3.1 - HISTOPATHOLOGY (NEW)

Clinical Pathology

Pathology

involves mostly the examination of bodily fluids for various laboratory tests that help diagnose conditions such as infections, anemia, and diabetes

Anatomic Pathology

structural changes

Pathology

focuses on tissue examination through both histopathologic and cytopathologic techniques, it is primarily concerned with identifying _ _ in tissues and cells to diagnose diseases

Histopathology

Can be defined as a branch of anatomic pathology which deals with the examination of a biopsy or surgical specimen to spot signs and characteristics of diseases in tissues

Routine Biopsy (Routine Histopath)

Cytology (Pap’s Smear)

Fine Needle Aspiration Biopsy (FNAB)

Histopathology

3 Areas in Histopathology:

Patient full name

Birthdate

Specimen label

Cold Ischemia Time

Histopathology

Routine Histopathology

Check for identifiers: (4)

Cold Ischemia Time

Histopathology

Routine Histopathology

Check for identifiers: 

time specimen is obtained to time it is submerged to formalin

Grossing

Histopathology

Routine Histopathology

Involves a careful examination and description of the specimen that will include the appearance, the number of pieces and their dimensions

3-4 mm

Histopathology

Routine Histopathology

Grossing

Specimen sections should be cut into small pieces with a measurement of _-_ _ to fit in the tissue cassette

Tissue processor

Histopathology

Routine Histopathology

Tissue Processing

an automated instrument that prepares tissue samples for microscopic examination

paraffin wax

Histopathology

Routine Histopathology

Tissue Processing

Tissue Processor — Allows the specimens to be infiltrated with a sequence of different solvents finishing in molten _ _

Tissue Transfer (open system, dip & dunk)

Histopathology

Routine Histopathology

Tissue Processing

2 Types of Tissue Processor by their Operation:

physically move tissue baskets from one station to another, submerging the tissues in a series of different reagents

Fluid Transfer (closed system, enclosed)

Histopathology

Routine Histopathology

Tissue Processing

2 Types of Tissue Processor by their Operation:

the tissue remains in a single process chamber or retort, while the reagents are pumped into and out of the chamber as needed

Fixation

Histopathology

Routine Histopathology

Most crucial/critical step in routine histopathology

autolysis

putrefaction

Histopathology

Routine Histopathology

Fixation

Primary Function: Preserve the morphology of cells by inhibiting _ (enzymatic activity) and _ (bacterial activity)

harden

protect

Histopathology

Routine Histopathology

Fixation

Secondary Function: To _ and _ the tissue

proteins

soluble, insoluble

Histopathology

Routine Histopathology

Fixation

• Stabilizes _

• Changes _ contents into _ substances

Additive Fixative

crosslinks

Histopathology

Routine Histopathology

Fixation

• enables proteins to combine chemically (_) with a fixative molecule and the protein then becomes insoluble

Non-additive Fixative

Histopathology

Routine Histopathology

Fixation

• fixative molecules does not combine with the protein, but causes the protein to become less intimate with water and becomes more reactive

Temperature

Size

Volume Ratio

Time

Choice of fixative

Penetration

pH

Osmolality

Histopathology

Routine Histopathology

Fixation

• Factors affecting fixation: (8)

Dehydration

Histopathology

Routine Histopathology

Removal of excess water/moisture from the tissue/specimen

increasing grades

Histopathology

Routine Histopathology

Dehydration

Must be done gradually in _ _ of the reagent (70-95-100%)

hard and brittle tissues

Histopathology

Routine Histopathology

Dehydration

Excessive dehydration results in ? that are difficult to section

soft and mushy

Histopathology

Routine Histopathology

Dehydration

Incomplete dehydration results to unfulfilled clearing action; ? blocks

fungal

Histopathology

Routine Histopathology

Dehydration

Water or moisture may attract _ accumulation in tissue during storage

Ethyl Alcohol

Methanol

Isopropanol

Histopathology

Routine Histopathology

Dehydration

• Commonly used reagent: _ _ (for routine Histopath)

