Pleural Fluid or Ascites Cytologie

HETTICH-Methods

Preparation of Pleural Fluid or Ascites with the HETTICH Cyto System

Besides mesothelial cells pleural fluid and ascites contain lymphocytes and eosinophil and neutrophil
granulocytes. Frequently the protein content amounts to about 30 g/l. The number of cells varies
strongly. The content of the samples we processed ranged from 30 to 5,000 cells/ml. Both fluids may be
mixed with blood, ascites may further be mixed with fat.

Procedure

The treatment is similar to that of cerebrospinal fluid.

Assemble the cyto insert, which consists of the cyto chamber, the filter card, the slide and the slide
carrier (B1*). Normally both fluids are cell-rich, wherefore we recommend to use 2 ml or 4 ml chambers.
Introduce the specimen into the chamber by pipette (B2*). Since the fluids are comparatively rich in
albumin, determination of the adequate amount of specimen is unproblematic.
We recommend: To fill specimens with a total number of maximally 100,000 cells into 2 ml chambers,
to fill specimens with a total number of maximally 200,000 cells into 4 ml chambers.
Centrifuge at an RCF of 490 (B3*) for 10 minutes.
With the UNIVERSAL line of centrifuges this corresponds to 2,000 rpm in the 6-place rotor. With models
UNIVERSAL 30 F and 30 RF use the "SOFT" key.
Pipette off the supernatant carefully (B4*).
Take off the cyto chamber (B4*).
Dry the preparation by centrifuging at an RCF of 1,100 (B4*) for 1 minute.
With the UNIVERSAL line of centrifuges this corresponds to 3,000 rpm in the 6-place rotor. With the
models UNIVERSAL 30 F and 30 RF use the "SOFT" key.
Remove the slide carrying the dried sediment and stain the preparation.
Preliminary treatment of specimens with a high rate of erythrocytes

A high rate of erythrocytes will affect the preparation negatively. Therefore, it is advisable to remove the
erythrocytes before preparing a specimen for cytological examination.

Below specified lysis buffer will cause the erythrocytes to burst:

8,29 g ammonium chloride (NH4CI),
1,0 g potassium bicarbonate (KHCO3),
0,037 g ethylene diamine tetraacetic acid (EDTA)

fill up to 1 l with distilled water and autoclave the buffer for better preservation.

Procedure:

Centrifuge the specimen at an RCF of 350 for 10 minutes.
Draw off the supernatant with a pipette and store it.
Add lysis buffer to the sediment (ratio: 1 volume of sediment to 10 volumes of buffer).
Suspend thoroughly, then allow the suspension to settle at room temperature for 10 minutes. The
liberated haemoglobin will turn the lysis buffer transparently red.
Stop the reaction by adding ten times the amount of physiological saline solution (0,9% NaCI) or
phosphate buffered saline solution (PBS).
Centrifuge at an RCF of 350 for 10 minutes.
Decant the supernatant, which contains the lysed erythrocytes, and dispose of it.
Resuspend the sediment in 10 ml 0,9% NaCI (PBS), stir well, centrifuge at an RCF of 350 for 10
minutes, then decant the supernatant.
Resuspend the sediment in the specimen`s supernatant, which has been kept, and mix well.
Prepare the specimen for cytological examination as described overleaf.
Take care not to expose the cells to lysis buffer too long, as they will otherwise burst as welll.

*Compare the correspondingly numbered assembly instructions in our leaflet "Perfect preparations-
with new HETTICH cyto-system all it takes is a turn".