UNIT 1. INTRODUCTION TO HISTOPATHOLOGY

Histopathology

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

Histopathologic techniques

Deals with the preparation of tissues for microscopic exam

Histotechnologist

what do you call a medtech that performs histopathologic techniques in the histolab; went special training

1. Numbering

2. Fixation

3. Decalcification (optional)

4. Dehydration

5. Clearing

6. Infiltration/ Impregnation

7. Embedding

8. Trimming

9. Section cutting

10. Staining

11. Mounting

12. Labeling

CONVENTIONAL TISSUE PROCESSING PROCESS

Numbering

Step that identifies the spx without writing the patient’s name on the specimen tag

Specimen type — Year (last 2 digits) — Accession # (4 digits)

How do you label specimens in the histolab

pencil

what is used to label spx

1. Surgical

2. Autopsy

3. Cytology

3 specimens processed in the histolab

Fixation

• 1st and most critical step

• prevents autolysis and stabilizes tissue to maintain cellular structures

tissue casette

in fixation, spx is placed in a ____________ and endorsed to an MT

10% neutral buffered formalin

• most common fixative

• diluted form

37% formalin (concentrated)

10% neutral buffered formalin is diluted from _____________________

• zonal fixation

• altered morphology

• affect staining characteristics

inadequate fixation leads to

decalcification

• process of removing Calcium salts from the spx

• ex: teeth, bone

ACIDS

• Nitric acid

• Formic Acid

• Hydrochloric acid

• Picric acid

• Acetic acid

• Citric acid

Decalcification agents

DEHYDRATION

• This step removes free water and unbound fixative from the tissue

Graded alcohols of increasing concentration

• ethanol,

• reagent alcohol,

• isopropanol,

• glycol ether

Dehydration agents

Over processing artifacts

Dehydration Artifacts:

• shrinkage, 'parched earth effect' and abnormal staining, dry brittle tissue

Incomplete dehydration

Dehydration Artifacts:

• impair penetration of clearing agent leaving the specimen soft and non-receptive to paraffin wax infiltration

Clearing

• this step displaces dehydrating solutions, making the tissue components receptive to the infiltrating medium

xylene

• 20 min

• 20 min

• 45 min

• most widely used clearing agents

• makes tissue transparent; removal of the alcohol, para maging receptive siya sa infiltrating medium and optical clarity.

• miscible with ethanol and paraffin wax

INFILTRATION/ IMPREGNATION

• This step permeates tissue with a support medium

paraffin wax

most common infiltrating medium

• 60°C = liquid

• 20°C =solid

Paraffin (liquid vs liquid)

4 mm thick

• 30 min

• 30 mon

• 45 min

To completely displace the clearing agent, a typical paraffin infiltration sequence of paraffin specimens not more than ______ would be:

Incomplete infiltration

INFILTRATION/ IMPREGNATION

• produces soft and crumbly tissues

• excessive shrinkage

• dry and brittle tissues

INFILTRATION/ IMPREGNATION

• too much time in high temperature wax leads to

Embedding

• step: orientation of the tissue sample in a support medium to create a tissue “block” suitable for sectioning

2-4° above its melting point of the paraffin wax

Embedding

• temperature

Tissues are embedded with the surface to be cut facing down in the mold

Embedding

• correct orientation

cold plate

Embedding

• Whole mold is placed on a _________ to solidify

Overheating

Embedding

• caused the hardening and distortion of tissues

paraffin wax (same as infiltration)

Embedding

• embedding medium

TRIMMING

• this step happens before section cutting

• Aims to completely expose the paraffin-embedded tissue in order to obtain a representative section

Section-cutting

• the step where embedded tissue are cut into sections that are thin enough to be placed on a slide using microtome

routine: 3-5 um

Section-cutting:

• size of each section

warm bath (5-10 °C)

Section-cutting:

• Ribbons are floated in ______________

• warm oven (60 °C) for 15 min

Section-cutting:

