Pleural or Ascitic Fluid

Slide preparations for the cytology of pleural fluid or ascitic fluid

In addition to mesothelial cells, pleural fluid and ascitic fluid also contain lymphocytes and eosinophilic and neutrophilic granulocytes. The protein content is frequently around 30 g/l. The cell content often varies considerably. Both fluids may contain blood, and ascitic fluid may also contain lipids.

Advantages of the Hettich Method

1. Cyto chambers for different cell contents and volumes
2. High cell yields
3. Good quality of the cells
4. Optimum spreading of the cells

Preparation

1. Preparation of samples with an elevated erythrocyte content

Since a high erythrocyte content will have a negative effect on the quality of the preparation, it is recommended that the erythrocytes be removed using a lysis buffer before derivation of the cyto preparation.

a) Preparation of the lysis buffer

The lysis buffer should be autoclaved to extend its shelf life.

b) Lysis of the erythrocytes

• Centrifuge the sample fluid for 10 minutes at 350 x g (corresponding to 1,700 min-1 in the 6-place rotor and 1,900 min-1 in the 4-place rotor).

• Pipette off the supernatant and set it aside.

• Resuspend the sediment in lysis buffer (1 part by volume sediment to 10 parts by volume buffer).

• Mix well and allow to stand for 10 minutes at room temperature. The hemoglobin liberated results in a transparent red coloration.

• Stop the reaction by adding 10 x the volume of physiological saline (0.9% NaCl) or phosphate-buffered saline.

• Centrifuge at 350 x g for 10 minutes.

• Decant the supernatant containing the lysed erythrocytes and discard it.

• Resuspend the sediment in 10 ml 0.9% NaCl (or PBS), mix it thoroughly and centrifuge it again for 10 minutes at 350 x g.

• Discard the supernatant and resuspend the sediment in the original sample supernatant that was set aside, and mix it well. A cyto preparation can now be derived from the sample.

2. Selection of suitable accessories

The 2 ml chamber (Cat. No. 1664) with sedimentation area of 60 mm2 and the 4 ml chamber (Cat. No. 1665) with sedimentation area of 120 mm2 are recommended for the centrifugation of pleural fluid and ascitic fluid. The 2 ml chamber is suitable for samples containing up to 100,000 cells and the 4 ml chamber for samples containing up to 200,000 cells.

3. Assembly of the cyto insert

Information on the assembly of the cyto insert is provided in our leaflet “Perfect preparations – with the Hettich cyto system all it takes is a turn”. For slide preparations from pleural fluid and ascitic fluid, it is generally necessary to use dry fixation. The cyto insert should, therefore, be assembled with the filter card (see B1 in the illustration). If the samples are infectious, then we recommend the use of our lid No. 1661 (see B2 in the diagram).

4. Centrifugation

a) Sedimentation

Fill the samples into the centrifugation tubes and centrifuge them for 10 minutes at 490 x g (corresponding to 2,000 min-1 with the 6-place rotor and 2,200 min-1 with the 4-place rotor).

b) Removal of the cell-free supernatant

The cell-free supernatant remains in the chamber after centrifugation and must be removed by careful aspiration.

It is crucial that the sediment is not disturbed while the supernatant is being removed, as this can affect the quality of the preparation and lead to cell loss. It is therefore recommended that the supernatant be removed using a Pasteur pipette with its tip just below the surface of the supernatant, moving downwards. The pipette should not come into contact with the slide – a small drop of liquid should be allowed to remain over the sediment!

c) Drying of the sediment

The sediment must be dried if the cells are to be stained using Giemsa stain. This requires a second centrifugation step. After removal of the supernatant, loosen the slide ring and remove it together with the chamber (see B4). Place the slide carrier with the slide and the filter card together with the suspension back in the centrifuge and centrifuge them for 1 minute at 1,100 x g (corresponding to 3,000 min-1 with the 6-place rotor and 3,400 min-1 with the 4-place rotor). The remaining liquid will be removed through centrifugal force and absorbed by the filter card. The cells will remain as a sediment on the slide. Their morphology will be intact and they will be well distributed over the surface. Evaporation artefacts such as leukocytes that are reduced in volume and the development of crystals will be avoided through the dry centrifugation.

d) Fixing and staining

The dry preparation can be fixed immediately and then stained.