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Basics - Libre Pathology

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

H&E is the standard...

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

In words:

• Blue is bad and pink is dead!^[1]

Note:

• There is a lengthy list of things that are blue and not "bad"... that why a pathology residency is years.
  • Lymph nodes are very blue... they aren't necessarily bad.
  • Reactive processes can be very blue... they aren't bad.

Pathology can be boiled down to:

1. What is it?
   • Biopsies.
2. Did I get it all?
   • Resections.
3. Did I get the right thing?
   • Most other things.

• Eosinophilic - pink.
• Hyperchromatic - blue.
• Amphophilic - bluish-red colour when referring to H&E stained section.^[2]
  • 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^[3][4]/loves silver/stains with silver.

This covers things like cribriform, hobnail, herring bone and many others.

There are several fancy terms:^[5]

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

Image:

• Karyolysis, pyknosis, karyorrhexis (WP).

• 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)

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).^[6]

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.

It is said that:^[7]

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:^[7]

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

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.

This should always be considered:

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

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.
• The above is a useful clinical classification. The problem is it isn't that useful for difficult cases as:
  • Germ cell tumours are often not distinctive.
  • Numerous epithelioid sarcomas can mimic carcinomas.
  • Spindle cell carcinomas can mimic sarcomas very well.
  • Neuroendocrine differentiation is not always readily apparent.
  • The modified general morphologic DDx of malignancy is better for approaching difficult tumours.

Memory device HMN GEM: hematologic, melanoma, neuroendocrine carcinoma, germ cell, epithelial, mesenchymal.

Factors to consider when attempting to group by morphology:

1. Cell shape (spindle cell, epithelioid, plasmacytoid, mixed).
2. Cell size (small or large) - size in relation to a neutrophil or red blood cell.
3. Cell cohesion - dyscohesive vs. cohesive.
   • If one sees several groups of 5+ cells... probably cohesive.
   • Presence of cell cohesion strongly disfavours lymphoma.
4. Cytoplasm - abundance (scant, moderate, abundant).
   • Eosinophilic cytoplasm disfavours lymphoma.
   • Oncocytic - possessing copious eosinophilic granular cytoplasm.
     • Benign lesions composed of oncocytes - oncocytoma
     • Oncocytic metaplasia (alteration of cytoplasm) can effect all or a part of a lesion.
     • Oncocytic neoplasms are common in the kidneys, thyroid and salivary glands.
     • Oncocytic change increases with age
     • May represent senescent accumulation of mitochondria in secretory epithelial.
5. Chromatin - coarseness (fine, granular).
6. Nucleoli - number (absent, present, multiple).
   • Large nucleoli (nucleoli seen with the 10x objective) pretty much exclude neuroendocrine.

Note:

• † 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.

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.

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.

• 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, site (location) very useful - esp. gonadal, midline, retroperitoneal.
• 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.

The above is more useful than the general clinico-histomorphologically motivated differential diagnosis of malignancy.

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

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

• Nucleus darker (hyperchromatic) - key feature.
• No nuclear membrane - key feature.
• In prophase chromatin may have a scalloped border/beaded border.^[9]

DDx:

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

• Mitoses and an atypical mitosis. (WC)

• Tripolar mitosis. (WC)

www:

• Mitoses (vetmed.vt.edu).
• Starburst mitosis (flicker.com).

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

Main article:

• 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.

• 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 metastasis 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.

• 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.

• SRCs - H&E stain. (WC/Nephron)

• SRCs - AL-PAS stain. (WC)

• SRC - H&E stain. (WC)

www:

• Signet rings (engravingarts.com).

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).^[10]

The ABCs of pink:

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

• Cardiac amyloid. (WC/Nephron)

• Fibrin in a thrombus. (WC/Nephron)

• Collagen in an ovarian fibroma. (WC/Nephron)

• Smooth muscle. (WC/Polarlys)

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. (???)

• AKA brown/black 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, amalgam tattoo).
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,^[11]can be considered the sixth (black) pigment.
  • Gentisic = jen-TIS-ik.^[12]

• Prussian blue (iron stain) for hemosiderin.
• Fontana-Masson stain (or Melan A) for melanin.
• PAS stain^[13] or Kluver-Barrera for lipofuscin.

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

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

R classification:^[14]

• "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,^[15] five year survival was found to be 60%, 31% and 0% for R0, R1, and R2 resections respectively.

