Basics

This article serves as an introduction to anatomical pathology and discusses the basics.

Pathology simplified

H&E is the standard...

• Too much PINK = DEAD (necrosis).
• Too much BLUE = BAD.

In words:

• Blue is bad and pink is dead!

Note:

• Lymph nodes are very blue... they aren't necessarily bad.

Terms

Very common

• Eosinophilic - pink.
• Hyperchromatic - blue.
• Amphophilic - bluish-red colour when referring to H&E stained section.
  • Amphophilic means stains with both acidic & basic dyes.
  • Images: amphophilic material - arrow (pancreaticcancer2000.com), amphophilic cytoplasm in prostate carcinoma (webpathology.com).

Less common

• Hobnail - basement membrane area < area exposed to luminal surface.
• Storiform - spiral appearance or cartwheel pattern
  • Images: Storiform pattern (upenn.edu), Storiform pattern (ispub.com).
• Plexiform - web-like formation.

Nuclear destruction words

There are several fancy terms:

• Karyolysis = nuclear fading/dissolution.
• Pyknosis = nuclear shrinkage.
• Karyorrhexis = nuclear fragmentation.

Image:

• Karyolysis, pyknosis, karyorrhexis (wikimedia.org).

DDx in medicine

Mnemonic CINE-TV-DATE:

• Congenital.
• Inflammatory.
• Neoplastic.
• Endocrine.
• Trauma.
• Vascular.
• Degenerative.
• Autoimmune.
• Toxic.
• Everything else (iatrogenic, idiopathic, psychiatric).

In diagnostic pathology, most stuff falls into the neoplastic category.

Basic pathologic DDx of malignancy

Malignancy

Epithelial (Carcinoma)

Mesenchymal (Sarcoma)

Germ cell tumour

Neuroendocrine carcinoma

Lymphoid (Lymphoma)

Malignant melanoma

Notes:

• Melanoma, i.e. malignant melanoma, is a separate category as it can look like almost anything under the microscope.
• Lymphoma includes leukemia.

Morphologic grouping

Factors to consider when attempting to group by morphology:

1. Cell cohesion - dyscohesive vs. cohesive.
   • If one sees several groups of 5+ cells... probably cohesive.
   • Presence of cell cohesion strongly disfavours lymphoma.
2. Cell size - in relation to a neutrophil or red blood cell.
3. Cytoplasm - abundance (scant, moderate, abundant).
   • Eosinophilic cytoplasm disfavours lymphoma.
4. Chromatin - coarseness (fine, granular).
5. Nucleoli - number (absent, present, multiple).
   • Large nucleoli (nucleoli seen with the 10x objective) pretty much exclude neuroendocrine.

Probable category by morphology:

• Carcinoma = cohesive, relatively large (>~2X neutrophil), +/-nucleolus, +/-gland formation (circular structures), often moderate to abundant cytoplasm.
• Sarcoma = cohesive, composed of spindle cells (cells taper at both ends, nucleus oval/cigar-shaped).
• Germ cell tumour = appearance often similar to carcinoma.
• Neuroendocrine carcinoma = cohesive, fine granular chromatin and no nucleolus.
• Lymphoma = dyscohesive, relatively small (usually <=2X neutrophil diameter), usu. scant basophilic (blue) cytoplasm.
• Melanoma = classically pigmented, often a prominent red nucleolus, a mix of spindle cells and epithelioid cells, mix of cohesive and dyscohesive cells.

Dyscohesive vs. cohesive

Deciding cells are dyscohesive vs. cohesive is important, as it is a strong determinant of whether one is dealing with a lymphoid lesion or not.

	Cell spacing	Cell membrane	Cytoplasm, abundance	Cytoplasm, staining
Cohesive	equal spacing or 3-D clusters or intracellular bridges	visible & opposed (in >50% of cells)	scant to abundant	any
Dyscohesive	unequal spacing, thin space surrounds cell	not apparent	usually scant	usually basophilic
Value/utility	equal or 3-D clusters suggests cohesive, pericellular space/rim suggests dyscohesive	visible opposed membrane r/i cohesive	abundant usu. cohesive	eosinophilic usu. cohesive

Strong predictors of cohesive:

• Intracellular bridges.
• 3-D clusters.
• Nuclear moulding.

Weak predictors of cohesive:

• Eosinophilic cytoplasm.
• Abundant cytoplasm.
• >2 X RBC diameter (most lymphoma smaller).

