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

Histopathology in Practice

Sample Reception, Dissection,Processing, Embedding.

Della ThomasSenior Biomedical Scientist

Independent Histopathology Services

della.thomas@unilabs.com

Histology

The science concerned with the microscopic structure of tissues and organs in relation to their function.

Also called microanatomy.

Specimen Reception

Specimen Unpacking

Mickey MouseRt nasal polyp

Dr. Makewell26/11/1318/11/28DOB:

Specimen QC

MOUSEMr. MICKEY18/11/281 2 3 4 5 6Dr. Makewell26/11/13 8am

C/O loss of function - ability to sniffout cheeseO/E: nasal polyp? nature.

Rt nasal polyp

M

Specimen Labelling

Sample documentingAssign the

specimen to a pathologist

MOUSEMr. MICKEY18/11/281 2 3 4 5 6Dr. Makewell26/11/13 8am

C/O loss of function - ability to sniffout cheeseO/E: nasal polyp? nature.

Rt nasal polyp

M

Cassetting

Specimen Dissection - the stage

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

Cut-up dictation

• “Morphology” computer system

• “Dragon” dictation system

• Hands-free

• Just scan the barcode and away we go!!

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

Category A SpecimensBreast core biopsies

Endometrium

Colonic series

Category B Specimens

Cervical LLETZ

Small skin biopsy

Small lipoma

Category C Specimens

Appendix

Gallbladder

Haemorrhoids

Prepuce

Category D Specimens

Salivary gland tumour

Large Intestine (Crohn’s)

Skin with markers

Pigmented skin lesions

C/D

C/D

Category E Specimens

Testis (seminoma)

Thyroid (medullary Ca)

Breast cancer

Uterus (endomet. Ca)

Specimen Dissection

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

Doctors’ handwriting!!

“dysplasia”?OR “lymphoma”?

Breast Ca....”Steroids”?OR “Stomach”?

“Nothing” suspiciousOR “Notably” suspicious?

Simple transfer

Prostate cores

Cervical biopsy

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”

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.

Simple dissection

Specimen: Skin from back

Clinical details: Sebaceous cyst

Simple dissection

Unlock the pathologyFistula-in-ano

Unlock the pathology

Gallbladder

Unlock the pathologyPosterior view

Cystoprostatectomy

Inking• Resection margins

• Embedding instructions

• Orientation

• Distinguish between samples

• Identify the cut surface

• Acetic Acid

Inking resection marginsPigmented 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 markerClinical Data:Suture marker at 3 o’clock.

“12 o’clock” markerClinical Data:Tag suture at 12 o’clock.

?

“12 o’clock” marker

Mapping on a larger scale

Red = RightGreen = Left

ProstatePosterior bladder

Anterior bladder

Ureter

Cystoprostatectomy

Cassette sizes

Calcified / firm tissue

• Femoral head

• Bone Marrow Trephine

• Ethmoid mucosa / nasal polyps (cartilage)

• Nail

• Hardened cysts

Most common:

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 Hydroxide/Ammonium Oxalate) - very time consuming, but accurate

• Physical manipulation - not very accurate, may damage the specimen, but simple and inexpensive

Cutting instructions

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

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

ProcessingFormalin Alcohol Xylene Wax

Formalin

Alcohol

Xylene

Wax

Processing programs

EmbeddingThe immortalisation of tissue presentation

Embedding CentreMolten wax

Cold platesWax dispenser

Wax flow adjuster

Heated chambersUsed lids

Hot surface

Embedding tools

Embedding processDispense wax Align tissue

Cool in place Cassette on

Embedding processID bead Top-up wax

Cool plate

Leave to set

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!!

Final block preparation

Imagination

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Thank you