TECHNICAL

PROCEDURES
HISTOPATHOLOGY
Marc Delvin C Quero Medical Technology Intern

IN

HISTOTECHNICIAN
A histotechnician (HT) prepares human body tissue for examination by other laboratory professionals (ASCP, 2012)

TECHNICAL PROCEDURES

TECHNICAL PROCEDURES

CYTOLOGY OF

FLUID EFFUSION

TECHNICAL PROCEDURES

CYTOLOGY OF FLUID EFFUSION 1. Always check clinical abstract for complete data. 2. Check if the specimen label matches that of the request. 3. Gross examination of the specimen. a. Volume b. Color c. Transparency

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CYTOLOGY OF FLUID EFFUSION

4. Put preservative consisting of 70% to 95% alcohol in 1:1 ratio. 5. Make 6 test tube sample aliquot. 6. Decant supernatant.

7. Recover all sediments

a. Cell block b. 4-6 smears 8. Stain

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CYTOLOGY OF FLUID EFFUSION

TECHNICAL PROCEDURES

CYTOLOGY OF FLUID EFFUSION

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CYTOLOGY OF FLUID EFFUSION

TECHNICAL PROCEDURES

PAPANICOLAOUS STAIN

TECHNICAL PROCEDURES

PAPANICOLAOUS STAIN / PAPS STAIN This polychrome staining method and its modifications consist of a nuclear stain and two counterstains.

Hydration prepares the cell sample for uptake of the nuclear dye (hematoxylin).
Dehydration prepares the cell sample for uptake of counterstains (OG 6 and EA 50). Dehydration and clearing solutions result in cellular transparency and prepare the cell samples for the final step: mounting and coverslipping.

TECHNICAL PROCEDURES

PAPANICOLAOUS STAIN / PAPS STAIN

ALCOHOL

10 dips MINWATER 10 ALCOHOL MIN 10 15 10 DIPS dips ALCOHOL ALCOHOL EA-50 10 10 dips10 MIN DIPS10 dips dips 10

HEMATOXYLIN

WATER

ALCOHOL

OG-6

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FROZEN SECTION

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FROZEN SECTIONS

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FROZEN SECTIONS
1. Preliminary gross description and dissection by the Pathologist.

2. Imprint and crush smears are done and processed as rapid FS

pap stains 3. Representative tissue samples are taken from cryo-embedding 4. Process as FS staining 5. Pathologist to release result to surgeon by telephone and

writes and signs out FS diagnosis on FS result form to be sent

ASAP to OR.

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FROZEN SECTION STAINING

Air dry the sections on the slide

95% alcohol

Eosin (3-5 dips)

Formol alcohol (30 seconds)

Wash in water

H & E staining

Wash in water (3 dips)

Harris Hematoxylin

(2 mins)

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HEMATOXYLIN-EOSIN

STAINING

TECHNICAL PROCEDURES

HEMATOXYLIN EOSIN STAINING

100% XYLENE DISTILLED DISTILLED 80% 95% ACID XYLENE HEMATOXYLIN WATER WATER ALCOHOL ALCOHOL ALCOHOL ALCOHOL 10AMMONIA 95% 15 MIN70%dips10 dips DISTILLEDdips DISTILLED 80% 95% 2-3 dips dips dips 10 10 ALCOHOL ALCOHOL EOSIN 10 dips WATER WATER 10 XYLENE ALCOHOL ALCOHOL WATER

10 dips

10 DIPS 10 DIPS DIPS 10 DIPS 5 DIPS 10 2 10 10 DIPS DIPS DIPS dips 10

TECHNICAL PROCEDURES

PREPARATION OF AMMONIA WATER

1. 1, 000 cc of water and 3 cc of ammonia 2. 80% = 840 cc etOH + 160 cc d.H20 3. 70% = 740 cc etOH + 260 cc d.H20 4. 50% = 540 cc etOH + 460 cc d.H20

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PREPARATION OF HARRIS HEMATOXYLIN

1. 5 grams Harris Hematoxylin dissolved in 50 mL of etOH. 2. Weigh 100 grams ammonium aluminum sulfate, dissolve in 1000 mL of water. 3. 2.5 grams of mercuric oxide

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PREPARATION OF HARRIS HEMATOXYLIN

a. 100 grams Ammonium aluminum sulfate in 100 mL boiling water. Stir until crystal dissolves. b. Add the hematoxylin for 15 min

c. Remove from the fire and add mercuric oxide gradually

d. Put back in boiling water until the color is intensely violet

e. Stock in closed colored bottle.

