commonly used fixatives are formalin (formaldehyde solution), alcohol, and Boulin’s solution. Fixatives slowly penetrate the histological specimen. It hardens and preserves the tissues. It also protects the tissues during other steps of processing. Selection of fixative depends upon types of tissues and diagnostic test to be performed on the histological specimen.

2. Processing and embedding: Histological specimen shall undergo series of processing such as fixation, dehydration, clearing and embedding in paraffin wax.

   Dehydration: Most of the histological specimens have water. Removal of moisture is called dehydration. Ethanol is miscible with water. Ethanol is used to dehydrate histological specimens. Ethanol is mixed with histological specimens in the series of increasing concentration in following orders 70% Ethanol for 15 minutes, 90% ethanol for 15 minutes, and 100% ethanol for 15 minutes. Process is continued till all moisture from histological specimens is removed.

   Clearing: Clearing is carried out to remove ethanol and wax from the histological specimens. A solvent is used that dissolve with ethanol and wax. It is called clearing agent. Clearing agent increases histological specimens’ transparency and optical clarity. Xylone is used as clearing agent.

   Wax infiltration (Tissue embedding): In this step, wax enter tissue sample. It provides support and stability to tissue sample. It allows easy cut of tissue sample into thin section without distortion. Paraffin wax based histological wax is most commonly used in histopathology. Melted Paraffin wax is used in the further processing of the histological specimen.

3. Storage: Storage of Histological specimen slide is most crucial to preserve tissue samples. After fixation and processed Histological specimen slides are stored in a slide folder or cabinets under controlled temperature and humidity. This prevents damage or degradation of Histological specimen slide. Access to Histological specimen slides should be restricted to authorised persons only.

1.3 Various Terms associated with staining:

Solvents, Mordants, Metachromasia, Accelerators, Progressive and regressive staining

1. Solvents: Solvent is used in staining process to dissolve or dilute the staining agent. Solvents are used to extract dye or stain from tissue or cells. Or to carry dye or stain into tissue or cells to be studied under a microscope. Several types of solvents are used in the staining technique. Selection of stain depends upon the nature of the dyes/staining agent. Examples:

a. Water: It is the universal solvent. It is used as a base to prepare stain solution. It is useful to dilute water-soluble dyes or stains.

b. Alcohol: Ethanol or methanol is commonly used alcohol as a solvent in the staining technique. It is useful to dissolve both water-soluble stains and alcohol-soluble stains. It is frequently used in Gram staining and acid-fast staining methods.

c. Xylene is a clear colorless liquid commonly used as a solvent in histological staining. Such as hematoxylin stain, and eosin stain. It helps to clear and dehydrate the tissue sample

d. Acetone: It is a volatile solvent used in microbiology staining procedures. It helps to fix and dehydrate the cells or tissues. It is commonly used in the Ziehl-Neelsen staining technique for acid-fast staining.

e. Dimethyl sulfoxide (DMSO): It is an organic solvent used in fluorescence staining techniques. It enhances the penetration of fluorescent dyes into the cells.

2. Mordants (Dye fixative): Mordant is an agent to increase the affinity of the dye or stain to the target cells or tissues. It helps to improve the intensity of the staining reaction. It allows better visualization and easy differentiation of cellular structures under a microscope.

It undergoes a complexation or precipitation reaction. It forms an insoluble complex or precipitate. This insoluble product binds with the dye or stain. This complex selectively binds to specific cellular or tissue components. It helps to improve the stability and affinity of the stain complex.

Several mordants are used in the microbiology and histopathology. The selection of mordant depends upon the staining techniques. Examples

a. Gram’s Iodine: It is a commonly used mordant in the Gram staining technique and differential staining technique. It forms a complex with the crystal violet dye. It helps to fix crystal violet dye within gram-positive bacteria cells.

b. Schiff's reagent: It is used as a mordant in histopathology staining technique. It is an ingredient of Periodic acid Schiff (PASS) staining to act as a mordant. It enhances the binding of the PAS dye to glycogen and mucopolysaccharides. It develops a strong magenta color.

c. Tannic acid: It is used in histopathology staining technique as a mordant. It is an ingredient of Mallory’s trichrome staining to act as a mordant. It helps to intensify the staining of collagen fibers. It forms an insoluble complex with aniline dyes used in the staining procedure.

d. Ferrous sulfate: It is used in histopathology staining technique as mordant. It is an ingredient of Prussian blue staining to act as a mordant. It forms a complex with potassium ferrocyanide. It intensifies the blue coloration in tissue containing iron deposits.

3. Metachromasia: Metachromasia is a phenomenon in histopathology staining technique in which dye/stain changes its original color in tissue or cellular components. Colour development is opposite to the color expected from stain/dye.

