Histopathology in Practice
Sample Reception, Dissection,
Processing, Embedding.
Della Thomas
Senior Biomedical Scientist
I...
Histology
The science concerned with the microscopic
structure of tissues and organs in relation to
their function.
Also c...
Specimen Reception
Specimen Unpacking
Specimen QC

Mickey Mouse
Rt nasal polyp
DOB:

MOUSE
Mr. MICKEY

18/11/28
26/11/13

Dr. Makewell

Rt nasal polyp
C/O loss ...
Specimen Labelling
Sample documenting
Assign the
specimen to a
pathologist

MOUSE
Mr. MICKEY
Rt nasal polyp
C/O loss of
function - ↓
ability ...
Cassetting
Specimen Dissection - the stage
Dissecting props
•
•
•
•
•
•
•
•
•

Cutting board
Forceps
Ruler
Scalpel / knives / saw
Inks / dyes
Cassettes / lids
Biopsy...
Cut-up dictation
• “Morphology” computer
system
• “Dragon” dictation system
• Hands-free
• Just scan the barcode and
away ...
Specimen Categories

A
B
C

Specimens only requiring transfer from container
to tissue cassette.

D
E

Dissection and samp...
Category A Specimens
Breast core
biopsies

Endometrium

Colonic series
Category B Specimens

Small lipoma

Small skin
biopsy
Cervical LLETZ
Category C Specimens
Prepuce

Gallbladder

Haemorrhoids

Appendix
Category D Specimens
Pigmented skin
lesions

Large Intestine
(Crohn’s)

C/D
C/D

Skin with markers

Salivary gland
tumour
Category E Specimens
Breast cancer

Thyroid (medullary
Ca)
Testis (seminoma)

Uterus (endomet. Ca)
Specimen Dissection
What does the pathologist
need to know?
Provide good descriptions - say what you see!!
• Shape
• Colour
• Texture
• Dimens...
Doctors’ handwriting!!
“dysplasia”?
OR “lymphoma”?

Breast Ca....”Steroids”?
OR “Stomach”?

“Nothing” suspicious
OR “Notab...
Simple transfer

Cervical biopsy

Prostate cores
Specimen Sampling
• Literally, taking a sample of the tissue
• Representative
• Generally, fewer blocks required if the ti...
Sampling rules: prostate chips
• If the chippings weigh 12g
or less, the entire
specimen must be
processed
• If the chippi...
Simple dissection
Specimen:
Skin from back
Clinical details:
Sebaceous cyst
Simple dissection
Unlock the pathology
Fistula-in-ano
Unlock the pathology

Gallbladder
Unlock the pathology
Cystoprostatectomy

Posterior view
Inking
• Resection margins
• Embedding instructions
• Orientation
• Distinguish between samples
• Identify the cut surface...
Inking resection margins
Pigmented lesion

Cervical cone
Embedding Instructions

Ink dots instruct the embedder to embed
the tissue a certain way.
Orientation - which way up?
• Anatomical
Orientation - which way up?
• Clockface
3 o’clock marker
Clinical Data:
Suture marker
at 3 o’clock.
“12 o’clock” marker
Clinical Data:
Tag suture at
12 o’clock.

?
“12 o’clock” marker
Mapping on a larger scale
Cystoprostatectomy
Prostate

Ureter

Red = Right
Green = Left

Posterior Anterior
bladder bladde...
Cassette sizes
Calcified / firm tissue
Most common:

• Femoral head
• Bone Marrow Trephine
• Ethmoid mucosa / nasal polyps (cartilage)
• ...
Softening
• For bony / hard tissue:
10% Formic Acid
• For nail:
Phenol or hair removal
cream.
Firm tissue testing methods
• X-ray - Expensive / ? bench space, but
very accurate
• Chemical end-point test (Ammonium
Hyd...
Cutting instructions
•
•
•
•
•
•
•

Levels
Special stains
Unstained Sections
Serial sections
Alopecia protocol
MM sentinel...
Specimen Storage
• Ventilated storage units
• Largest buckets lower shelves
• Units are in date (week) order
• 5-weeks’ wo...
Processing
Formalin → Alcohol → Xylene → Wax
Wax

