3.2 - HISTOPATHOLOGY (OLD)

10% Neutral Buffered Formalin

preserve

harden

protect

Tissue Processing

Fixation

?

Primary: To _ cells and tissues as close to the original as possible Secondary: To _  tissues To _ the tissue from trauma of further handling

Formol Nitric Acid

calcium ions

lime salts

Tissue Processing

Decalcification

?

Removal of ? or ? from the organic extracellular matrix, calcified collagen, and surrounding tissues of bones

Ethyl Alcohol

water

Tissue Processing

Dehydration

?

Removal of intracellular and extracellular _ from tissues after fixation

Xylene

dehydrating agent

refractive index

Tissue Processing

Clearing

?

Removal of _ _ from tissues, making tissue transparent and increase _ _

Paraffin Wax

clearing agent

Tissue Processing

Impregnation

?

Remove _ _ from tissues and fill up cavities and tissue spaces

Paraffin Wax

solidify

56-60

Tissue Processing

Embedding

?

Allow infiltrated tissue to _ Melting point: _-_ C

Rotary Microtome

3-5

Tissue Processing

Trimming

?

Thickness of tissue: _-_ mm

Hematoxylin

Eosin

nucleus

cytoplasm

Tissue Processing

Staining

Primary: _ Secondary: _

Hematoxylin – stains _ Eosin – stains _

Canada Balsam or Eukitt

Tissue Processing

Mounting

(2) (SLMC)

Diamond Pencil or Lead Pencil

Tissue Processing

Labelling

(2)

Routine Biopsy

Cytology

Fine Needle Aspiration Biopsy

Three Areas in Histopathology Section

(3)

3-5

Three Areas in Histopathology Section

• Routine Biopsy (Routine Histopath)

  • Turn Around Time: _-_ Working days

Gross Examination

Routine Histopathology

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

Cell Block

Routine Histopathology

Gross Examination

All biopsy specimens will be gross by the resident pathologist except OTHER BODY FLUIDS or what is called a _ _

Thermo Scientific Excelsior

paraffin wax

Routine Histopathology

Tissue Processing

• Tissue Processor: _ _ _

• Allows the specimen to be infiltrated with a sequence of different solvents finishing in molten _ _

10% Neutral Buffered Formalin

70% Alcohol

80% Alcohol

95% Alcohol

100% Alcohol

Xylene

Water

12

Routine Histopathology

Tissue Processing

• The processor contains:

  • (7)

• The processor also contains Paraffin Wax

• Time of Tissue Processing: _ hours

Embedding

Routine Histopathology

After processing the specimens are placed in an embedding center where they are removed from their cassettes and placed in wax-filled molds

Leica EG 1150H

Routine Histopathology

Embedding

_ _ _ – Embedding Machine

Small

Medium

Large

Routine Histopathology

Embedding

• Three Sizes of Mold Plates:

  • _ – for colonic polyp, renal biopsy, skin punch biopsy

  • _ – gallbladder, thyroid, mammotome, prostate strips

  • _ – myoma (big cuts), breast mass, etc.

Leica EG 1150C

Routine Histopathology

Embedding

_ _ _ – cold plate used in Histopath SLMC QC

Trimming/Cutting/Sectioning

Routine Histopathology

Embedding

• A histotech must “deeper” first the specimen why? To expose the tissue!

• After deeper, the next step is sectioning. It is wherein a histotech will make a ribbon of sections

LEICA RM 2245

LEICA RM 2235

Routine Histopathology

Embedding

• Microtome: _ _ _ (semi-automated) and _ _ _

Sakura Tissue Tek

Routine Histopathology

Staining

• _ _ _ – automated stainer

hematoxylin and eosin (H&E)

blue

pink

Routine Histopathology

Staining

• The routine stain used universally as a starting point in providing essential structural information, is the _ and _ stain. With this method cell nuclei are stained _ and cytoplasm and many extra-cellular components in shades of _

Giemsa Stain

Alcian Blue

Reticulin Stain

Perl’s Stain

PAS

Routine Histopathology

Staining

• Special Stains:

  • _ _ – for gastric polyp; detection of H. pylori detection

  • _ _ – for esophageal mucosa; Active Inflammation (pH – 2.7)

