Biopsy and specimen handling

Cytology versus histology

• Cytology looks at loose cells (fine-needle aspiration, brushings, fluids, smears). It shows what the cells look like but not how they are arranged.
• Histology looks at a piece of tissue with its architecture intact, so it can show invasion of a capsule, vessel or basement membrane, the grade, and the pattern of a lymphoma.
• The principle that answers most questions: if the diagnosis depends on invasion or architecture, cells alone are not enough. This is why FNAC cannot separate follicular adenoma from follicular carcinoma (the difference is capsular or vascular invasion), cannot subtype most lymphomas and cannot tell in-situ from invasive breast cancer.
• FNAC is still excellent when cell type alone settles the question: thyroid nodules (papillary carcinoma has diagnostic nuclear features), metastatic carcinoma in a node, TB lymphadenitis (caseation, AFB, GeneXpert), salivary lumps and cysts.

Choosing the biopsy

Remember Cells for type, tissue for invasion: FNAC says what it is, core or excision says how far it has gone.

Special biopsy routes

• Sentinel lymph node biopsy: the sentinel node is the first node draining the tumour, found with a radioisotope (technetium-99m) and/or blue dye. In early breast cancer with clinically and ultrasound-normal axillary nodes its purpose is to stage the axilla. If it is negative, axillary clearance and its lymphoedema are avoided. It is also used in melanoma.
• Liver biopsy: percutaneous biopsy goes through the liver capsule, so bleeding risk rises with coagulopathy, low platelets and ascites (the liver is pushed away and the tamponade is lost). With gross ascites and a raised INR (for example 2.2) the safest route is transjugular: the needle passes from the internal jugular vein into a hepatic vein, so any bleeding returns into the circulation. Giving plasma rarely makes percutaneous biopsy safe. Transjugular access also allows hepatic venous pressure gradient measurement.
• Bone marrow: aspirate (cytology, flow cytometry, cytogenetics) plus trephine core (cellularity, infiltration, fibrosis), from the posterior superior iliac spine. The sternum is used for aspirate only in adults; the proximal tibia in infants. A dry tap suggests myelofibrosis or marrow infiltration.
• Renal biopsy: split for light microscopy (formalin), immunofluorescence (fresh) and electron microscopy (glutaraldehyde).
• Neck node biopsy in the posterior triangle: the spinal accessory nerve lies superficially there; injury weakens trapezius and causes shoulder drooping and pain.
• Amyloid: abdominal fat pad aspirate or rectal biopsy; stained with Congo red, giving apple-green birefringence under polarised light.

Lesions not to biopsy directly

• Testicular tumour: never through the scrotum (changes lymphatic drainage, seeds the scrotum); do inguinal orchidectomy.
• Phaeochromocytoma and carotid body tumour: risk of catecholamine crisis or haemorrhage; diagnose by biochemistry and imaging.
• Hydatid cyst: spillage causes anaphylaxis and seeding.
• Vascular lesions such as haemangioma; and a sarcoma should not be biopsied by a non-specialist through a track that will not be excised.

Fixation and specimen handling

• Standard fixative is 10% neutral buffered formalin (about 4% formaldehyde). It cross-links proteins and stops autolysis. Use about 10–20 times the specimen volume, fix promptly (breast within 1 hour so receptor staining is reliable) and open large specimens, because formalin penetrates only about 1 mm per hour.

• The lab only changes the fixative if it is told: writing "query gout" (or "?TB", "?lymphoma") on the request is what triggers the correct handling.
• Orientation: sutures (for example short superior, long lateral), ink or pinning on cork, with a diagram on the form, so margins can be reported.
• Avoid crush (forceps) artefact and excessive diathermy at margins; use separate containers for separate sites.

Exam trap Formalin ruins a specimen for culture, frozen section, flow cytometry and gout crystals; decide the test before the specimen goes into the pot.

Frozen section

• Fresh tissue is frozen in a cryostat, cut and stained with H&E in about 20 minutes while the patient is still under anaesthetic.
• Uses: confirm malignancy when the answer changes the operation, check resection margins, confirm lesional tissue is present, identify tissue (parathyroid versus lymph node), and Mohs surgery.
• Limits: freezing artefact, sampling error, fatty tissue cuts poorly, and it cannot reliably diagnose follicular thyroid carcinoma. The final diagnosis rests on paraffin sections; tell the lab in advance.

Processing, stains and immunohistochemistry

• Fixation, dehydration, clearing and paraffin embedding, sections of 3–5 micrometres, then H&E (haematoxylin nuclei blue, eosin cytoplasm pink). Bone needs decalcification, which delays reporting.

Labelling and the request form

• Label the container, not the lid, with at least three identifiers (full name, date of birth, hospital or NHS number), plus site, side, date and time. Unlabelled or mismatched specimens are rejected.
• The form needs requesting clinician and contact, specimen type and site, clinical details and working diagnosis, previous histology, the specific question, urgency, relevant drugs and infection risk (TB, HIV, hepatitis B and C).
• Pre-analytical errors (wrong patient, wrong tube, delay, haemolysis, wrong fixative) are the commonest laboratory errors. ABO-incompatible transfusion usually starts with a sample labelling error, so crossmatch samples are hand-labelled at the bedside.
• Blood order of draw: culture bottles, citrate (coagulation), serum, heparin, EDTA, fluoride oxalate. EDTA carry-over gives falsely high potassium and low calcium.

How FCPS asks about this

• FNAC limitation in a thyroid nodule: cannot distinguish follicular adenoma from carcinoma; needs histology for capsular or vascular invasion.
• Biopsy that removes only part of a large lesion: incisional biopsy; take the edge of an ulcer, not the necrotic centre.
• Purpose of sentinel node biopsy in node-negative early breast cancer: to stage the axilla and avoid unnecessary clearance.
• Liver biopsy with ascites and INR above 2: transjugular route.
• Fixing a suspected gouty tophus: absolute alcohol, because urate dissolves in formalin; write "query gout" on the form.
• Specimens sent fresh: frozen section, TB culture, lymphoma, karyotype; glutaraldehyde for electron microscopy; 10% formalin at 10–20 times specimen volume for routine histology.
• Nerve at risk in posterior triangle node biopsy: spinal accessory nerve; amyloid confirmed by Congo red on rectal or fat pad biopsy.