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

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

1. Histopathology in Practice
Sample Reception, Dissection,
Processing, Embedding.
Della Thomas
Senior Biomedical Scientist
Independent Histopathology Services
[email protected]

2. Histology
The science concerned with the microscopic
structure of tissues and organs in relation to
their function.
Also called microanatomy.

3. Specimen Reception

4. Specimen Unpacking

5. Mickey Mouse
Rt nasal polyp
Dr. Makewell
26/11/13
18/11/28
DOB:
Specimen QC
MOUSE
Mr. MICKEY
18/11/28
1 2 3 4 5 6
Dr. Makewell
26/11/13 8am
C/O loss of
function - ¯
ability to sniff
out cheese
O/E: nasal polyp
? nature.
Rt nasal polyp
M

6. Specimen Labelling

7. Sample documenting
Assign the
specimen to a
pathologist
MOUSE
Mr. MICKEY
18/11/28
1 2 3 4 5 6
Dr. Makewell
26/11/13 8am
C/O loss of
function - ¯
ability to sniff
out cheese
O/E: nasal polyp
? nature.
Rt nasal polyp
M

8. Cassetting

9. Specimen Dissection - the stage

10. Dissecting props
•Cutting board
•Forceps
•Ruler
•Scalpel / knives / saw
•Inks / dyes
•Cassettes / lids
•Biopsy pads / tissue
•Filter bags
•Weighing scales

11. Cut-up dictation
• “Morphology” computer
system
• “Dragon” dictation system
• Hands-free
• Just scan the barcode and
away we go!!

12. Specimen Categories
A
Specimens only requiring transfer from container
to tissue cassette.
B
Specimens requiring transfer but with standard
sampling, counting, weighing or slicing.
C
Simple dissection required with sampling
needing a low level of diagnostic assessment
and/or preparation.
D
Dissection and sampling required needing a
moderate level of assessment.
E
Specimens requiring complex dissection and
sampling methods

13. Category A Specimens
Breast core
biopsies
Endometrium
Colonic series

14. Category B Specimens
Cervical LLETZ
Small skin
biopsy
Small lipoma

15. Category C Specimens
Appendix
Gallbladder
Haemorrhoids
Prepuce

16. Category D Specimens
Salivary gland
tumour
Large Intestine
(Crohn’s)
Skin with markers
Pigmented skin
lesions
C/D
C/D

17. Category E Specimens
Testis (seminoma)
Thyroid (medullary
Ca)
Breast cancer
Uterus (endomet. Ca)

18. Specimen Dissection

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. Doctors’ handwriting!!
“dysplasia”?
OR “lymphoma”?
Breast Ca....”Steroids”?
OR “Stomach”?
“Nothing” suspicious
OR “Notably” suspicious?

21. Simple transfer
Prostate cores
Cervical biopsy

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. 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
•For every 5g over 12g, one
more cassette must be
processed
Prostate chips (19g)
Sampled in 8 cassettes:
First 12g = 6 cassettes
19g = 7g over 12g
1 cassette per 5g over = 2
more cassettes.
Sampled in 8 cassettes:
First 12g = 6 cassettes
19g = 7g over 12g
1 cassette per 5g over = 2
more cassettes.

24. Simple dissection
Specimen:
Skin from back
Clinical details:
Sebaceous cyst

25. Simple dissection

26. Unlock the pathology
Fistula-in-ano

27. Unlock the pathology
Gallbladder

28. Unlock the pathology
Posterior view
Cystoprostatectomy

29. Inking
•Resection margins
•Embedding instructions
•Orientation
•Distinguish between samples
•Identify the cut surface
•Acetic Acid

30. Inking resection margins
Pigmented lesion
Cervical cone

31. Embedding Instructions
Ink dots instruct the embedder to embed
the tissue a certain way.

32. Orientation - which way up?
•Anatomical

33. Orientation - which way up?
•Clockface

34. 3 o’clock marker
Clinical Data:
Suture marker
at 3 o’clock.

35. “12 o’clock” marker
Clinical Data:
Tag suture at
12 o’clock.
?

36. “12 o’clock” marker

37. Mapping on a larger scale
Red = Right
Green = Left
Prostate
Posterior
bladder
Anterior
bladder
Ureter
Cystoprostatectomy

38. Cassette sizes

39. Calcified / firm tissue
•Femoral head
•Bone Marrow Trephine
•Ethmoid mucosa / nasal polyps (cartilage)
•Nail
•Hardened cysts
Most common:

40. Softening
•For bony / hard tissue:
10% Formic Acid
•For nail:
Phenol or hair removal
cream.

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. Cutting instructions
•Levels
•Special stains
•Unstained Sections
•Serial sections
•Alopecia protocol
•MM sentinel lymph node
•Hirschprung’s protocol

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. Processing
Formalin ® Alcohol ® Xylene ® Wax
Formalin
Alcohol
Xylene
Wax

45. Processing programs

46. Embedding
The immortalisation of tissue presentation

47. Embedding Centre
Molten wax
Cold plates
Wax dispenser
Wax flow adjuster
Heated chambersUsed lids
Hot surface

48. Embedding tools

49. Embedding process
Dispense wax Align tissue
Cool in place Cassette on

50. Embedding process
ID bead Top-up wax
Cool plate
Leave to set

51. Embedding tips
• Ink dots: usually used to instruct the
embedder to embed the tissue a specific
way. Make sure you know your own
lab’s protocol.
• 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!!
• 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. Final block preparation

53. Imagination

54. www.slideshare.net/DellaThomas
[email protected]
www.micro2tele.com
@dellybean (Della Thomas)
@HistoQuarterly (Histology Blog)
www.facebook.com/histoquarterly
Thank you