  • _ _ – for reticular fibers

  • _ _ – iron 

  • _ – for glycogen and carbohydrates

Trichrome

Gomori Methenamine Silver Stain

AFB / Fite Faraco Stain

Weigert’s Stain

Routine Histopathology

Staining

Special Stains:

• _ – collagen

• _ _ _ _ – fungal

• _/_ _ _ – acid fast bacilli

• _ _ – for elastin

Mounting

Routine Histopathology

The sections are covered with a glass coverslip and are then sent a pathologist who will view them under a microscope to make an appropriate diagnosis and prepare a report

Frozen Section

Branches of Routine Histopathology

Branch of routine histopathology that is rapid and immediate

Leica CM1860 UV

Branches of Routine Histopathology

Frozen Section

_ _ _ – cryostat machine

15-30

Skin Immunofluorescence

Renal Immunofluorescence

Cryomatrix

Branches of Routine Histopathology

Frozen Section

• Leica CM1860 UV – cryostat machine

  • Turn around time – _-_ minutes

  • Renal Immunofluorescence and Skin

  • _ _ – IgG, IgA, IgM, C3, and Fibrinogen

  • _ _ – IgG, IgA, IgM, C3, Fibrinogen and C1q. It uses acetone before washing

  • _ – counterpart of paraffin in frozen section

1 minute

20

2-3 minutes

20

10

Branches of Routine Histopathology

Frozen Section

• Staining Procedure in Frozen Section:

1. Absolute alcohol – ?

2. Water – _ dips

3. Hematoxylin – ?

4. Water – _ dips

5. Ammonia – _ dips

6. 95% Alcohol – 5 dips

7. Eosin – 15 dips

8. 95% Alcohol – 20 dips

9. Absolute Alcohol – 20 dips

10. Xylene – 20 dips

    • Xylene – clearing agent

    • Formol Nitric Acid – decalcifying agent

    • Ammonia Water – bluing agent

5

15

20

20

20

Xylene

Formol Nitric Acid

Ammonia Water

Branches of Routine Histopathology

Frozen Section

• Staining Procedure in Frozen Section:

1. Absolute alcohol – 1 minute

2. Water – 20 dips

3. Hematoxylin – 2-3 minutes

4. Water – 20 dips

5. Ammonia – 10 dips

6. 95% Alcohol – _ dips

7. Eosin – _ dips

8. 95% Alcohol – _ dips

9. Absolute Alcohol – _ dips

10. Xylene – _ dips

    • _ – clearing agent

    • _ _ _ – decalcifying agent

    • _ _ – bluing agent

Invasive Ductal Carcinoma

Branches of Routine Histopathology

Immunohistochemistry

Breast Panel

• If the breast biopsy is _ _ _, the patient’s specimen must undergo Breast Panel

Breast Panel

Branches of Routine Histopathology

Immunohistochemistry

• Contains Estrogen Receptor Assay (ERA), Progesterone Receptor Assay (PRA) and CERB or the Her-2-neu. Sometimes other doctors add Ki-67

FISH (Fluorescence In Situ Hybridization)

Hormonal Therapy

Oral Therapy

Chemotherapy

Branches of Routine Histopathology

Immunohistochemistry

Breast Panel

• CERB is Equivocal – next test is _

• ERA and PRA is Positive – patient will undergo _ _

• CERB is Positive – the patient will undergo _ _

• ERA, PRA, CERB is Positive – patient will undergo _

Ventana Benchmark XT

Leica Bondmax

Branches of Routine Histopathology

Immunohistochemistry

Breast Panel

• Machines for breast panel (2)

Dehydrating

Water – 95% Alcohol – 100% Alcohol – Xylene

Branches of Routine Histopathology

Immunohistochemistry

• _ Procedure – removal of water

  • ?

• Rehydrating Procedure – addition of water

  • Xylene – 100% Alcohol – 95% Alcohol – Water

Rehydrating

Xylene – 100% Alcohol – 95% Alcohol – Water

Branches of Routine Histopathology

Immunohistochemistry

• Dehydrating Procedure – removal of water

  • Water – 95% Alcohol – 100% Alcohol – Xylene

• _ Procedure – addition of water

  • ?