• ECOG - score from 1-5 for performance status.^[16]
  • 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.

Pathologist have a great lifestyle 'cause tissue takes long to fix; the penetration of tissue by formalin is 1 mm/hour.^[17]

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.

Main article:

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

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

• 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.

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 modern microscopes, 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.

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

There is no universal standard; however, there is a push to standardize by the Association of Directors of Anatomic and Surgical Pathology,^[18] among others.

Main article:

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.^[19] Pilots have been using checklists since the 1930s.

Site, operation/procedure:
- Tissue type diagnosis.

Example:
Gallbladder, cholecystectomy:
- Acute cholecystitis.

Tissue cutting terms - these often vary from lab-to-lab:^[20]

• 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.

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

1. ↑ Streutker, C. 8 June 2013.
2. ↑ URL:http://pancreaticcancer2000.com/page1.htm. Accessed on: 3 June 2010.
3. ↑ URL: http://www.merriam-webster.com/medical/argyrophilic. Accessed on: 29 August 2011.
4. ↑ URL: http://en.wiktionary.org/wiki/argyrophilic. Accessed on: 29 August 2011.
5. ↑ http://upload.wikimedia.org/wikipedia/en/5/51/Nuclear_changes.jpg
6. ↑ Arashiro, RT.; Teixeira, MG.; Rawet, V.; Quintanilha, AG.; Paula, HM.; Silva, AZ.; Nahas, SC.; Cecconello, I. (Jul 2012). "Histopathological evaluation and risk factors related to the development of pouchitis in patients with ileal pouches for ulcerative colitis.". Clinics (Sao Paulo) 67 (7): 705-10. PMC 3400158. PMID 22892912. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3400158/.
7. ↑ ^{7.0 7.1} S. Boerner. 12 September 2011.
8. ↑ URL: http://www.medterms.com/script/main/art.asp?articlekey=25657. Accessed on: 18 January 2010.
9. ↑ URL: http://www.microbehunter.com/wp/wp-content/uploads/2009/lily_prophase.jpg and http://www.microbehunter.com/2009/12/06/mitosis-stages-of-the-lily/. Accessed on: 3 November 2010.
10. ↑ URL: http://moon.ouhsc.edu/kfung/jty1/Com08/Com801-1-Diss.htm. Accessed on: 3 November 2010.
11. ↑ Mitchell, Richard; Kumar, Vinay; Fausto, Nelson; Abbas, Abul K.; Aster, Jon (2011). Pocket Companion to Robbins & Cotran Pathologic Basis of Disease (8th ed.). Elsevier Saunders. pp. 20. ISBN 978-1416054542.
12. ↑ URL: http://dictionary.reference.com/browse/gentisic+acid. Accessed on: 11 January 2012.
13. ↑ Kovi J, Leifer C (July 1970). "Lipofuscin pigment accumulation in spontaneous mammary carcinoma of A/Jax mouse". J Natl Med Assoc 62 (4): 287–90. PMC 2611776. PMID 5463681. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2611776/pdf/jnma00512-0077.pdf.
14. ↑ URL: http://www.informedicalcme.com/colon-cancer/tnm-stage-groupings/. Accessed on: 27 March 2012.
15. ↑ Larsen SG, Wiig JN, Dueland S, Giercksky KE (April 2008). "Prognostic factors after preoperative irradiation and surgery for locally advanced rectal cancer". Eur J Surg Oncol 34 (4): 410–7. doi:10.1016/j.ejso.2007.05.012. PMID 17614249.
16. ↑ Oken MM, Creech RH, Tormey DC, et al. (December 1982). "Toxicity and response criteria of the Eastern Cooperative Oncology Group". Am. J. Clin. Oncol. 5 (6): 649–55. PMID 7165009.
17. ↑ Gross rounds. 14 August 2009.
18. ↑ URL: http://www.adasp.org/papers/position/Standardization.htm
19. ↑ Soar J, Peyton J, Leonard M, Pullyblank AM (2009). "Surgical safety checklists". BMJ 338: b220. PMID 19158173. http://bmj.com/cgi/pmidlookup?view=long&pmid=19158173.
20. ↑ URL: http://www.mailman.srv.ualberta.ca/pipermail/patho-l/2002-July/016955.html. Accessed on: 18 October 2011.

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

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