  • _ – for Pap’s

  • _ – never absolute, always w/ 1% H2O

Decalcification

Histopathology

Routine Histopathology

Process of complete removal of calcium or lime salts from tissues (bones, teeth, calcified tissues)

after, before

Histopathology

Routine Histopathology

Decalcification

Should be done _ Fixation and _dehydration

End Point Decalcification

Histopathology

Routine Histopathology

Decalcification

the most important step in the decalcifying process; It must always be carefully monitored

Physical/Mechanical Method

Chemical Method

X-Ray/Radiographic Method

Histopathology

Routine Histopathology

Decalcification

3 Basic Methods in Determining the Endpoint

Acid Method

Histopathology

Routine Histopathology

Decalcification

Methods of Decalcifying

Most widely used

Nitric Acid

Histopathology

Routine Histopathology

Decalcification

Methods of Decalcifying

1. Acid Method

   • Most widely used

   • _ _ –most commonly used and is the fastest, recommended for routine purposes

Chelating Agents

Histopathology

Routine Histopathology

Decalcification

Methods of Decalcifying

• Combines with calcium ions and other salts forming complexes and facilitates removal of calcium

EDTA

immunohistochemistry

slow

Histopathology

Routine Histopathology

Decalcification

Methods of Decalcifying

1. Chelating Agents

• Combines with calcium ions and other salts forming complexes and facilitates removal of calcium

• _ – most common, can be used in _ procedures, causes less damage to tissues, considered a _ decalcifying agent

Ion Exchange Resins

Histopathology

Routine Histopathology

Decalcification

Methods of Decalcifying

Ammonium form of resins hastens decalcification by removing calcium ions, keeping the solution free of calcium and speeds up reactions

Ammonium form

Histopathology

Routine Histopathology

Decalcification

Methods of Decalcifying

1. Ion Exchange Resins 

   • _ _ of resins hastens decalcification by removing calcium ions, keeping the solution free of calcium and speeds up reactions

Electrophoresis

Histopathology

Routine Histopathology

Decalcification

Methods of Decalcifying

Process where calcium ions are attracted to negative electrode and removed from the decalcifying sol’n

small specimens

Histopathology

Routine Histopathology

Decalcification

Methods of Decalcifying

1. Electrophoresis

• Process where calcium ions are attracted to negative electrode and removed from the decalcifying sol’n

• Satisfactory for _ _ – has a strong potential for tissue destruction and total loss of cellular detail and stainability

Clearing

Histopathology

Routine Histopathology

Removal of dehydrating agent from the tissue and is replaced with an intermediate solvent that is miscible with both ETHANOL and PARAFFIN WAX

De alcoholization agents

Histopathology

Routine Histopathology

Clearing

primary purpose is to remove the alcohol and to make the tissue receptive to the infiltration medium

Incomplete dehydration

Histopathology

Routine Histopathology

Clearing

soft and mushy tissues

Prolonged in clearing

Histopathology

Routine Histopathology

Clearing

hard and brittle tissues

increasing the refractive index

Histopathology

Routine Histopathology

Clearing

Other main function: To make the tissue transparent by ? for us to be able to view the specimens under microscope

Xylene

Histopathology

Routine Histopathology

Clearing

Commonly used reagent: _

Xylene

Histopathology

Routine Histopathology

Clearing

most widely used clearing agent

15-30

cloudy

Histopathology

Routine Histopathology

Clearing

• Xylene – most widely used clearing agent

  • Most rapid clearing agent (_-_ mins), however intolerant with water left in the tissue

  • Xylene turns _ in the presence of water

Toluene

1-2

Histopathology

Routine Histopathology

Clearing

• _ – does not overharden tissue and can store tissue overnight without harm

  • Clearing time (_-_ hours)

  • May be a substitute for xylene or benzene but is more flammable and volatile

Benzene

15-60

aplastic anemia

Histopathology

Routine Histopathology

Clearing

• _ – very fast acting (_-_ mins) and does not overharden tissues however, it hardens muscle, tendon, and uterus more than toluene

