Appendix 4. Fine needle aspirate cytology (FNAC)


FNAC is used to obtain material from lymph nodes. The material is expressed onto slides and prepared for examination.

Two smears will be prepared with Giemsa stain1 2 to look for caseum, granuloma, giants cells, and epithelioid cells or histocytes and 1 or 2 will be prepared with Ziehl-Neelsen (ZN) stain to look for acid-fast bacilli (AFB).

Equipment

– Needle 23G (in very few cases, it would be possible to use 19G)
– 10 ml syringe
– 2 slides for Giemsa + one or 2 slides for ZN stain
– 10% polyvidone iodine, sterile gauze, gloves

Technique

– Disinfect the area.
– With the needle attached to the syringe, insert the needle deep into the lymph node.
– After the needle has entered the mass, pull back on the syringe plunger to create a vacuum.
– Rapidly move the needle in a to-and-fro fashion to allow material entering the needle.
– When blood or material appears in the needle hub the aspiration should be stopped. Try to aspirate as much as possible of materials, the amount of materials that has been aspirated would have effect on the specificity and sensitivity of diagnosis.
– Release the negative pressure before to take out the needle from the lymph node. Do not continue sucking while taking out the needle, this will avoid aspiration of materiel into the barrel of the syringe and avoid mixing the sample with the possible peripheral blood in the skin.

Slide preparation

Slide should be identified prior to the aspiration and prepared immediately after the aspiration.
– Detach the needle from the syringe immediately after the aspiration.
– Fill the syringe with air (needle is still detached).

Prepare the smear as follow:

Giemsa
• Reattach the needle to the syringe and carefully release one small drop of sample onto one end of the slide by pushing down the plunger of the syringe (if the drop is placed in the middle of slide it would be difficult to make smear afterwards).
• Put another slide over the sample.
• Slide the two slides against each other, in opposite directions, to spread the sample out completely between them. Do not press the slides together forcefully, to avoid crushing the cells.
• Allow to air dry.
• Fix the smears by methanol when they are completely dry.
• Proceed to Giemsa staining.

Ziehl-Neelsen
• Place a small drop of ganglion aspirate on the slide.
• Make a smear that is neither to thin or too thick.
• Allow to air dry.
• Fix the smear by flame when it is completely dry.
• Proceed to ZN staining.

Reading after Giemsa staining

On each slide, one or several of the following aspects can be found:
– Caseation necrosis (caseum): a uniform, acellular, pinkish substance.
– Granuloma: cluster of epithelioid cells and lymphocytes scattered through out smear with or without caseous necrosis.
– Epithelioid cells: elongated, often semi-lunar cells with a fine granular nuclear chromatin surrounded by pink cytoplasm.
– Giant cells: huge multinuclear cells.


Notes:
– It would be better to look for granuloma and necrosis with the 10x and 40x power of microscope then to look for epithelioid cells and giant cells with 100x power.
– Observation of smear requires a competent reader with skills in cytology. Slides have to be sent to a referral cytopathology laboratory for quality control or confirmation.
– The quality of the specimen and the preparation are essential. The smear is to be done by skilled technicians.



Footnotes
Ref Notes
1

The golden standard of diagnosis for TB on tissue samples is hematoxylin-eosin stain, but Giemsa stain can be used as an alternative in remote areas with limited equipment.