Metachromasia commonly develops in basic stains such as toluidine blue, methylene blue, or crystal violet in histopathological staining. These stains have positive charges that bind to negatively charged cellular components such as acidic polysaccharides or proteoglycans. This causes a color shift. This is called metachromatic staining.

The color change in Metachromasia due to interaction between stain and specific cellular component is called metachromatic shift or metachromatic effect. This metachromatic effect depends upon the capacity of the stain to form complex or aggregate with specific cellular components. The formation of this complex or aggregate changes the light absorption or emission property. This alters the color of the stain.

Metachromasia is useful in histopathological examination because it provides valuable information about the composition and distribution of specific cellular components.

4. Accelerators: Accelerators in staining techniques are the chemicals or methods to speed up the staining process and improve the quality of the stained sample. They are used to enhance the penetration of stain into cells or tissues, to increase the intensity of staining, and to reduce overall staining time. Accelerators are used in various steps of staining techniques such as fixation, permeabilization, and staining.

• · Fixation accelerators: Fixation is the initial step of the staining technique. Fixation preserves specimens and prevents decay of specimens, and cellular structure of specimens. Heat and chemicals are most commonly used as accelerators to speed up the fixation process. Heat fixation requires slow heating of the specimen to promote protein denaturation and cross-linking. It ensures better preservation of cellular components.

• Permeabilization accelerators: Permeabilization allows stains to penetrate into cells or tissues effectively. Some cells or tissues have natural barriers to stop stains. Accelerators such as detergent or enzymes are used to disrupt impermeable barriers and increase the penetration of stains into cells or tissues. Detergents such as Triton X-100 or Tween 20 are commonly used to solubilize lipid membranes of cells. This increases penetration stains into cells or tissues. Enzyme accelerator such as proteinase K or trypsin is used to digest protein. This also increases stain penetration into cells or tissues.

• Staining accelerator: Staining accelerator increases the rate of binding of stains to specific cellular components. It enhances their visibility. The following are used as staining accelerators:

  • Heat acceleration: Heating to stained slide increases the rate of binding of dyes with cellular components. Heat increases the diffusion of dyes into cells or tissues, and improves the binding affinity of stain with cellular components. and reduces the staining time

  • Microwave acceleration: Microwaving of slides during the staining process reduces staining time. Microwaving increases temperature and accelerates chemical reactions during the staining process. It increases stain penetration into cells and tissues. It improves the intensity of

  • Enzymatic acceleration: Some stains require enzymes to stain cells The addition of enzymes such as peroxidize or alkaline phosphatise can accelerate these reactions. This results in fast and intense staining.

5. Progressive staining: It is used in histology and microbiology to stain cellular components with varying degrees of intensity. It allows stain solution to act on the cells or tissues for a specified duration. During this duration, staining intensity slowly increases till the desired contrast is achieved.

Progressive staining is commonly used in several staining techniques such as hematoxylin and eosin staining (H&E Staining). This staining process differentiates nuclei from the cytoplasm of cells. The following steps are followed in the progressive staining.

• Stain Application: Apply the staining solution onto the specimen. The stain has a strong affinity with some cellular components while a weak affinity with other cellular components in the cell.

• Staining period: Incubate the sample with stain solution for a specific period. Stains may defuse and bind with cellular components at a slow rate. Stain intensity increases with time. Staining duration depends upon the nature of cells' cellular components or staining requirements.

• Continuous monitoring: The staining process is continuously monitored either visually or under a microscope. This determines the staining duration.

• Termination of staining: The staining process is terminated after achieving the required staining intensity. Staining is terminated by rinsing the specimen with a suitable solvent. Rinsing removes excess stain solution.

6. Regressive staining: Progressive staining is frequently used in microbiology. Regressive staining is not used in microbiology. It is mainly used in histology. It is used for selective staining of specific cellular or tissue components to develop optimal contrast and visibility. Adjustment of staining intensity and optimal contrast is possible. It allows selective visualization of specific structures. Regressive staining involves the over-staining of the entire stain followed by the removal of excess staining solution. This stains the cellular components up to the desired intensity. The following steps are followed in the regressive staining

Stain application: Apply stain solutions on the specimen to saturate the specimen.

• Excessive staining: Incubate the stained specimen for a longer duration for optimal staining. Ensure heavy staining of each component of the specimen.

• Differentiation: Remove excess stain solution from the specimen. This is called differentiation or decolorisation. A decolorizing solution is used to remove excess stain solution. The desired staining of cellular components is achieved by this differentiation/decolorization step.