• Excessive exposure may be very toxic and carcinogenic, affecting blood and bone marrow (_ _)

Chloroform

Histopathology

Routine Histopathology

Clearing

• _ – for tough tissues (skin, fibrinoid, decalcified tissue, nervous tissue, lymph nodes and embryos) it causes minimum shrinkage and hardening than xylene

  • Does not make tissue transparent; Toxic through long exposure

Universal Solvents

Histopathology

Routine Histopathology

Reagents that avoid the use of two solutions; _ _ perform both the dehydrating and clearing steps

Dioxane

Tertiary butanol

Tetrahydrofuran

Histopathology

Routine Histopathology

Universal Solvents

• _ – faster dehydrant

• _ _ – expensive

• _ – best among the universal solvents

  • Useful in the reprocessing of inadequately dehydrated and cleared tissue

  • Deparaffin → Clearing → Water

Impregnation (Infiltration)

Histopathology

Routine Histopathology

Process in where the clearing agent is completely removed

Fill the tissue cavities and hold cells and intercellular structures in their proper state while thin sections are cut

soft and mushy

Histopathology

Routine Histopathology

Impregnation (Infiltration)

Inadequate impregnation: Tissue becomes ? resulting in poor sectioning

Paraffin

Celloidin

Gelatin

Plastics

Histopathology

Routine Histopathology

Impregnation (Infiltration)

Types of Impregnation Medium (4)

Paraffin Wax

Histopathology

Routine Histopathology

Impregnation (Infiltration)

Most common and best medium for routine tissue processing

56-60

Histopathology

Routine Histopathology

Impregnation (Infiltration)

Paraffin Wax

Melting point: _-_ C

increases

Histopathology

Routine Histopathology

Impregnation (Infiltration)

Paraffin Wax

Melting point _ – paraffin becomes harder and better supports tissue. Thinner sections can be obtained but ribboning is more difficult

decreases

Histopathology

Routine Histopathology

Impregnation (Infiltration)

Paraffin Wax

Melting point _ – paraffin becomes soft and provides less support for hard tissues. Thin sections are difficult to obtain, but ribboning becomes easier

Celloidin

Histopathology

Routine Histopathology

Impregnation (Infiltration)

purified form of nitrocellulose, recommended for large hollow or hard tissue/organs which tends to collapse

Gelatin

histochemical and enzyme

Histopathology

Routine Histopathology

Impregnation (Infiltration)

Rarely used

When dehydration is to be avoided, for _ and _ studies also for delicate/friable specimens and frozen sections

Plastic (epoxy resins)

Histopathology

Routine Histopathology

Impregnation (Infiltration)

usually for Electron Microscopy

Diamond knife

Glass knife

Histopathology

Routine Histopathology

Impregnation (Infiltration)

• Plastic (epoxy resins) – usually for Electron Microscopy 

  • _ _ – 60 to 90 nm thick can be cut

  • _ _ – 0.5 um sections for LM

Embedding/Casting/Blocking

Histopathology

Routine Histopathology

Involves enclosing the tissue in the infiltration medium at a temperature 5 to 10°C above the melting point and then cooled rapidly at -5°C allowing the medium to solidify

Orientation

Histopathology

Routine Histopathology

Embedding/Casting/Blocking

most important/critical step in Embedding

one block at a time

forceps metastasis

Histopathology

Routine Histopathology

Embedding/Casting/Blocking

• Why Critical?