Weak predictors of dyscohesive:

• Pericellular space/rim.
• Scant cytoplasm.
• Basophilic cytoplasm.

Histomorphologic classification

Types of cells:

• Spindle cell:
  • Tapered at both ends.
  • Suggests mesenchyme, i.e. sarcoma, compatible with melanoma and some carcinomas.
• Plasmacytoid cell.
  • Resemble a plasma cell: eccentric nucleus, "clockface" chromatin pattern, scant basophilic cytoplasm.
• Epithelioid cell.
  • Looks like epithelium - cell borders touch neighbouring cells so that the cells collectively form a barrier.
• Small round blue cell tumour:
  • Small cells with scant cytoplasm.
    • "Small" is classically 2x a "resting lymphocyte" diameter.
      • Diameter of a "resting lymphocyte" ~ diameter of a red blood cell (RBC) ~ 8 micrometres.
      • Most carcinoma cells are 3-4x the size of a RBC.

Finding the elements

Mitoses

• Nucleus darker (hyperchromatic) - key feature.
• No nuclear membrane - key feature.
• In prophase chromatin may have a scalloped border/beaded border.

DDx:

• Apoptotic cell -- has nuclear condensation (pyknosis), eosinophilic cytoplasm.

Images:

• Mitoses (vetmed.vt.edu).
• Mitoses and an atypical mitosis (WC).
• Tripolar mitosis (WC).
• Starburst mitosis (flicker.com).

Phases of mitosis

• Prophase. - chromatin condenses to chromosomes.
• Metaphase - chromosome aligned.
• Anaphase - spindles separated.
• Telophase - reversal of prophase.

Neutrophils

• Little dots = the multilobular nucleus - key feature.
• Neutrophils are often found with friends, i.e. lymphocytes, plasma cells.

DDx of little specs:

• Nuclear debris - apoptotic cell.
  • Apoptotic cell -- has nuclear condensation (pyknosis), eosinophilic cytoplasm.

Notes:

• AKA PMNs - polymorphonuclearcyte, polymorphonuclear cell.
• You find PMNs by their nucleus; on a histologic section don't bother looking for the cell membrane (they are usually impossible to see).
• A collection of PMNs... think about necrosis and abscess.

Lymph node metstatsis

• Take a good to look at the tumour first.
• Tumour in a node is often better differentiated than the most poorly differentiated part in the primary site.
• Subcapsular space - the first place to look for mets.
• Lymph node metstasis are usually obvious.
• Histiocytes may be difficult to separate from tumour - esp. initially.
  • Histiocytes usually are in germinal centre, i.e. the node architecture helps,
  • Malignant cells have to have malignant features, i.e. the NC ratio is abnormal, there is nuclear pleomorphism.

See: Lymph node article for a detailed description of cell types in a lymph node.

Signet ring cells

Definition:

• Signet ring cells resemble signet rings (image).
  • They contain a large amount of mucin, which pushes the nucleus to the cell periphery. The pool of mucin in a signet ring cell mimics the appearance of a finger hole and the nucleus mimics the appearance of the face of the ring in profile.

Microscopy:

• Typically 2-3x the size of a lymphocyte.
  • Smaller than the typical adipocyte.
• Often have a cresentic-shaped nucleus, or ovoid nucleus.
  • Capillaries sectioned on their lumen have endothelial cells-- the nuclei of these are more spindled.
• SRCs are usually close to friend (another SRC)
  • This helps differentiate SRCs from capillaries sectioned on their lumen.
• The mucin is often clear on H&E... but maybe eosinophilic.

Stains:

• PAS stain.
• Alican blue-PAS stain.

Images:

• SRCs (H&E) by Nephron (WC).
• SRC AL-PAS stain (WC).
• SRC H&E stain (WC).

Comment:

• It has been said that there are two types of pathologists... those that have missed SRCs and those that will miss SRCs.

Necrosis

Main article: Necrosis

Features:

• Dead cells - pink (on H&E).
  • Anucleate cells ("Ghost cells")/outlines of cells - usu. subtle.
    • Fluffy appearance.
• +/-Neutrophils (very common).

DDx of necrosis:

• Fibrin.

Images (necrosis):

• Necrosis at the centre of a granuloma (sun.ac.za).
• Necrosis (biomedical-engineering-online.com).
• Necrosis (nature.com).
• Necrosis (ouhsc.edu).
• Necrosis in cat scratch disease (WC).
• Necrosis in histiocytic necrotizing lymphadenitis (WC).
• Necrosis in SLE lymphadenopathy (WC).