• Glass slides containing specimen are placed in __________________________

• tearing

• ripping

• creases

• folding of sections

section-cutting common artifacts

Staining

• this step uses chemicals or dyes that will bind or have affinity for certain components of the cells and extracellular components

Hematoxylin & Eosin

• H = nucleus = blue

• E = cytoplasm = pink

most commonly used satin

MOUNTING

• Tissues are impregnated with transparent medium that has an index of refraction close to glass slide and tissue

Canada balsam

Most common mounting medium

LABELING

• the last step

• Can be automated or manual

AUTOMATED TISSUE PROCESSING

• efficiently processes large tissue volumes

• replaces manual processing for better speed and consistency

starts with a water-based tissue specimens → passes thru dehydration agents (ethanol) → clearing agents (xylene) → finally infiltrated with molten paraffin wax (hydrophobic, water-immiscible

AUTOMATED TISSUE PROCESSING FLOW

1. Fixation

2. Dehydration

3. Clearing

4. Infiltration

AUTOMATED TISSUE PROCESSING 4 STEPS

approx. 6 hrs

often run overnight

AUTOMATED TISSUE PROCESSING

• total time

• Carousel design

• Single chamber

TYPES OF AUTOMATED TISSUE PROCESSORS

Carousel system

TYPES OF AUTOMATED TISSUE PROCESSORS

• older design

• cassettes in a cage are agitated vertically through a series of glass beakers with different solutions

Single chamber system

TYPES OF AUTOMATED TISSUE PROCESSORS

• modern design

• solutions are pumped in/out

• more controlled and efficient

• shorter TAT

• Tissue Density & Thickness

• Agitation

• Temperature

• Vacuum Pressure

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

DENSITY

• spongy tissues (lungs) = ↑ infiltration

• hard/dense tissues = ↓ infiltration

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

• spongy tissues (lungs) =

• hard/dense tissues =

THICKNESS

• thicker/larger tissues = ↓ infiltration

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

• _________________ need more time for reagents to diffuse

AGITATION

• faster

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

• increase flow of fresh reagents around tissues → ____

vertical/rotary oscillation

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

• agitation in automated processors

37-45 °C

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

• optimal temp range

• short term use only

• speeds penetration

too high temp = tissue shrinkage, hardening, brittleness (especially affects collagen)

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

• too high temp =

too low temp = reagents become more viscous = slower diffusion; longer processing

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

• too low temp =

2-3°C above

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

• Keep wax __________ its melting point

Vacuum (reduce pressure)

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

• speeds infiltration

• removes trapped air

• good for porous tissues (lungs)

• improves quality

• reduces time for dense, fatty tissues

High pressure

FACTORS THAT IMPACT THE DURATION OF TISSUE PROCESSING AND EXTENT OF INFILTRATION:

• Helps force viscous media into dense/hard specimen

F

DOES NOT shorten wax infiltration time; depends mainly on viscosity

T or F

Vacuum shorten the wax infiltration time of specimens.

needle biopsies & blood specimens;

fatty specimens

Generally, ____________________________ should be incubated conservatively, whereas ________________ can be processed for longer than average.

1. Fine needle aspiration

2. Core needle biopsy

3. Incisional biopsy

4. Excisional biopsy

5. Punch biopsy

6. Shave biopsy

7. Curettings

TYPES OF BIOPSY SPECIMEN (7)

Fine needle aspiration

TYPES OF BIOPSY SPECIMEN (7)

• simplest, least invasive test

• uses the smallest needle to remove cells from the area of abnormality

• guided by ultrasound

Core needle biopsy

TYPES OF BIOPSY SPECIMEN (7)

• removes not only cells, but also small surrounding tissue

• additional info to assist in the examination of the lesion

Incisional biopsy

TYPES OF BIOPSY SPECIMEN (7)

• Takes out even more surrounding tissues

• takes out some of the abnormality, but not all

Excisional biopsy

TYPES OF BIOPSY SPECIMEN (7)

• removes the entire area

Punch biopsy

TYPES OF BIOPSY SPECIMEN (7)