  • Certain techniques need to be practiced to achieve precise diagnosis for the pathologist

  • Example:

    • Hard tissue, such as bone, section more easily if embedded diagonally in the mold and not parallel with the mold edges

    • One should practice ? to avoid confusion when there is an interruption while embedding

    • Embedding forceps should be wiped with gauze between blocks to avoid “_ _”

Trimming

Histopathology

Routine Histopathology

Process of removing excess wax after embedding

Coarse Trimming

Histopathology

Routine Histopathology

Trimming

2 Types of Trimming:

sides, tips and the bottom of the tissue are trimmed to remove the bulk of the paraffin surrounding the block forming a truncated pyramid

Coarse Trimming

Histopathology

Routine Histopathology

Trimming

2 Types of Trimming:

Achieved by making continuous, rapid cuts with the microtome, usually at a thicker setting, to expose the tissue

Fine Trimming

Histopathology

Routine Histopathology

Trimming

2 Types of Trimming:

careful and precise cutting of thin layers from the block until the paraffin is just a few millimeters thick around the tissue of interest

Sectioning/Cutting

Histopathology

Routine Histopathology

Process where in a tissue block is placed and cut into uniformly thin slices (sections) using a microtome to facilitate studies under the microscope

Rotary

Histopathology

Routine Histopathology

Sectioning/Cutting

Types of Microtome

most common type for routine histopathology

Uses a stationary blade; while the block is moved in a vertical motion to initiate sectioning

Sliding (or Base-Sledge)

Histopathology

Routine Histopathology

Sectioning/Cutting

Types of Microtome

features a knife that moves on a track while the tissue block is clamped in place

Well-suited for large and hard blocks

Often preferred for cutting hard tissues( bone, or teeth)

Cryostat (Freezing)

Histopathology

Routine Histopathology

Sectioning/Cutting

Types of Microtome

a specialized version of a rotary microtome, used to cut unfixed, fresh, or frozen tissue sections for rapid diagnosis

Ultramicrotome

Histopathology

Routine Histopathology

Sectioning/Cutting

Types of Microtome

designed for extreme precision, an ultramicrotome cuts extremely thin sections

Required for transmission electron microscopy

It often uses a glass or diamond knife

4 to 6 micra

10 to 15 micra

Histopathology

Routine Histopathology

Sectioning/Cutting

Type of Section	Usual Thickness
Paraffin Section	?
Celloidin Section	?
Frozen Section	Approximately 10 micra TAT: 15-30 mins
Ultrathin Section (used for EM)	Semi-thin: 0.5 to 1 micron Ultrathin: 50 to 120 nm

10 micra, 15-30 mins

0.5 to 1 micron, 50 to 120 nm

Histopathology

Routine Histopathology

Sectioning/Cutting

Type of Section	Usual Thickness
Paraffin Section	4 to 6 micra
Celloidin Section	10 to 15 micra
Frozen Section	Approximately ? TAT: ?
Ultrathin Section (used for EM)	Semi-thin: ? Ultrathin: ?

Staining

Histopathology

Routine Histopathology

Process of applying dyes on the sections to observe the architectural pattern of the tissue and physical characteristics of the cell

Direct Staining

Histopathology

Routine Histopathology

Methods of Staining:

process of giving color to sections using aqueous or alcoholic dyes

Indirect Staining

Histopathology

Routine Histopathology

Methods of Staining:

action of the dye is intensified by application of another agent or a mordant

Mordant

Histopathology

Routine Histopathology

Methods of Staining:

a link or a bridge between tissue and dye to make staining reaction possible

Accentuators

Histopathology

Routine Histopathology

Methods of Staining:

hastens the speed of staining by increasing staining intensity and selectivity

Progressive Staining

Histopathology

Routine Histopathology

Methods of Staining:

staining in a definite sequence

Solutions are applied for a specific period of time until sufficient color is attained

NO DECOLORIZATION or washing is required (H&E Frozen Section)

Regressive Staining

Histopathology

Routine Histopathology

Methods of Staining:

section is over stained to ensure all the target structures are thoroughly stained

Excess stain is then removed or decolorized from the unwanted parts of the tissue

This process allows for precise control over the final stain intensity

Hematoxylin

Histopathology

Routine Histopathology

Routine Histopath Staining

Most valuable and most widely used nuclear staining reagent

primary stain

basic

Haematoxylon campechianum

Hematein

Histopathology

Routine Histopathology

Routine Histopath Staining

Hematoxylin

The _ _ involved in routine H/E

A _ dye that binds to the acidic components of the cell

Extracted from the logwood _ _, which has no staining properties

_ is the active coloring agent of hematoxylin; Formed from hematox oxidation process (ripening)

