Preparation for biopsy:
Needles: Standard disposable 27-22 guage, 30-50mm long needles are suitable for superficial, palpable lesions. 25- guage needles are mostly used. Yield with such needles is however less, samples are however adequate and smears are of better quality due to lesser admixture with blood. 27 gauge needles are used mainly for children and for sensitive areas. Needles of 23-22 guage give best yield but thinner needles are more efficient. Larger needles are used if cell volume is more important than the smear quality for example to provide material for ancillary tests. 22 guage are most suitable for deep biopsies. Core needle biopsy (CNB) can provide tissue from fibrotic hypocellular lesions from which the yield of FNB is often inadequate. CNB has greater risk and trauma. FNA is a fine needle biopsy. Good quality disposable 5-20ml plastic syringes that produce a good negative pressure and fit into a syringe holder.
Sterile Containers: Sterile containers need to be used which have the balanced salt solution. Special cell cultures are required at times.
Slides: Glass slides thoroughly cleaned and free from grease. The aspirate can be smeared between 2 standard microscopic slides. Air dried smears are used.
Fixatives: Routine wet fixative of smears; either 70-90% ethanol in coplin jars is used. Carnoy's fixative has advantage of lysing red blood cells; glutaraldehyde and 10% buffered formalin should be available if tissue fragments for paraffin embedding are obtained.
Stains and Microscope: Differential quick stain or haematoxylin-eosin stain is used for proper viewing through the lightweight portable microscope.
Patient Preparation: A formal written consent is taken from the patient before any biopsy test. Pre biopsy sedation is rarely required. Atropine is recommended in preparation for transpleural biopsy to prevent unlikely risk of vasovagal reflex. Spray anaesthesia of a local anaesthetic is useful.
Biopsy Procedures: Insertion of needle in vertical position, this is needed for the better control and positioning of the needle.
Aspiration:The negative pressure created does not tear cells but merely holds the tissue against the sharp cutting edge of the needle, which cuts softer tissues.
Fibrous stromal cells are poorly represented in the aspirate, whereas cellular or myxoid stromal material is easily sampled. A maintained negative pressure must be released before the needle is withdrawn. Even so, a good part of aspirate is drawn up into the hub of needle and the aspirate must be rinsed with fluid to recover specimen.
Fine needle without aspiration: This technique is based on observation that the capillary pressure in a fine needle is sufficient to keep the detached cells inside lumen of the needle. This gives an excellent feel of consistency of tissues. This method suited well for biopsy of thyroid and other vascular tissues.
Failure to obtain sample: If the needle misses the target tangentially thus aspirating cells from the benign region but not from malignant region then it gives a scant yield.
Processing the sample is the next essential step.
Direct smearing: An aspirate is dry if it has creamy consistency and has numerous cells in a small amount of tissue fluid. A wet aspirate consists of smaller number of cells suspended in fluid or blood.
Macroscopial appearances of smears
(a) Optimal smear of dry sample carcinoma of prostate. Cell clusters seen as blue dots.
(b) Smear of wet sample where blood is at top end and cell clusters concentrated and evenly spread in thin mid portion.
(c) Poorly presented smears.
If smears are large they should be transferred to many slides and haematoxylin-eosin is used for immunostaining. Good cell fixation depends on rapid drying this is enhanced by moderate heat.
Indirect smearing: Thin fluid samples are the best processed by centrifugation on cytocentrifuge. Millipore or nucleopore film is an alternative but has been less satisfactory. Optimal pressure of cells and nuclear shapes are important in diagnosis of malignant lymphoma. Hank's balanced salt solution with addition of 10-20% ideal.
Tissue fragment and cellblocks: Fragments are the best assemble with a drop of blood adding thrombin to produce a clot. Tissue fixed in 5-10% buffered isotonic formalin and processed.
Fixation and staining: Two different methods for fixation and staining are used in FNAC. Air drying followed by heamatol stain such as MGG, alcohol fixation and staining according to Pap or with H & E. Air drying is a sensitive method to produce a thin and even film of material on side.
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