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

Staining

• 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).
• Argyrophilic means has an affinity for silver/loves silver/stains with silver.

Morphologic patterns

Name of pattern	Meaning	DDx (incomplete/abbrev. list)	Image
Hobnail	basement membrane area < area exposed to luminal surface	angiosarcoma, clear cell carcinoma	angiosarcoma - high mag. (WC)
Storiform	spiral appearance or cartwhell pattern	solitary fibrous tumour, DFSP, dermatofibroma, UPS	DFSP - intermed. mag. (WC),SFT - intermed. mag. (WC)
Fascicular	the long axis of the (spindle) cells are perpendicular to one another in adjacent bundles of cells	leiomyoma, leiomyosarcoma	leiomyosarcoma - intermed. mag. (WC), Hemangiopericytoma (upenn.edu)
Plexiform	web-like formation	plexiform neurofibroma, MPNST, plexiform lesion of pulmonary hypertension	plexiform lesion of Pulm. HTn (flickr.com)
Cribriform	pierced with small holes	cribriform DCIS, cribriform HGPIN, cribriforming in a tubular adenoma with high-grade dysplasia, endometrioid endometrial carcinoma	cribriform DCIS (breastpathology.info)
Solid or Sheeting	no architecture - back-to-back cells with no pattern apparent / no spaces between cells	solid DCIS, poorly-differentiated malignancy (carcinoma, sarcoma, lymphoma)	solid DCIS (breastpathology.info)
Micropapillary	nipple like projections without a fibrovascular core (papillary = nipple-like)	micropapillary DCIS, micropapillary HGPIN	micropapillary DCIS (breastpathology.info)
Papillary	nipple-like projection with a fibrovascular core	papillary thyroid carcinoma	papillary RCC - high mag. (WC)
Flat	board-like, does not have a projection above the surface	flat DCIS	flat DCIS 1 (breastpathology.info), flat DCIS 2 (breastpathology.info)
Herring bone	like herring bone (technique) for climbing a hill in cross country skiing; books on a shelf, where they have partially fallen over -- on the one shelf to the left and the one below to the right	fibrosarcoma, synovial sarcoma, MPNST	MPNST intermed. mag.(WC), MPNST - high mag.(WC)
Trabecular or cords	trabecula = little beam; quasi-linear arrangement of cells	normal liver lobule, Sertoli cell tumour	Sertoli cell tumour - low mag. (WC), PTC - intermed. mag. (WC).
Nested (nesting)	islands of cells with a circular outline	neuroendocrine tumours	neuroendocrine tumour - low mag. (WC)
Biphasic / multiple patterns	multiple patterns or cell types; e.g. spindle cells and epithelioid cells	synovial sarcoma, malignant melanoma, pleomorphic adenoma, angiomyolipoma, chondroid syringoma	DSRCT - high mag. (WC)

Nuclear destruction words

There are several fancy terms:

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

Image:

• Karyolysis, pyknosis, karyorrhexis (WP).

Erosions and ulcers

• Ulcer = lesion through skin or mucous membrane.
• Erosion = limited to the mucosa - superficial ulceration.
  • In dermatopathology - through the epidermis.

Image:

• Ulcers and erosions - schematic (WC).

Microscopic - erosion

Features - require 1 and 2:

1. Loss of epithelium.
2. Vital response at site of lost epithelium.
   • Neutrophilic infiltrate.
   • +/-Fibrin.
   • +/-Cellular debris.

Image:

• Mucosal erosion (nih.gov).

The general differential diagnosis

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.

Features of malignancy

Cytologic features of malignancy

It is said that:

1. It is the nuclear abnormalities that make a cell malignant.
2. The cytoplasm that gives one clues as to the cell of origin.

Nuclear features and malignancy:

Feature	Strength in predicting malignancy?
Large nuclear size	weak
Nuclear-to-cytoplasmic ratio	strong
Nuclear pleomorphism	weak
Nucleoli shape (angulated, spiked, complex)	strong
Nucleoli size	weak - generally; strong if like in a RS cell
High nucleoli number	weak negative; finding favours benign
Chromatin hyperchromasia	weak
Chromatin granularity	strong
Nuclear membrane irregularities	strong (clefting, flat edges, sharp angles), scalloped (suggests benign)
Mitoses	weak §
Atypical mitoses	strong

§ mitoses are seen in poorly differentiated tumour and regeneration. High mitotic rate in the context of unremarkable nuclear morphology is usually not malignant.

Other features

In the context of soft tissue lesions, it is said that the two most important features of malignancy are:

1. Necrosis.
2. High vascularity.

Notes:

• Benign soft tissue lesions may have marked nuclear atypia and abundant mitotic activity.

Basic pathologic differential diagnosis of malignancy

This should always be considered:

Malignancy

Primary

Metastatic

Q. Why?
A. (1) The site of the tumour can considerably change the differential diagnosis. (2) The management is usually totally different.

A clinically motivated histomorphologic classification of malignancy:

Malignancy

Epithelial (Carcinoma)

Mesenchymal (Sarcoma)

Germ cell tumour

Neuroendocrine carcinoma

Hematologic

Malignant melanoma

Notes:

• Malignant melanoma, also melanoma, is a separate category as it can look like almost anything under the microscope.
• Hematologic includes lymphoma, leukemia, plasma cell neoplasms and others.

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, moderate basophilic cytoplasm, +/-"clockface" chromatin pattern (clumping of chromatin at the periphery of the nucleus), +/-perinuclear hof (crescentic cytoplasmic clearing adjacent to the nucleus; represents abundant Golgi apparatus).
• Epithelioid cell.
  • Looks like epithelium - cell borders touch neighbouring cells so that the cells collectively form a barrier.
• Small round blue cell tumour/lymphoid:
  • 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.
• Small lymphoid (small cell lymphoma).
  • "Small" in the context of lymphoid is classically ~1x a "resting lymphocyte" diameter.
  • Often not malignant by cytology.