• Monitoring: Closely monitor the differentiation step under a microscope

• Termination: Terminate the differentiation step after achieving the desired level of contrast. Terminate the differentiation step by rinsing the specimen by using a suitable solvent or buffer.

1.4 Theory of staining (Routine)

Staining is a technique in microbiology and histopathology to enhance the visualization of microorganisms and tissues under a microscope. Specific dyes or stains are used to bind with cellular components to improve contrast and identification of structure.

Gram staining and acid-fast staining are commonly used to stain microorganisms. Hematoxylin and eosin stains (H & E Stains) and some special stains are commonly used in histopathology.

Gram stain is mainly used to differentiate gram-positive bacteria and gram-negative bacteria. Gram stain uses crystal violet as a dye, iodine as a mordant, alcohol or acetone as a decolorizing agent, and safranin as a counterstain. Stain interacts with cellular components. Gram-positive bacteria accept the color of crystal violet and appear purple. Gram-negative bacteria do not accept crystal violet color. The decolorization step washes crystal violet from gram-negative bacteria. Gram-negative bacteria accept the color of safranin to appear pink or red.

Acid-fast stain is used in microbiology to identify acid-fast bacteria. These bacteria do not accept the color of conventional staining solution. Carbol fuchsin is used as a dye in the acid-fast stain technique. The application of heat or steam facilitates the penetration of crabolfuchsin into bacteria cells. Acid-fast bacteria retain the color of carbon fuchsin even after the use of a decolorizing agent. Acid-alcohol is used as decolorising agent. Non-acid fast bacteria do not retain the color of carbon fuchsin after the decolorization step. An acid-fast stain is used to differentiate acid-fast bacteria from non-acid-fast bacteria.

Hematoxylin and eosin stains (H & E Stains) technique is used in histopathology. Hematoxylin is a basic dye that stains acidic components of cells such as nucleic acid. It develops a blue-purple color. Eosin is an acidic dye that stains basic components of cells such as cytoplasm and extracellular components. It develops a pink color. Hematoxylin and eosin stains (H & E Stains) techniques increase visualization of cellular details. cellular architecture and pathological conditions.

Special stains are used in histopathology to visualize specific cellular components. Example: Periodic acid fast Schiff (PAS) stain to visualize fungal cell walls and carbohydrates such as glycogen or in the cell.

Use of controls in staining and their significance: Controls in the staining technique are reference standards against the staining procedure. It is used to compare observations and staining patterns. Controls in staining techniques are used to ensure the accuracy, reliability, and consistency of staining results. Commonly, the following types of controls in staining techniques are used.

1. Positive control: Positive controls in staining techniques contain the target organism or structure to be stained. It is used to confirm that the staining procedure is working correctly and interacting with targets appropriately.

2. Negative control: Negative control in the staining technique does not contain the target organism or structure. It is used to assess background staining. A negative control should produce no staining or minimal background staining. It helps to observe stain is specific to the target.

3. Reagent control: Reagent control in staining techniques uses staining reagents without the presence of a specimen. It ensures staining reagents do not develop unexpected staining patterns. Reagent controls also help to identify any problems in the use of staining reagents such as contamination or degradation of staining reagents.

4. Quality control: Quality control measures are used to verify the consistency and accuracy of staining techniques. It helps to identify any problem that affects staining results.

1.5 Principle, mechanism and various steps of routine stain (Haematoxylin and Eosin): Deparaffinization, Hydration, Nuclear Staining, Differentiation, Blueing, Counterstaining, Dehydration, Clearing and Mounting, Results.

Hematoxylin and eosin stains (H & E Stains) technique is most widely used staining technique in histopathology. There are several steps in H & E Stain technique. The followings are the principle and mechanism of H & E Stain technique steps:

1. Tissue preparation: Preparation of tissue section requires suitable medium (paraffin) to embed tissue ant to cut it into slices by using a microtome. Mount these tissue sections on a clean sterile glass slide. This is paraffin embedded tissue section.

2. Deparaffinization: Deparaffinization is the removal of the paraffin medium from the paraffin-embedded tissue section. Deparaffinization is carried out by dipping paraffin-embedded tissue sections into xylene or a xylene substitute. This step removes paraffin from xylene. Paraffin is soluble in xylene.

3. Hydration: The deparaffinized tissue section is hydrated to remove organic solvents and to make tissue suitable for the staining process. Alcohol of various strengths is used for this purpose such as ethanol 95%, ethanol 70%, and ethanol 50%. The deparaffinized tissue section is dipped in this alcohol strength in decreasing order. Alcohol has ability to mix with water and remove organic solvents from the Deparaffinized tissue section.