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PREPARATION OF SOLUTIONS Alcoholic Eosin Soln

Eosin and water soluble

Distilled water

1.0 gm
20.0 cc

95% alcohol

80.0 cc

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PREPARATION OF SOLUTIONS Acid Alcohol

70% alcohol
Conc. HCl

1000 cc
10.0 cc

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PREPARATION OF SOLUTIONS Nitric Acid

Formalin
Conc. HNO3

100 cc
15.0 cc

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PREPARATION OF SOLUTIONS 1% Acetic Acid

Glacial Acetic Acid

Distilled water

1.0 cc
99.0 cc

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PREPARATION OF SOLUTIONS Phosphate Buffered Saline

Distilled water
NaCl

800.0 cc
170.0 gm

KPO4 (dibasic)
KPO4 (monobasic)

28.0 gm
49.0 gm

*Add distilled water to make 1.0 liter

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IHC

TECHNICAL PROCEDURES

IHC
Microscopic localization of specific antigens in tissues by staining with antibodies labeled with fluorescent or enzymatically processed dyes. combines histological, immunological and biochemical techniques Antigen: molecular target Antibody Primary: Ig raised against the molecular target Monoclonal Polyclonal Secondary: Ig raised against species of primary antibody; usually conjugated with a reporter Reporter: substance that aids in visualization (fluorophore or enzyme)

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IHC
Specimen type Archived specimens Paraffin blocks
Must heat and process through xylenes and alcohols

Fixation - aldehydes
Sectioning - microtome

Deparaffinization xylene; decreasing concentration of alcohol (100,95,70)

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IHC
Antigen Retrieval HIER
Use MW/steamer/pressure cooker ~ 20 minutes, slow cool

Citrate 6.0 Tris-EDTA 9.0 EDTA 8.0 PIER Proteinase K Trypsin Pepsin Improving Antibody penetration- Detergents; Triton X;Tween

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IHC
Blocking - avoids non-specific reaction ACTIVITY block Inhibits endogenous substances that mimics reporter activity (enzyme system) SERUM block Inhibits binding of Primary IgG on the slide surface Visualization

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ER/PR HER2 NEU

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ER/PR HER 2 NEU

ER+: About 80% of breast cancers are estrogen-receptor positive. ER+/PR+: This means that the cells have receptors for both hormones, which could be supporting the growth of the breast cancer. ER+/PR-: This means that estrogen, but not progesterone, may be supporting the growth and spread of the cancer cells. ER-/PR+: This means that the hormone progesterone is likely to support the growth of this cancer. ER-/PR-: If the breast cancer cells do not have receptors for either hormone, the cancer is considered estrogen-receptor-negative and progesterone-receptor-negative (or hormone-receptor-negative). About 25% of breast cancers fit into this category.

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ER/PR HER 2 NEU ER/PR positive?

HORMONAL REDUCTION THERAPY

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ER/PR HER 2 NEU ER/PR negative?

HER 2 positive?
Her2 expression is associated with a diminished prognosis (e.g., higher risk of recurrence)

TRASTUZUMAB

TECHNICAL PROCEDURES

ER/PR HER 2 NEU

1. Cut paraffin sections at 3.0 m.

2. Melt paraffin by placing slides in a 58C oven for 30 minutes

or in a 37C oven overnight; dewax in xylene. 3. Rehydrate slides in decreasing ethanol grades. 4. Block endogenous peroxidase by using a 6% solution of hydrogen peroxide in water (3.0 minutes, room temperature).

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ER/PR HER 2 NEU

5. Place slides in target retrieval solution and heat at 90C in a

vegetable steamer for 10 minutes.

6. Block endogenous biotin by using the biotin-blocking reagent.

7. Incubate with primary antibodies, ER-1D5 (dilution 1:25) and

PR-636 (dilution 1:100), 22 minutes at room temperature 8. Add the linking solution; biotinylated antimouse immunoglobulin; incubate for 22 minutes.

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ER/PR HER 2 NEU

9. Add streptavidin-peroxidase conjugate and incubate for 22

minutes.
10. Place slides in diaminobenzidine solution for 10 minutes. 11. Apply 1% cupric sulfate (1.0 minute, room temperature) to intensify signal; counterstain with 0.2% fast green (2.0 seconds). 12. Dehydrate in increasing grades of ethanol, clear in xylene, and mount.