Xylene

Formalin

Alcohol
Processing programs
Embedding
The immortalisation of tissue presentation
Embedding Centre
Molten wax
Used lids

Heated chambers

Wax flow adjuster
Hot surface

Cold plates

Wax dispenser
Embedding tools
Embedding process
Dispense wax

Cool in place

Align tissue

Cassette on
Embedding process
ID bead

Top-up wax

Leave to set

Cool plate
Embedding tips
• Always keep your eye on the tissue:
even for the most careful embedders,
tissue can ping like tiddlywinks...
Final block preparation
Imagination
Thank you
www.slideshare.net/DellaThomas
della.thomas@unilabs.com
www.micro2tele.com
@dellybean (Della Thomas)
@HistoQuart...
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Histopathology In Practice - Sample Reception, Dissection, Processing, Embedding.

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A talk about the daily operations of a histopathology laboratory - from specimen reception through to tissue embedding.

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Histopathology In Practice - Sample Reception, Dissection, Processing, Embedding.

  1. 1. Histopathology in Practice Sample Reception, Dissection, Processing, Embedding. Della Thomas Senior Biomedical Scientist Independent Histopathology Services della.thomas@unilabs.com
  2. 2. Histology The science concerned with the microscopic structure of tissues and organs in relation to their function. Also called microanatomy.
  3. 3. Specimen Reception
  4. 4. Specimen Unpacking
  5. 5. Specimen QC Mickey Mouse Rt nasal polyp DOB: MOUSE Mr. MICKEY 18/11/28 26/11/13 Dr. Makewell Rt nasal polyp C/O loss of function - ↓ ability to sniff out cheese O/E: nasal polyp ? nature. 18/11/28 123456 Dr. Makewell 26/11/13 8am M
  6. 6. Specimen Labelling
  7. 7. Sample documenting Assign the specimen to a pathologist MOUSE Mr. MICKEY Rt nasal polyp C/O loss of function - ↓ ability to sniff out cheese O/E: nasal polyp ? nature. 18/11/28 123456 Dr. Makewell 26/11/13 8am M
  8. 8. Cassetting
  9. 9. Specimen Dissection - the stage
  10. 10. Dissecting props • • • • • • • • • Cutting board Forceps Ruler Scalpel / knives / saw Inks / dyes Cassettes / lids Biopsy pads / tissue Filter bags Weighing scales
  11. 11. Cut-up dictation • “Morphology” computer system • “Dragon” dictation system • Hands-free • Just scan the barcode and away we go!!
  12. 12. Specimen Categories A B C Specimens only requiring transfer from container to tissue cassette. D E Dissection and sampling required needing a moderate level of assessment. Specimens requiring transfer but with standard sampling, counting, weighing or slicing. Simple dissection required with sampling needing a low level of diagnostic assessment and/or preparation. Specimens requiring complex dissection and sampling methods
  13. 13. Category A Specimens Breast core biopsies Endometrium Colonic series
  14. 14. Category B Specimens Small lipoma Small skin biopsy Cervical LLETZ
  15. 15. Category C Specimens Prepuce Gallbladder Haemorrhoids Appendix
  16. 16. Category D Specimens Pigmented skin lesions Large Intestine (Crohn’s) C/D C/D Skin with markers Salivary gland tumour
  17. 17. Category E Specimens Breast cancer Thyroid (medullary Ca) Testis (seminoma) Uterus (endomet. Ca)
  18. 18. Specimen Dissection
  19. 19. What does the pathologist need to know? Provide good descriptions - say what you see!! • Shape • Colour • Texture • Dimensions • Weight • Distances from margin(s) • Orientation markers • Cut surface appearance etc.... • Keep your fingers crossed for good clinical history
  20. 20. Doctors’ handwriting!! “dysplasia”? OR “lymphoma”? Breast Ca....”Steroids”? OR “Stomach”? “Nothing” suspicious OR “Notably” suspicious?
  21. 21. Simple transfer Cervical biopsy Prostate cores
