2026 case study AP

steps to histopathology

 

1. Specimen reception

2. Fixation

3. Gross examination and cutting

4. Tissue processing

5. Embedding

6. Sectioning - microtomy

7. Staining

8. DCM

9. Microscopy

sputum

Mucus in airways in response to infection

bronchial washings

Normal saline washed over mucosa and suctioned back through bronchoscope into sterile container

bronchial brushings 

Cytology brush rubbed against slide to dislodge cells

Bronchoalveolar lavage (BAL) 

Most distal parts of bronchial tree sampled using normal saline

Fine needle aspiration – transthoracic

Surpasses ribcage

Surpasses ribcage

Fine needle aspiration – transbronchial (TBNA) 

Central/mediastinal structures (lymph nodes, tumours) 

steps for immunohistochemistry

1. Section cutting

2. Antigen retrieval

3. Blocking

4. Primary antibody

5. Secondary antibody

6. Visualisation

Oestrogen receptor

malignant cells that have receptors for oestrogen, once bound is a cascade of events, divide and grow, driving force of cancer, key for engine

Progesterone receptor

only when staining pattern is paired up with ER

OR+, PR-: more aggressive clinical course than double pos

HMW cytokeratin

squamous epithelia; skin and esophagus

low MW cytokeratin

glandular epithelium

fundamentals to in situ hybridisation

1. Fix tissue and mount sections on slide 

2. Pre-treat and denature DNA/RNA (makse single stranded) 

3. Probe DNA > searches for needle

4. Needs a way to be visualised > fluorescent label in FISH attached to probe DNA

5. Denaturation

   1. Breaking the hydrogen bonds (heat/chemical) between the double stranded probe and target DNA, so that it becomes single stranded

6. Hybridisation: labelled probe attaches to the complementary sequence of the target DNA,

7. detection

8. Fluorescence microscope for fluorescent probe

steps to FISH

1. Sections cut at 4-5um on charged slides

2. Slides baked at 60 degrees

3. Dewaxing

4. Pre-treatment

a. Heat/pressure cooker

b. Enzyme treatment

c. (opens up molecular structure)

5. Dehydration in gradient alcohols

6. probe mix added to tissue

7. Denaturation

8. Place in hybrider (14-72 hours)

9. Washes

10. DAPI counterstain

11. View under fluorescence microscope

HER2 gene

Oncogene on chromosome 17

HER2 drug

Transtuzumab (herceptin) = anti-HER 2 antibody

EGFR

• Epidermal growth factor receptor

• Transmembrane receptor plays a role in intracelllular signalling pathways

• Mutations in EGFR gene affect the EGFR tyrosine kinase domain

ALK gene marker

Gene fusion (e.g., EML4–ALK) → constitutive activation

NSCLC, adenocarcinoma

cytoplasmic staining

ROS1 gene marker

Fusion with partners → continuous signalling

NSCLC

cytoplasmic/membranous staining

HER2 gene marker

Gene amplification → receptor overexpression

breast cancer

EGFR gene marker

Point mutations or overexpression → constant activation of downstream signalling

NSCLC

TTF-1

Positive in lung adenocarcinoma, some SCLC

nuclear staining.

Napsin A

Positive in lung adenocarcinoma

granular cytoplasmic staining.

CK7

Positive in lung, breast, and thyroid adenocarcinomas cytoplasmic staining.

CK20

Positive in colorectal and intestinal-type gastric adenocarcinoma

cytoplasmic staining.

CEA

Positive in adenocarcinomas (lung, colon, pancreas, breast)

cytoplasmic/membranous staining.

Ber-EP4

Positive in adenocarcinoma; negative in mesothelioma.

membranous staining;

EMA

Positive in epithelial tumours (adenocarcinoma, mesothelioma, SCC)

membranous or cytoplasmic staining.

Synaptophysin

Positive in SCLC

fine granular cytoplasmic staining.

CD56 (NCAM)

Positive inSCLC;

membranous staining.

Chromogranin A

Positive in SCLC;

coarse granular cytoplasmic staining.