Differential diagnosis by site

Main article: Short power list

It is essential to have a concept of what is common. The short power list gives a short differential diagnosis for the common sites.

Main article: Long power list

The long power list is a longer list for the common sites.

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

Main article: 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

Main article: Lymph node metastasis

• 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.
  • There are of course exceptions, e.g. small cell carcinoma, invasive lobular carcinoma.
• Histiocytes may be difficult to separate from tumour - especially for the novice.
  • Histiocytes may be found in the germinal centres, i.e. the node architecture helps.
  • Malignant cells, generally, have to have malignant features, i.e. the NC ratio is abnormal, there is nuclear pleomorphism.
• Several things can mimic metastases - see Lymph node metastasis.

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

Signet ring cell carcinoma

Main article: Signet ring cell carcinoma

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

Microscopic:

• Cells resemble signet rings:
  • 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.
  • The nucleus mimics the appearance of the face of the ring in profile.
• Cells typically 2-3x the size of a lymphocyte.
  • Smaller than the typical adipocyte.
• Often have a crescent-shaped 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.

DDx:

• Fat atrophy.

Stains:

• PAS stain.
• Alican blue-PAS stain.

Images:

• Signet rings (engravingarts.com).
• SRCs (H&E) (WC/Nephron).
• SRC AL-PAS stain (WC).
• SRC H&E stain (WC).

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

1. Collection of cells that have abundant bubbly cytoplasm - most useful feature.
   • Image: Granulomas showing abundant bubbly cytoplasm (WC).
2. 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).
3. Necrosis - too much pink (on H&E stained sections).
   • Image: Granuloma with necrosis (WC).

Notes:

• Small round collection of lymphocytes - without a capsule (as seen in lymph nodes).
  • If there are no macrophages... it's a lymphoid nodule.

As a list

Features:

1. Foamy/bubbly cytoplasm, abundant - low power.
2. Epithelioid morphology - cell borders near indistinct - key feature.
3. "Footprint" pattern nuclei/bean-shaped nuclei - key feature.
   • Macrophages usu. have an ovoid nucleus.
4. +/-Nucleoli, small.
5. +/-Fibrosis.
6. +/-Palisading at edge.

DDx

• Reactive germinal centre - especially if there are abundant surrounding lymphocytes.

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, infections in immunosuppressed individuals.

Etiologic classification

1. Infectious, e.g. tuberculosis, MAC, leprosy, syphilis, cat-scratch disease, schistosomiasis, fungal infection.
2. Neoplastic, e.g. seminoma, Lennert lymphoma, ruptured ovarian teratoma.
3. Autoimmune/inflammatory, e.g. Wegener's granulomatosis, Churg-Strauss syndrome.
4. Allergic, e.g. hypersensitivity pneumonitis.
5. Foreign body, e.g. pulmonary talcosis, silicosis, berylliosis.
6. Drug reaction.
7. Idiopathic, e.g. sarcoidosis.

Notes:

• Memory device: DNF AAII = drug reaction, neoplasm, foreign body, allergy, autoimmune, idiopathic, infection.
• A long DDx for granulomas is complied by James.

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 burnetii (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).

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).

Images:

• Collagen - ovarian fibroma (WC).
• Amyloid - cardiac amyloidosis (WC).

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 (brown) granular crap

DDx of granular stuff/pigment:

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 BH MILF = Bile, Homogentisic acid, Melanin, Iron (hemosiderin), Lipofuscin, Foreign material.
• Homogentisic acid found in alkaptonuria,can be considered the sixth (black) pigment.
  • Gentisic = jen-TIS-ik.

Stains that can help sort it out

• Prussian blue (iron stain) for hemosiderin.
• Fontana-Masson stain (or Melan A) for melanin.
• PAS stain or 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

Tumour remaining

Main article: Surgical margins

R classification:

• "RX resection" = residual tumour cannot be assessed.
• "R0 resection" = clean margin macroscopically & microscopically.
• "R1 resection" = microscopic tumour left.
• "R2 resection" = macroscopic tumour left.

Surgeons use this terminology. Essentially, it is the margin status. It is nice when the surgeon's assessment and the pathologist's are in agreement.

Note:

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

Clinician talk

Performance status

• 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

Note:

• Most of the resident microscopes, at U of T, have an eye piece diameter of 22 mm. Therefore, the field diameter at 40 X is approximately 22 mm / 40 X ~= 0.55 mm and the field of view is pi/4*(0.55 mm)^2 = 0.2376 mm^2.

Pathology reports

Main article: Pathology reports

The key point in report writing is that the report should be precise, complete and easy-to-understand.

Standards

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

Checklists

Main article: CAP checklists

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

Pathologists will probably 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. Pilots have been using checklists since the 1930s.

Standard diagnostic notation

Site, operation/procedure:
- Tissue type diagnosis.

Example:
Gallbladder, cholecystectomy:
- Acute cholecystitis.

Lab talk

Tissue cutting terms - these often vary from lab-to-lab:

• Recut = cut off the top of the block.
• Serial sections = make several cuts off the top of the block and look at all of 'em.
• Level = trim the block ~30 micrometres --throw away trimmed tissue-- and then cut a section to look at.
• Deeper = trim the block ~100 micrometres --throw away trimmed tissue-- and then cut a section to look at.

See also

• Granulation tissue.
• No truth in names.
• Blood work.
• Quality.

External links

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