TOPIC 1: Introduction to Histopathology

Histopathology

A branch of pathology that involves the examination of tissued and cells at a microscopic level to diagnose diseases and understand their underlying structural changes

Histopathologic techniques

Deals with the preparation of tissued for microscopic examination

Autopsy - Non living

Biopsy/ Surgical- Living

Cytology- Cells

Type of Specimen in Histopathology

1. Adhere to policies and procedures

2. Report problems immediately to the pathologist

3. Perform proper histopathologic techniques

4. Submit best quality slides to pathologist with attention to proper numbering, labeling, sequence of slides corresponding to SP request

5. Assist the pathologist in maitaing quality practice in the histo lab

Role of Histotechnologist

1. Numbering

2. Fixation

3. Decalcification ( Optional)

4. Dehydration

5. Clearing

6. Impregnation

7. Embedding

8. Trimming

9. Section cutting

10. Staining

11. Mounting

12. Labelling

12 steps of Convention/Manual Tissue Processing

Numbering

Process of identifying the specimen without writing the name on the specimen

Fixation

• First and most critial step

autolysis and stabilizes

Fixation prevent _____ and______ tissue

10% Neutral Buffere Formalin

Most common fixatinve

37%

What is the concentration of pure formalin

Decalciication

Optional process done when the tissue sample has high level of calcium

Nitric Acid (Most common)

• Hydrochloric Acid

• Formic Acid

• Picric Acid

• Acetic Acid

• Citric Acid

Most common agents for decalcification and give the 5 other agents

Dehydration

Removes the water and unbound fixative from the tissue

increasing

Dehydration uses alcohol with ______ concentration

Ethanol

Most common Dehydrating Agent

Over Processing

Artifact-shrinkage, parched eath effect and abnormal staining dry brittle tissue

Incomplete dehydration

Impair penetration of clearing agent leaving the specimen soft and non receptive to parafic wax infiltration

Clrearing

Displaces dehyrating solution, making the tssue component receptive to the infltrating medium

Xylene

Xylene 20 min

Xylene 20 min

Xylene 45 min

Most wide used clearing agent and its 3 different immersions

Impregnation or Filtration

permeated tissue with support medium

Paraffin wax

liquid ad 60 degress celcius and allowed to cool at 20 degrees celcius and is the most common infiltratiing medium

60 degrees celcius- liquid

20 degrees celcius- solid

Degrees at which paraffine in liquid and solid

4 mm

Paraffin wax 30 min

Paraffin wax 30 min

Paraffin wax 40 min

WHat thickness the specimen and its time exposed to paraffine wax

Incomplete Infiltration

soft and crumbly tissue is caused by -__

Hard and Brittle tissue

Too much time in high temperature wax- _____

Embedding

Orientation of the tissue sample in a support medium to create a tissue block suitable for sectioning

2-4 degreees above

Temperature in relation to paraffines wax’s melting point

Colde plate

Where is cut mol palced to solidify?

overheating

hardening and distortion of tissues in embedding

Trimming

Happens before section cutting and aims to completely expose the paraffin-embedded tissue in order to obtain a representative section

Section-cutting
- Tissue Ribbon

Embedded tissure are cut into section that are thin enought to be placed on a slide using a microtome.

what are is its product

3-5 um

Thickness of tissue ribbon

5-10 degress ceciusAfte

Ribbon are floated in a warm bath with what temperature?

60 C

15 min

After placing on glass slides the specimen in placed in what temperature and what time in the oven?

• tearing

• ripping

• creases

• holes

• folding

after cutting in the rotary microtome, what are the common artifacts?

Staining

use chemicals or dyes that will bind or have affinity for certain components of the cells and extraellular components

hamatoxylin and eosin

Most commonly used staining techniques

Mounting

Tissues are impregnated with tranparent cover that has an index of refraction close to the glass slide and tissue

canada balsam

What is the most common mounting medium

Automated tissue processing

Efficiently processes large tissue volumes: replaces manual processing for better speed and consistency

waterbased tissue specimen → Dehydrating agents → Clearing Agents → Infiltration

In automated tissue processing what is the entire process flow

6 hrs

what is the approximate totalt time of automated tissue processing

Carousel system

Older design automated tissue processing where cassettes in a cage are agitated vertically through a series of glass beakers with different solutions

Single Chamber

Modern design tissue processing where solution are pumped in and is more controlled and efficient

• Tissue density and thickness

• Agitation

• Temperature

• Vaccuum and pressure

Factor that impact the duration of tissue processing and extent of infiltration

faster
longer

Spongy bone infiltrated _____ compared to hard/dense tissued who take ____

Agitation

increases flow of fresh reagent around tissued resulting in faster exchange

Vertical or Rotary Oscillation

what is the mechanism of agitation

• Pack tissues loosely in baskets/cassetes

• Align cassette perforations with fluid flow

• Avoid tight packing or setting at the bottom — it reduces surface area and slows processing

Best practices for Agitation

37-45 C

Optimal temperature in automated tissue processing

Tissue shrinkage

Hardening

Brittleness

What does too hgih of temperature cause?

