Objectives: Aetiological diagnosis of visceral leishmaniasis (kala-azar) by microscopic examination and culture and demonstration of the organism.

Aspiration may be contraindicated in patients with haemorrhagic disorders, local infection (skin, peritoneum, liver abscess, lung, pleura) close to the puncture site, or hydatid disease.

Test material: Splenic aspirate.

Collection time: Preferably in the morning, as the patient should be under close supervision for the next 10-12 hours.

Equipment: Two glass slides, tubed culture media (NNN and Schneider's Drosophila media), syringe, needle, antiseptic solution, cotton wool sponges, plaster.

Procedure: 1. Before the examination have the following laboratory results checked:

- thrombocyte (platelet) count > 10⁵/mm³ (10¹¹/L);

- coagulation time, whole blood (Lee and White) < 10 min.;

- bleeding time (Duke method) < 5 min.;

- prothrombin time (Quick) > 40% of normal value;

- thromboplastin coagulation time < 10 sec. longer than control value.

2. Clean two glass slides and label with patient's name, number, date, and "splenic aspirate." Have ready culture media (1 tube each of NNN and Schneider's Drosophila media) labelled like the slides. Allow the culture media to warm to room temperature. Attach the needle to the syringe. Place all items on the table at bedside.

3. Explain the procedure to the patient. Palpate the spleen and outline its margins on the patient's abdomen with a pen. For safety, the spleen should be palpable at least 3 cm below the costal margin on expiration. Disinfect the skin at the site of aspiration two times and allow it to dry in between.

4. With the 21-gauge needle attached to the 5-mL syringe, just penetrate the skin, midway between the edges of the spleen, 24 cm below the costal margin. Aim the needle cranially at an angle of about 45° to the abdominal wall.

5. To perform the actual aspiration, pull the syringe plunger back approximately 1 mL to apply suction, and with a quick in-and-out movement push the needle into the spleen to the full needle depth, and then withdraw it completely, maintaining suction throughout. The insertion should be timed with breathing so that the diaphragm is not moving.

6. In uncooperative young children, have two assistants hold the child (arms folded across the chest with shirt raised in the patient's line of vision and pelvis held firmly). Carry out the aspiration as a single stage procedure, using the same landmarks, angles, and suction as in steps 3 to 5, all in one, quick motion.

7. Only a tiny amount of splenic material is obtained, but this is adequate for culture and smear. Slowly pull the plunger back to 2-3 mL, and, using the sterile technique insert the needle into a tube of culture medium. Briskly push the plunger into the barrel to expel the contents of the needle into the side walls of the tube. If necessary, repeat once or twice until the splenic material is visible in the tube. Replace cap on tube and invert to wash the splenic material off the side of the tube. Repeat this procedure with the second tube of culture medium. A sterile technique is essential.

8. Expel additional material gently onto glass slides, holding the needle tip on the surface of the slide. Immediately spread evenly with the needle using a linear (not circular) motion. The smear should be not quite as thick as a thick blood film for malaria. Remove the needle and use it to obtain additional material from the tip of the syringe and spread it on slides. Further material may be found on the end of the plunger and dabbed directly on to a slide and spread. Allow the slides to dry.

9. Write time of aspirate on the patient's chart and instructions: "Record pulse and blood pressure half-hourly for 4 hours, then hourly for 6 hours. Patient to remain in bed for 12 hours." See that the patient understands these instructions. Enter the procedure in the notes and sign.

Storage: Room temperature, should not be refrigerated.

Transportation: The slides and media should be brought to the laboratory immediately.

Reporting: The microscopic examination is available within 24 hours; the culture may take up to 14 days.