4. Nuclear staining: Hematoxylin is used as a dye to stain nucleic acid in the nucleus. Hematoxylin is a basic dye that stains acidic components in tissues such as the nucleus. Stained structure appears bluish-purple. Mechanism: A negative charge of an acidic structure attracts positively charged Hematoxylin. This causes staining of nucleic acid in the nucleus and relatively unstained cytoplasm.

5. Differentiation and bluing: Differentiation is a process to remove excess dye from the tissue section. This develops proper contrast and differentiation of tissue components. It is carried out by using an acid alcohol solution such as 1% hydrochloric acid in ethanol 70%. Acid alcohol solution removes excess hematoxylin from the cytoplasm. It does not affect the color of nucleic acid.

6. Bluing: Bluing is a step to treat the tissue section with a weak alkaline solution such as Scott’s tap water substitute or commercial bluing reagent after the differentiation step. The purpose of bluing is to change the color of the hematoxylin-stained nucleus from red to blue. This step neutralizes the remaining acid in the tissue section. It also stabilizes hematoxylin stain and increases color intensity.

7. Counterstaining: Eosin is used as counter staining solution. Eosin is an acidic dye that stains basic components of the tissue section such as cytoplasm and connective tissues. It develops a pink or red color in a stained structure. The tissue section is dipped in an eosin solution. Positively charged basic components of the cell attract eosin molecules. This step helps to differentiate cytoplasm and connective tissues from nuclei inside the tissue section.

8. Dehydration and Clearing: Dehydrate tissue sections using graded alcohol after eosin staining. The purpose of dehydration is to remove excess water and make the tissue section ready for the mounting step. It is just the opposite of hydration.

9. Mounting: It is the last step. Place a cover slip over the stained tissue section. Place mounting medium, such as synthetic resin. It sticks the coverslip with the glass slide. It also preserves the stained tissue section to be examined under a microscope.

10. Report: Submit a report of tissue section examination under a microscope.

1.6 Automation: Use of automatic stainer and cover-slipper

Automatic stainer and cover slipper are essentials to automate and streamline the staining and coverslipping process for histological slides. Automatic stainer and cover slipper improve efficiency, consistency, and quality control in laboratory workflow.

1. Automatic stainer: An automatic stainer is designed to automate the staining process of histological slides. Automatic stainer has the following advantages over manual staining of histological slides.

a. Standardised staining: An automatic stainer eliminates errors in staining due to variations in the manual staining process. It accurately controls the duration, intensity, and sequence of staining steps. It results standerdised staining in all histological slides.

b. Time efficiency: An automatic stainer efficiently reduces the time to prepare stained slides. It can process multiple slides in one go. This is beneficial to handle a large number of slides in busy medical laboratories.

c. Enhanced reproducibility: An automatic stainer accurately controls the timing and application of staining reagents. All slides receive the same treatment. Thus it minimises variation in staining of slides.

d. Workflow optimization: An automatic stainer optimizes the workflow in the laboratory, minimizes repetition of work, and the laboratory technician shall have enough time to perform other works in the laboratory.

e. Quality control: An automatic stainer has built-in software to control, monitor, and record staining parameters such as the use of reagents, temperature, incubation period, etc.

2. Automatic cover slipper: An automatic cover slipper is a device to place a coverslip on stained tissues on glass slides automatically. Cover slip is used to protect stained tissues on glass slides during microscopic examination. An automatic cover slipper has the following advantages:

a. Consistent cover slipping: An automatic cover slipper maintains uniformity and consistency in coverslipping. It applies cover slip on stained tissue with controlled pressure and accuracy. This minimizes the risk of air bubbles, uneven mounting, or damage to stained tissues.

b. Time efficiency: Manual cover slapping is a dedicated and time-consuming process. It can handle multiple slides at one time. It reduces the time required to place a coverslip on stained tissues on glass slides, improves the efficiency of the laboratory, and reduces the labor cost.

c. Improved slide quality: Properly aligned cover slip and properly placed cover slip improve the quality of stained slides. Anmatic cover slipper removes excess mounting medium present on the tissue. This further enhances the stained slides' clarity.

d. Occupational safety: Mounting media and adhesive agents are used to place a coverslip on stained tissues. These mounting media and adhesive agents are toxic and may harm laboratory technicians during manual use. An automatic cover slipper reduces the exposure of laboratory technicians to mounting medium and adhesive agents. This reduces health risks to laboratory technicians.

e. Slide preservation: An automatic cover slipper places the coverslip on stained tissues properly. This protects the stained tissues from damage, dust, and humidity. This prevents dryness of the mounting medium, maintains the integrity of stained tissues, and extends the shelf life of stained tissues on slides to prevent dryness.

Author: Dr Pramila Singh