Granulomas

• Granulomas can be elusive to the novice.
• Plural of granuloma was granulomata; granulomas (an anglicized version) is, however, now generally accepted.

Definition of granuloma

• Many definitions exist.
• The term is used rather loosely by clinicans.
  • Radiologists occasionally call small lung nodules "granulomas".

Strict pathologic definition

Robbins definition:

• Chronic inflammatory reaction characterized by the focal accumulation of activated macrophages, often with an epithelioid appearance.
  • "Epithelioid" cells = cells whose morphology resembles that of epithelial cells; the cells appear to adhere to one another.

Adams definition - it's short & sweet:

• A compact collection of macrophages.
  • The macrophages must form a small ball/cluster of cells, i.e. touch one another.

Other pathologic definitions include the presence of:

• Plasma cells.
• Lymphocytes.
• Epithelioid macrophages.

Notes:

• The textbook answer for what is a granuloma is: "A collection of epitheliod macrophages."
  • Granulomas are often associated with lymphocytes.

Features that assist one in finding granulomas

• Collection of cells that have abundant bubbly cytoplasm - most useful feature.
  • Image: Granulomas showing abundant bubbly cytoplasm (WC).
• Necrosis - too much pink (on H&E stained sections).
  • Image: Granuloma with necrosis (WC).
• Multinucleated giant cells - these are easy to identify if you've seen some before.
  • Individual/singular multinucleated giant cells are not diagnostic of a granuloma... but should raise one's suspicion of one being present.
  • Image: Granulomas with multinucleated giant cells in sarcoidosis (WC).
• Small round collection of lymphocytes - without a capsule (as seen in lymph nodes).
  • If there are no macrophages... it's a lymphoid nodule.

Classification of granuloma

Histologic classification

1. Necrosing (also caseating).
   • More likely to be infectious.
   • Examples: Tuberculosis (TB).
2. Non-necrosing.
   • Less likely to be infectious.
   • Examples: Crohn's disease, sarcoidosis, drug reaction.

Whether necrosis is present in a granuloma is affected by the immune function, e.g. a HIV/AIDS patient may have non-necrosing granulomata due to TB.

Notes:

• A few people differentiate between caseating (fragments of recognizable tissue) and necrosing (dead debris only).
• Infectious non-necrosing infections: Mycobacterium avium complex (MAC), cryptococcus, immunosuppressed individual.

Etiologic classification

1. Infectious, e.g. tuberculosis, MAC, fungal infection.
2. Neoplastic, e.g. seminoma.
3. Autoimmune, e.g. Wegener's granulomatosis, Churg-Strauss syndrome.
4. Allergic, e.g. hypersensitivity pneumonitis.
5. Foreign body, e.g. pulmonary talcosis.
6. Drug reaction.
7. Idiopathic, e.g. sarcoidosis.

Memory device: DNF AAII = drug reaction, neoplasm, foreign body, allergy, autoimmune, idiopathic, infection.

Lung granulomata

There are many causes.

Infectious:

• Myocbacterial: Tuberculosis, MAC, other.
• Fungal: Histoplasmosis, Cryptococcosis, Blastomycosis, Coccidioidomycosis.
• Aspiration pneumonia.

Non-infectious:

• Pneumoconioses/hypersensitivity pneumonitis: Talcosis, Berylliosis.

Idiopathic/autoimmune:

• Sarcoidosis,
• Wegener's granulomatosis,
• Churg-Strauss disease,
• Rheumatoid nodules.

Special granulomas

Fibrin ring granuloma

• Classically associated with Q fever.
  • DDx: infections (Coxiella burneti (causes Q fever), CMV, EBV + others), drug reaction, malignancy (e.g. Hodgkin lymphoma).
• Appearance:
  • Epithelioid macrophages (i.e. a granuloma) surrounding a fibrin ring with a clear (lipid-filled) vacuole at its center.
    • Images:
      • FRG (gooword.com).
      • FRG (pathconsultddx.com).

Common morphologic problems

DDx of pink stuff (on H&E)

The ABCs of pink:

• Amyloid.
• Blood clot (organized); fibrin.
• Collagen (fibrous tissue).
• Smooth muscle cells (SMCs).

Smooth muscle cells (SMCs) vs. fibrous tissue

Fibroblasts (fibrous tissue):

• Wavy nuclei with pointy ends.
• Less nuclei.

SMCs:

• Elliptical nuclei.
• More nuclei.