Eosin

Histopathology

Routine Histopathology

Routine Histopath Staining

Most widely used counterstain, cytoplasmic stain in routine H/E staining

An acidic dye that binds to positively charged (basic) components within the cells and tissues

Regressive

Histopathology

Routine Histopathology

Routine Histopath Staining

Principle Method used: _

Bluing Agent

Histopathology

Routine Histopathology

Routine Histopath Staining

a mildly alkaline solution to convert the hematoxylin dye from a reddish color to a desirable blue or purple color

Bluing

Acid alcohol

Ammonium water

Histopathology

Routine Histopathology

Routine Histopath Staining

_ helps to reveal and enhance the detail within the nucleus, which is crucial for pathologists to interpret the tissue

_ _ – decolorizer

_ _ – bluing agent

Giemsa stain

Alcian Blue stain

Periodic Acid Schiff stain

Histopathology

Routine Histopathology

Routine Histopath Staining

SPECIAL STAINS
Some special stains involved in SLMC-QC
_ _	Detection of H. pylori
_ _ _	Acidic mucins
_ _ _ _	Glycogen
Silver stain (GMS)	Fungal
Fite Faraco (AFB)	Acid Fast Bacteria
Perl’s stain	Iron
Reticulin stain	Reticulin fibers

Silver stain (GMS)

Fite Faraco (AFB)

Perl’s stain

Reticulin stain

Histopathology

Routine Histopathology

Routine Histopath Staining

SPECIAL STAINS
Some special stains involved in SLMC-QC
Giemsa stain	Detection of H. pylori
Alcian Blue stain	Acidic mucins
Periodic Acid Schiff stain	Glycogen
_ _	Fungal
_ _	Acid Fast Bacteria
_ _	Iron
_ _	Reticulin fibers

Mounting

Histopathology

Routine Histopathology

Process of applying a cover slip to the tissue slide using a mounting medium

Canada balsam

Histopathology

Routine Histopathology

Mounting

• Resinous Mounting Media

  • Natural resin:

    • _ _ – acidic and causes fading of some stains

    • Taking months to harden and tends to yellow with age

Eukitt

Histopathology

Routine Histopathology

Mounting

• Resinous Mounting Media

  • Synthetic resin:

    • _

      • Hardens quickly

      • Neutral in action

      • Does not yellow with age

Pencil (lead/graphite)

diamond pencil

Histopathology

Routine Histopathology

Labelling

_ for frosted slides and _ _ for non frosted slides

Never use an inked pen because staining has alcohol in its process that may remove the labels

Cytology

Introduction to Cytology/Cytopathology

study of microscopic appearance of cells

type

morphology

inappropriate cell

Introduction to Cytology/Cytopathology

• Goal:

  • To identify the _ of cells 

  • Detect changes in the _ of the cell 

  • Detect the presence of _ _

Cytology

Introduction to Cytology/Cytopathology

• _ is used as a “screening tool”

  • Healthy individuals who are at risk of a particular disease

  • E.g.: Cervical cytology (primary screening tool)

    • CIN (Cervical Intraepithelial Neoplasia)

Diagnostic Cytology

Introduction to Cytology/Cytopathology

• used for symptomatic patients (w/ suspicion of having malignant disease)

Exfoliative Cytology

Aspiration Cytology

Introduction to Cytology/Cytopathology

Preparation Techniques

CLASSIFICATION OF SPECIMEN COLLECTION
_ _	_ _
Collection of cells that have spontaneously shed from the surface of a tissue	Forcible removal of cells from lesion or masses using a needle and syringe
(Malignant cells: One of cell characteristic is the loss of adhesiveness)

Non-invasive

Minimally invasive

Introduction to Cytology/Cytopathology

Preparation Techniques

• Techniques used:

  • _-_ – easy to collect (urine, sputum)

  • _ _ – bronchial washings, FNAB