Cerebrospinal Fluid Preparation

Slide preparation of cerebrospinal fluid for cytological examination

In liquor diagnostics, conventional liquor cytology plays a vital role in the detection and classification of infectious inflammatory diseases of the CNS, in the detection of bleedings in subarachnoid spaces and the discovery of neoplastic cells. Also, cytological preparations are used for follow-up tests and therapy control.¹

As cerebrospinal fluid usually contains few cells and little albumin, cells start suffering damage a few hours after punction. Therefore, liquor samples taken to prepare slides for cytological examination should not be older than 2 hours.²

Not only is the age, but also the processing of the samples of vital importance for the diagnostic value of the preparations. Proper preparations of cerebrospinal fluid require optimal cell recovery and the preclusion of selective cell loss. The morphology of the cells needs to be maintained to enable correct identification of the different cell populations.

ADVANTAGES OF THE HETTICH METHOD

1) Immediate processing of the liquor samples

• • Suitable for samples of various cell content and different volumes up to 8 ml
  • Use of the cell-free supernatant for further analyses

2) Excellent preparation quality

• • Optimal preservation of the morphology
  • Optimal spreading of the cells
  • Optimal yield
  • Preparation

PREPARATION

1) Sample preparation

Often the quality of preparations of cell and albumin poor liquor samples is not satisfactory. We, therefore, recommend adding human serum or albumin solution. The addition of albumin to samples with cell counts below 10/µl and albumin rates below 3000 mg/l is considered vital for the stabilization of cells. To guarantee an albumin concentration above 3000 mg/l, 50 µl normal serum with a total protein content of approx. 70 g/l is added to a liquor sample of 400 µl. Autogenic serum is not required.³The use of coated slides can also increase cell yield. For routine and conventional staining it is recommended to use slides coated with PolysineTM, for the immunocytological detection of a cellular surface marker, particularly tumor markers it is recommended to use Super Frost® Color slides.⁴(PolysineTM and Super Frost® are registered trademarks of the Gerhard Menzel Glasbearbeitungswerk GmbH & Co. KG).

2) Suitable accessories

The available quantity and the cell content of the samples determine the adequate chamber. With liquor samples, we generally recommend the use of 1 ml and 2 ml chambers with sedimentation areas of 30 mm² or 60 mm².

3) Assembly of the cyto insert

How to assemble a cyto insert can be learned from our leaflet “Perfect preparations – with the HETTICH cyto-system all it takes is a turn.” Slide preparations of liquor cerebrospinalis usually require dry fixation. For this reason, the insert should be assembled with a filter card (see illustration B1 in the above-mentioned leaflet). When working with infectious samples, the inserts should be closed with hygienically sealed lid no. 1661 (see illustration B2 in the above-mentioned leaflet).

CENTRIFUGATION

a) Sedimentation

Centrifuge the cyto chambers for 3 minutes at 275 x g (this corresponds to 1,500 min-1 with the 6-place rotor and 1,700 min-1 with the 4-place rotor). During centrifugation, the cells sediment onto the slide.

b) Removal of the cell-free supernatant

After centrifugation, the cell-free supernatant is still within the chamber and needs to be removed except for a small remainder. When draining the chamber, take care not to stir the sediment. Otherwise, the quality of the preparation will suffer, and cells may get lost. We advise using a Pasteur pipette for removing the supernatant. Do not position the tip of the pipette on the slide, but follow the liquid level with it carefully downwards. Do not drain the chamber completely, but leave a drop of the supernatant on the sediment! If no albumin has been added to the sample, the recovered supernatant may be used for biochemical analyses.

c) Drying of the sediment

With the most frequently performed Giesma method of staining, the sediment needs to be dry. Dry the sediment by centrifuging it a second time. After the supernatant has been removed, twist off the fastening ring and lift it off with the chamber (see illustration B4). Put the cyto suspension with slide carrier, slide, and filter card back into the centrifuge and spin for 1 minute at 1,100 x g (this corresponds to 3,000 min-1 with the 6-place rotor and 3,400 min-1with the 4-place rotor). The residual supernatant is spun off by centrifugal force and absorbed by the filter card. The cells which constitute the sediment stay on the slide. They are well preserved and ideally spread. By drying the preparation by centrifugation, evaporation artifacts like shrunk leucocytes or crystallisations can be avoided.

d) Fixing and staining

The dry preparation can now be fixed and stained immediately.

ORDERING INFORMATION

• ROTOFIX 32A
• UNIVERSAL 320
• UNIVERSAL 320 R

Selection of accessories 

• 4-place rotor 1624
• 6-place rotor 1626
• Cyto suspension 1660
• Lid fitting onto 1660 1661
• Slide carrier with fastening ring 1662
• Cyto chamber 1 x 2 ml (60 mm2) 1664
• Cyto chamber 1 x 4 ml (120 mm2) 1665
• Labora-system frame for six slides 1285

1. Kluge et al., Stellenwert der praktischen (klassischen) Liquorzytologie im Gesamtspektrum der Liquordiagnostik, in Kluge et al. (Hrsg.) Atlas der praktischen Liquorzytologie, Stuttgart 2005, p. 2-3.
2. Ibidem, p. 7.
3. Ibidem, p. 8.
4. Recommended by the Liquor Laboratory of the Institute for Clinical Chemistry and Laboratory Diagnostics (Prof. Dr. Kluge, Dr. Roskos) of the Clinical Centre of the University of Jena.
5. The complete range of Hettich cyto accessories is listed in our brochure on cyto centrifugation, which can be ordered free of charge.