• Reagent becomes viscous

• Slower diffusion

• longer processing

What does to low of a temperature cause?

speed filtration

What does vaccum do in automated tissue processing

Help force viscous media into dense/hard specimen

What does High pressure do in automated tissue processing

Fine Needle Aspiration

TYPE OF BIOPSY SPECMEN

• simples, least invasice test and uses the smallest needle to remove cells ffrom the area abnormality

TYPE OF BIOPSY SPECMEN

Care Needle Aspiration

TYPE OF BIOPSY SPECMEN

• removes not only cells, but also a small surrounding issue, this provide additional information to assist in the examination of the lesion

Incisiona Biopsy

TYPE OF BIOPSY SPECMEN

• Takes out even more surrounding tissue. It takes out some of the abnomality, but not all

• The docotor will slice into the lesion and remove only a portion of i

Excisional Biopsy

TYPE OF BIOPSY SPECMEN

• Removes the entire area in question

Punch Biopsy

• 3-4 cm

TYPE OF BIOPSY SPECMEN

• Considered the primary technique for obtaining diagnostic full-thickness skin specimen

• This technique involves the use of a circular blade that is rotated down through the epidermis and dermis, and into subcutaneous fat, yielding _____ (Give also the usual radius/diametes) cm cylindrical core of tissye sample

Shave Biopsy

TYPE OF BIOPSY SPECMEN

• Small fragment of tissue are shaved from the surface

Curettings

TYPE OF BIOPSY SPECMEN

• Tissue is scooped or spooned to remove the tissue or growth from the body cavity such as endometrium or cervical canal

Teasing and Dissociation

METHOD OF FRESH TISSUE EXAMINATION

• process wherein selected tissue specimen is immersed in a watch glass containing isotonic sald solution, carefully dissected or separated and examine dunder the microscope

Squash preparation or Crushing

• 1 mm

METHOD OF FRESH TISSUE EXAMINATION

• process where small pices of tissue no more than ___( give the size) in diameter and placed in amicroscopic slide and forcibly compressed with another slide or with a coverglass, vital dyes are placed at the slide and coverslip junction and absorbed through capillary action

Smearing

METHOD OF FRESH TISSUE EXAMINATION

• technques useful in cytological examination particularly for cancer diagnosis

Streaking

METHOD OF FRESH TISSUE EXAMINATION

• Type of smearing in that make use of an applicator stick or platinum loop applied in a direct zigzag line

Spreading

METHOD OF FRESH TISSUE EXAMINATION

• Type of smearing in which material is tranferred in a clean slide and gently spread into a moderatley thick film by teasing using an applicator stick recommended for preparations of fresh sputum, broncuak aspirated and thick mucoid secretions

Pull apart

METHOD OF FRESH TISSUE EXAMINATION

• Type of smearing where two slides are pulled apart with a single uninterrupted motion useful in preparation of thick secretion such as serous fluid, conc, sputum, enzymatic lavage samples from git and blood smear

Touch Preparation

METHOD OF FRESH TISSUE EXAMINATION

• Type of smearing in whcih freshly cut tissue is brought into contact and pressed on the sruface of a clean glass slide

Frozen section

• -20 C

• Fresh and Unfixed

METHOD OF FRESH TISSUE EXAMINATION

• Normaly utilized when a rapid diagnosis of the tissue in question is required

• Teachnique in chihc tissue is frozen rapidly at ___ ( give the temp) and sections are cut and stained commonly used for rapid diagnosis especially in operative surgery for the pathology to help the surgeon in choosing his next plan of action

• For frozen section, the tissue dhould be sent to the laboratory ___ and ___ (Fill this in as well)

• Rapid pathologic diagnosis during surgery

• Diagnostic and Research for enzym histochemistry

• Demonstration of lipid (Fat cells) and carbohydrates

• Immunoflourescent and immunohistochemical staining

Applicaiton of Frozen Section

• Laboratory worker should always be informed about frozen section before hand

• Deal with tissue urgently upon arrival in the laboratory

• Cryostat should preferably remain on at all time to maintain its tempreature of -20 C

Pracaution in using frozen section

Cold knife procedure

METHOD OF PREPARING FROZEN SECTION

• Uses cold knife in a controleld cold environment

• Knife: - 40 to - 60 C

• Tissue: - 5 to - 10 C

• Environment: 0 to 10 C

• Optimum conditions for sectioning in cold knie procedure

Cryostat procedure

• - 18 to - 20 C

METHOD OF PREPARING FROZEN SECTION

• Refrigeratrd apparatus used in fresh tissue microtomy consist of an insulated rotary microtome housed in an electrically driven refrigerated chamber and maintained at temperature near - 20 C where microtome knide, specimen and atmostphere are kept at the same temperature

• _____- Give the optimum temperature of the machine

Liquid Nitrogen

TYPES OF CRYOSTAT USED

• Generally most widely used

• Most rapid- used in histochemistry and during introoperational procedure

Isopentane

TYPES OF CRYOSTAT USED

• Useful for muscle tissue

Carbondioxide gas

TYPES OF CRYOSTAT USED

• Cold knife procedures

Aerosol Spray

TYPES OF CRYOSTAT USED

• Freezing small pieces except muscle