Pap Smear/Papanicolaou Stain/Gynecytology

Cytology

Detection of Cervical Cancer

Conventional

Autocyte

Tripath Imaging

Cytology

Pap Smear/Papanicolaou Stain/Gynecytology

• Two types of Pap Smear:

  • _ – made on slide; Manual procedure

  • _ – made from prepstain vial; Automated procedure

_ _ – automated pap smear machine

density gradient

2000, 10

300

600

50

10-15

Cytology

Pap Smear/Papanicolaou Stain/Gynecytology

• How to Process Pap Smear?

1. Label the vial with its corresponding access number of the patient

2. Put _ _

3. Put the rack in the tripath imaging for extracting the specimen from the prepstain vial

4. Put all the vials inside the centrifuge at _ rpm for _ minutes

5. While waiting for the centrifuge to stop, add _ uL of distilled water inside the well

6. After centrifugation, decant the fluid and put _ uL of distilled water

7. Disturb small amount of precipitate (inside the vial), aspirate _ uL of specimen and put it in the well

8. Stand the specimen for _-_ minutes

Superficial Cell

Intermediate Cell

Parabasal Cell

Cytology

Pap Smear/Papanicolaou Stain/Gynecytology

• Three Basic Cells of Pap Smear:

1. _ _ – pyknotic cells with pink cytoplasm

2. _ _ – nucleus is bigger than the superficial and has blue green cytoplasm

3. _ _ – blue green cytoplasm with enlarged nucleus

Fine Needle Aspiration Biopsy

It is primarily used for the detection of Thyroid Papillary Carcinoma

For deep seated tumor cells

95% alcohol

Buffered formalin

Fine Needle Aspiration Biopsy

• Fixative: _ _ for the slides

• Fixative (aspirated tissue): _ _

Urine Decoy Cells

10

100

5

Fine Needle Aspiration Biopsy

• Notes to Remember:

  • _ _ _ – test to detect decoy cells from the patient with virally infected epithelial cells

    1. 1:1 ratio of reagent and sample

    2. Centrifuge the specimen for _ minutes.

    3. Decant the fluid

    4. Aspirate _ uL of urine and put it on Thermo Shandon slides

    5. Centrifuge the slides in the cytocentrifuge for _ minutes

Thermo Scientific Revos

Machines in Histopathology

First 4 steps of tissue processing is done here

2

Machines in Histopathology

Thermo Scientific Revos

Each step (duration is _ hours)

Pressure - 1000

Agitation

Temperature - 30-35

Machines in Histopathology

Thermo Scientific Revos

• 3 factors:

  • _ – _

  • _

  • _ – _-_ C

Histocore Arcadia H and Histocore Arcadia C

Machines in Histopathology

Embedding Machine

Leica RM2235

Machines in Histopathology

Rotary Microtome

Fine Wheel

Coarse Wheel

2-3

Machines in Histopathology

Leica RM2235

• _ _ – 1 rotation is 1 um

• _ _ – one rotation is 5 um; larger

• Tissue Thickness – _-_ um

Epredia Automated Stainer

Machines in Histopathology

For staining

Ammonia water

Machines in Histopathology

Epredia Automated Stainer

Bluing agent: _ _

Biobase

Machines in Histopathology

Mounting fume hood

Ventana Benchmark Ultra

Machines in Histopathology

For IHC

Hologic ThinPrep 5000 Processor

Machines in Histopathology

For Autocyte Pap Smear

Filtration

20

Machines in Histopathology

Hologic ThinPrep 5000 Processor

• Principle: _

• Workload: _ specimen cups

Ascitic fluid, Pleural fluid

Coagulum

Additional Notes:

• Cell Block

  • Specimens: (2), etc. – need to make a cassette

  • _ – used specimen in the yellow cassette

  • If there is no coagulum, put the fluid in the conical tube and centrifuge it  it will have a sediment after centrifugation and that is what you’re going to put in the cassette

95% Ethyl Alcohol

Gross → Cell block → Stain

Additional Notes:

• FNAB

  • _ _ _

  • _ → _ _ → _

Bone Marrow & CSF

Urine

Additional Notes:

• Bone Marrow

  • _ _ & _ – stain in Hema section

  • _ – stain in Histopath section