case study COMPLETE

Routine H&E (Progressive)

Haematoxylin (binds DNA/RNA) → Scott’s tap water (blueing) → Eosin (counterstain).

Mechanism: haematoxylin binds acidic structures, bluing converts to blue lake, eosin binds basic proteins.

Results: nuclei blue-black, cytoplasm pink, RBCs orange/red, fibrin deep pink.

Routine H&E (Regressive)

Haematoxylin → Scott’s tap water → 1% acid alcohol (differentiate) → Eosin.

Mechanism: overstain, remove excess with acid alcohol, reblue, counterstain.

Results: same as progressive (nuclei blue-black, cytoplasm pink).

PAS (Periodic Acid–Schiff)

Periodic acid → Schiff’s reagent → haematoxylin → Scott’s water.

Mechanism: oxidises carbohydrates to aldehydes, Schiff’s reacts → magenta.

Results: glycogen & carbs magenta, nuclei blue.

Alcian Blue (pH 2.5)

Acetic acid → Alcian blue → Nuclear fast red.

Mechanism: cationic dye binds anionic acidic mucins (electrostatic).

Results: acid mucins blue, nuclei red.

AB–PAS (Combined)

Acetic acid → Alcian blue → Periodic acid → Schiff’s reagent → Haematoxylin.

Mechanism: Alcian blue first (acid mucins), PAS second (neutral mucins).

Results: acid mucins blue, neutral mucins magenta, nuclei pale blue.

Ziehl–Neelsen (Acid-Fast)

Carbol fuchsin → Acid alcohol → Methylene blue.

Mechanism: carbol fuchsin penetrates waxy mycolic coat, acid alcohol decolorises non–acid-fast.

Results: acid-fast bacilli red, background blue.

Gram Stain

Crystal violet → Lugol’s iodine → Acetone → Safranin/Twort’s.

Mechanism: CV binds peptidoglycan, iodine forms complex, Gram+ retain stain, Gram– lose during decolorisation.

Results: Gram+ dark violet, Gram– pink/red, nuclei red/mauve, cytoplasm light green.

Grocott Methenamine Silver (GMS)

Chromic acid → silver bath → Gold chloride → Sodium thiosulphate → Light green.

Mechanism: oxidises fungal carbs → aldehydes → reduce silver → black.

Results: fungi black, background green.

Perl’s Prussian Blue (Iron)

HCl + potassium ferrocyanide → Nuclear fast red.

Mechanism: ferric iron forms ferric ferrocyanide (Prussian blue).

Results: haemosiderin dark blue, nuclei red.

Congo Red (Amyloid)

Congo red → Mayer’s haematoxylin.

Mechanism: binds β-pleated amyloid fibrils.

Results: amyloid red, apple-green birefringence under polarised light, nuclei blue.

Gordon & Sweet’s Reticulin

Potassium permanganate (oxidise)→ Oxalic acid (bleach)→ Ferric ammonium sulphate (sensitiser)→ Wilder’s solution (impregnate) → Formalin (reduces)→ Sodium thiosulphate (remove excess).

Mechanism: oxidation, sensitisation, silver impregnation → visible black reticulin.

Results: reticulin black, collagen brown.

Masson’s Trichrome

Bouin’s fixative → Weigert’s haematoxylin → brilliant crocein → phosphotungstic acid → Aniline blue → Acetic acid.

Mechanism: acid dyes separate muscle/collagen by charge/size.

Results: nuclei black, collagen blue/green, muscle red.

Gomori’s Aldehyde Fuchsin

Aldehyde fuchsin → 95% alcohol → Picric carmine–picric acid.

Mechanism: stains elastic fibres, beta cells & mucin purple; counterstain colours collagen/muscle.

Results: elastic fibres & mucin deep purple, collagen blue-green, muscle yellow.

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

cytopathology checklist (cell)

• Presentation

• shape

cytopathology checklist (nucleus)

• Size

• Stain

• Position

• Chromatin

• Granularity

• Shape

• Border

cytopathology checklist (cytoplasm)

• Density

• Colour

• Texture

• Differentiation

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. Probe DNA > searches for needle

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

3. Denaturation

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

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

• Probe forms new bonds with complementary sequence

5. Detection

6. 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

describe features

fine, evenly distributed chromatin, intermediate cells 

describe features

fine, evenly distributed chromatin bronchial cells

describe features

coarse, irregularly distributed chromatin

describe features

coarse, irregularly distributed chromatin

describe features

coarse, evenly distributed chromatin

describe features

fine, evenly distributed chromatin

describe features

coarse, evenly distributed chromatin

describe cytoplasm

non-vacuolated

describe cytoplasm

finely vacuolated 

describe cytoplasm

dense

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