Remembering the above:

• SMCs are stretched; ergo, not wavy.
• Fibrous tissue is fibrous... more protein... less cells; ergo, less nuclei.
• Fibroblast = football-like.
• Cigar-shaped nuclei (SMCs) are affected by cigars (smoking causes vascular disease).

Notes:

• Schwann cells (found in nerve): nuclei = wavy appearance, thin. (???)

DDx of granular crap

DDx of granular stuff:

1. Lipofuscin - especially in old people.
2. Hemosiderin.
3. Bile - found in hepatocytes, yellow.
4. Foreign material (tattoo pigment, anthracotic pigment).
5. Melanin.

Notes:

• Granular stuff should prompt consideration of malignant melanoma.
• Memory device: Männer lieben feine BHs = Melanin, Lipofuscin, Foreign, Bile, Hemosiderin.

Stains that can help sort it out

• Prussian blue for hemosiderin.
• Melan A for melanin.
• Kluver-Barrera for lipofuscin.

Staining

Main article: Stains

Basic knowledge of stain is important. The above article starts with H&E and goes from there.

Immunohistochemistry

Main article: Immunohistochemistry

If the special stains don't help... there is immunohistochemistry.

Food and pathology

Main article: Pathology and food

Clinician talk

General surgeon talk

• "R2 resection" = macroscopic tumour left.
• "R1 resection" = microscopic tumour left.
• "R0 resection" = clean margin macroscopically & microscopically.

Generally, positive margins suck; in locally advanced rectal cancer survival, in one study, five year survival was found to be 60%, 31% and 0% for R0, R1, and R2 resections respectively.

Oncologist talk

• ECOG - score from 1-5 for performance status.
  • ECOG = Eastern Cooperative Oncology Group.

ECOG score:

• ECOG 0: healthy.
• ECOG 1: ambulatory, no strenuous activity.
• ECOG 2: limited to self-care in bed <50% of time.
• ECOG 3: difficult to care for self in bed >50% of time.
• ECOG 4: bed bound.
• ECOG 5: dead.

Pathology & pathologists

Fixation & lifestyle

Pathologist have a great lifestyle 'cause tissue takes long to fix; the penetration of tissue by formalin is 1 mm/hour.

Malignancy & inflammation

If there is lots of inflammation... and you're thinking cancer you should probably back-off, i.e. tend toward benign. Inflammation can make cells look more malignant than they might be if left alone.

Miscellaneous

Infectious stuffs

Main article: Microorganisms

Images: http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram3.htm

• Staphylococcus - in clusters.
• Streptococcus - in chains.

Microscopes

Main article: HPFitis

• Pathologists throw around the term high power field (HPF).
  • "HPF" has no agreed upon definition and, IMHO, should never be used without a non-ambiguous definition.

HPF generally refers to the area seen with the largest magnification objective (40x), i.e. the field at 400x (as the eye piece magnification is usually 10x). The field size varies significantly from microscope to microscope.

Estimating field of view

FOV = D_{eye piece} x 1/M_obj.

Where:

• FOV = field of view.
• D_{eye piece} = diameter of eye piece (this is usually inscribed on the side of the eye piece).
• M_obj = magnification of the objective.

Example:

• D_{eye piece} = 22 mm
• M_obj = 40x (largest magnification objective)

Applying the formula:

• FOV = 22 mm / 40
• FOV = 0.55 mm

Pathology reports

There is no universal standard; however, there is a push to standardize by the Association of Directors of Anatomic and Surgical Pathology, among others.

Standards lead to uniformity and consistency.

The closest I've found to a standard is laid-out in by Goldsmith et al..

Standards

As far as I know, the first papers on the topic of standards were written in 1992.

Checklists

The College of American Pathologists (CAP) has checklists for cancer - CAP protocols.

I suspect pathologists will use more checklists in the future... they are deemed effective in a number of places inside and outside of medicine. Surgeons know that checklists work and that they save lives. Airline pilots have been using checklists for years and years.

An excellent book about checklists is: The checklist manifesto by Harvard surgeon Dr. Atul Gawande.

Standard diagnostic notation

Site, operation/procedure:
- Tissue type diagnosis.

Example:
Gallbladder, cholecystectomy:
- Acute cholecystitis.

See also

• Granulation tissue.

External links

• CAP protocols - cap.org.
• Regressive histopathologic findings (altervista.org).
• Atlas of Granulomatous Diseases (granuloma.homestead.com).