  22. 22. Specimen Sampling • Literally, taking a sample of the tissue • Representative • Generally, fewer blocks required if the tissue looks uniform throughout (for benign cases) • Sampling “rules”
  23. 23. Sampling rules: prostate chips • If the chippings weigh 12g or less, the entire specimen must be processed • If the chippings weigh more than 12g, a minimum of 6 cassettes must be processed Prostate chips (19g) Sampled in 8 cassettes: First 12g = 6 cassettes • For every 5g over 12g, one more cassette must be processed 19g = 7g over 12g 1 cassette per 5g over = 2 more cassettes.
  24. 24. Simple dissection Specimen: Skin from back Clinical details: Sebaceous cyst
  25. 25. Simple dissection
  26. 26. Unlock the pathology Fistula-in-ano
  27. 27. Unlock the pathology Gallbladder
  28. 28. Unlock the pathology Cystoprostatectomy Posterior view
  29. 29. Inking • Resection margins • Embedding instructions • Orientation • Distinguish between samples • Identify the cut surface • Acetic Acid
  30. 30. Inking resection margins Pigmented lesion Cervical cone
  31. 31. Embedding Instructions Ink dots instruct the embedder to embed the tissue a certain way.
  32. 32. Orientation - which way up? • Anatomical
  33. 33. Orientation - which way up? • Clockface
  34. 34. 3 o’clock marker Clinical Data: Suture marker at 3 o’clock.
  35. 35. “12 o’clock” marker Clinical Data: Tag suture at 12 o’clock. ?
  36. 36. “12 o’clock” marker
  37. 37. Mapping on a larger scale Cystoprostatectomy Prostate Ureter Red = Right Green = Left Posterior Anterior bladder bladder
  38. 38. Cassette sizes
  39. 39. Calcified / firm tissue Most common: • Femoral head • Bone Marrow Trephine • Ethmoid mucosa / nasal polyps (cartilage) • Nail • Hardened cysts
  40. 40. Softening • For bony / hard tissue: 10% Formic Acid • For nail: Phenol or hair removal cream.
  41. 41. Firm tissue testing methods • X-ray - Expensive / ? bench space, but very accurate • Chemical end-point test (Ammonium Hydroxide/Ammonium Oxalate) - very time consuming, but accurate • Physical manipulation - not very accurate, may damage the specimen, but simple and inexpensive
  42. 42. Cutting instructions • • • • • • • Levels Special stains Unstained Sections Serial sections Alopecia protocol MM sentinel lymph node Hirschprung’s protocol
  43. 43. Specimen Storage • Ventilated storage units • Largest buckets lower shelves • Units are in date (week) order • 5-weeks’ worth of storage • Only authorised specimens are discarded after 5 weeks • Any outstanding cases are stored separately until further notice
  44. 44. Processing Formalin → Alcohol → Xylene → Wax Wax Xylene Formalin Alcohol
  45. 45. Processing programs
  46. 46. Embedding The immortalisation of tissue presentation
  47. 47. Embedding Centre Molten wax Used lids Heated chambers Wax flow adjuster Hot surface Cold plates Wax dispenser
  48. 48. Embedding tools
  49. 49. Embedding process Dispense wax Cool in place Align tissue Cassette on
  50. 50. Embedding process ID bead Top-up wax Leave to set Cool plate
  51. 51. Embedding tips • Always keep your eye on the tissue: even for the most careful embedders, tissue can ping like tiddlywinks. Make sure you see where it lands!! • Ink dots: usually used to instruct the embedder to embed the tissue a specific way. Make sure you know your own lab’s protocol. • Hide-and-seek: open lids / sponges carefully - tissue often sticks to them. ~ why it’s important for the embedder to know number of bits in the cassette • Cleanliness: always watch out for potential carry-over!!
  52. 52. Final block preparation
  53. 53. Imagination
  54. 54. Thank you www.slideshare.net/DellaThomas della.thomas@unilabs.com www.micro2tele.com @dellybean (Della Thomas) @HistoQuarterly (Histology Blog) www.facebook.com/histoquarterly

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