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

• use of circular blade that is rotated down through the epidermis and dermis, and into subcutaneous fat, yielding a 3-4 mm skin specimen

Shave biopsy

TYPES OF BIOPSY SPECIMEN (7)

• small fragment of tissue are shaved from a surface

Curettings

TYPES OF BIOPSY SPECIMEN (7)

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

1. Teasing/Dissociation

2. Squash/Crushing

3. Smearing

4. Froze section

METHODS OF FRESH TISSUE EXAMINATION (4)

Teasing/Dissociation

METHODS OF FRESH TISSUE EXAMINATION

• tissue spx is immersed in a watch glass containing isotonic salt solution (NSS)

• carefully dissected or separated and examined under the microscope

Squash/ Crushing

METHODS OF FRESH TISSUE EXAMINATION

• small pieces of tissue, not more than 1 mm in diameter, placed in a microscopic slide and forcibly compressed with another slide or with a cover glass

• vital dyes are placed at the slide and coverslip junction and absorbed through capillary action

Smearing

METHODS OF FRESH TISSUE EXAMINATION

• techniques useful in cytological exams

• particularly for cancer diagnosis

1. streaking

2. spreading

3. pull-apart

4. touch preparation

TYPES OF SMEARING (4)

streaking

TYPES OF SMEARING

• applicator stick or platinum loop

• direct or zigzag line

Spreading

TYPES OF SMEARING

• material is transferred in a clean slide and gently spread into a moderately thick film by teasing using an applicator stick

• fresh sputum

• bronchial aspirates

• thick mucoid secretions

spreading is recommended for preparations of

pull-apart

TYPES OF SMEARING

• 2 slides are pulled-apart with a single uninterrupted motion

• serous fluid

• conc. sputum

• enzymatic lavage

• sample from GIT

• blood smeaar

Pull-apart method is recommended for

touch preparation

TYPES OF SMEARING

• freshly cut tissue is brought into contact and pressed on the surface of a clean glass slide

Frozen section

METHODS OF FRESH TISSUE EXAMINATION

• normally utilized for rapid diagnosis, especially in intra-operative pathology to help surgeon in choosing his next plan of action

-20°C

Frozen section

• tissue is frozen rapidly at ______ and sections are cut and stained

fresh & unfixed

Frozen section specimen requirements

• rapid pathologic diagnosis during surgery

• diagnostic and research enzyme histochemistry

• demonstration of lipid (fat cells) and carbohydrates

• immunofluorescent and immunohistochemical staining

Applications of Frozen section

cryostat

-20°C

equipment used for frozen section and its temp

optimum cutting temperature (OCT) compound

embedding medium for frozen section

1. Cold Knife procedure

2. Cryostat procedure

FREEZING METHODS (2)

cold knife procedure

FREEZING METHODS

• use of cold knife in a controlled cold environment

• knife = -40 to -60°C

• tissue = -5 to -10°C

• environment 0 to -10°C

cold knife procedure optimum conditions

Cryostat

FREEZING METHODS

• Refrigerated apparatus used in fresh tissue microtomy consists of an insulated rotary microtome housed in an electrically driven refrigerated chamber

-18 to -20°C

FREEZING METHODS

• optimum working temp of cryostat

1. Liquid nitrogen

2. Isopentane

3. Carbon dioxide gas

4. Aerosol sprays

MOST COMMONLY USED MEDIUM/ REAGENTS IN FREEZING (4)

Liquid nitrogen

MOST COMMONLY USED MEDIUM/ REAGENTS IN FREEZING

• most rapid and most widely used

• histochemistry and intraoperative procedures

Isopentane

MOST COMMONLY USED MEDIUM/ REAGENTS IN FREEZING

• for muscle tissue

Carbon dioxide gas

MOST COMMONLY USED MEDIUM/ REAGENTS IN FREEZING

• cold knife procedure

Aerosol sprays

MOST COMMONLY USED MEDIUM/ REAGENTS IN FREEZING

• for small pieces of tissue except muscle tissue

tissue block

product in the embedding step