PD-L1

Positive in NSCLC and other tumours eligible for immunotherapy;

membranous staining on tumour cells.

P40

Positive in squamous cell carcinoma;

strong nuclear staining.

CK5/6

Positive in squamous cell carcinoma and mesothelioma;

cytoplasmic staining.

P63

Positive in squamous cell carcinoma and basal cells of prostate;

nuclear staining.

p16

Positive in HPV-related squamous cell carcinoma (cervix, oropharynx);

nuclear and cytoplasmic staining.

P504S / AMACR

Positive in premalignant and malignant prostate lesions;

cytoplasmic staining (pink/red).

34βE12

Positive in benign prostate basal cells and SCC; negative in prostate carcinoma.

cytoplasmic/membranous staining;

CDX2

Positive in colorectal and intestinal-type gastric adenocarcinoma;

nuclear staining.

MLH1, MSH2, MSH6, PMS2

Positive in normal tissue nuclei;

loss of nuclear staining indicates MMR deficiency (Lynch/MSI-high).

HER2 (IHC)

Positive in breast and gastric carcinoma;

complete membranous staining (3+ IHC score).

Ber-EP4 & EMA (Combined)

Positive in adenocarcinoma;

Ber-EP4 membranous, EMA cytoplasmic.

TTF-1 & Napsin A (Combined)

Positive in lung adenocarcinoma;

TTF-1 nuclear, Napsin A cytoplasmic.

baseline cells

endocervical cells

endocervical cells

pyknosis

nuclear degenration - karyorrhexis

nuclear degenration - karyolysis

nuclear degenration - karyolysis

nuclear and cytoplasmic degenration

regenerative activity

macrophages 

squamous cells

bronchial cells

metaplastic cells

parabasal cells

mucus producing cells - goblet cells

alveolar macrophages

squamous metaplasia 

squamous cell carcinoma

squamous cell carcinoma

squamous cell carcinoma

squamous cell carcinoma - tadpole cell

squamous cell carcinoma - pearl formation

SCC differential - atypical squamous metaplasia

SCC differential - reactive changes

small cell carcinoma (SCLC)

small cell carcinoma (SCLC)

small cell carcinoma (SCLC)

small cell carcinoma (SCLC)

small cell carcinoma (SCLC)

SCLC differential - reserve cell hyperplasia

adenocarcinoma

adenocarcinoma

adenocarcinoma

adenocarcinoma - mucin vacuole

adenocarcinoma - 3D group

adenocarcinoma - nucleoli

adenocarcinoma differential - macrophages 

adenocarcinoma differential - creola body

adenocarcinoma differential - goblet cell hyperplasia

non—small cell carcinoma (NSCLC)

non—small cell carcinoma (NSCLC)

non—small cell carcinoma (NSCLC)

non—small cell carcinoma (NSCLC)

non—small cell carcinoma (NSCLC)

pap stain - airdrying artefact

giemsa stain

affinity

movement of a dye from the dye bath onto a section

Cationic dyes (+) / basic

• Positive charge, binds to negatively charged tissue groups 

• Tissue components referred to as basophillic 

Anionic dyes (-) / acid

• Negative charge, bind to positively charged tissue groups 

• Tissue components are acidophilic  

Direct dyeing 

• Direct attachment of dye to tissue component by ionic bonding 

• Opposite charge 

Indirect dyeing  

• Attachment of dye to tissue using an intermediate substance called a mordant 

• Mordant: metal, has affinity for both the dye and tissue 

• Dye + mordant = dye lake 

Metachromasia 

Certain dyes which when attached to particular tissue groups, produce a colour different from the original dye

Dyeing by permeation  

Difference in permeabilty of tissye structures and uses dyes of different colour and molecular size 

Differential staining 

Two or more of the above methods 

Histochemical methods 

Reagents used react with specific tissue components to produce a colour insoluble product

Metallic impregnation

• Particular chemical groups in tissue have the ability to bind or reduce silver salts 

• Metal precipitates in or on particular tissue constituents  

Argentaffin reaction

causes silver to be deposit without using a chemical reducing agent (black)