HETTICH-Methods

Slide Preparation of Cerebrospinal Fluid for Cytological Examination



Advantages of the HETTICH-Method


Direct preparation of the specimen because:
Processing of volumes up to 8 ml.
Adaptable to specimens with widely varying cell contents.
Gain of cell-free supernatant for further analyses.

The result: Slide preparations of high quality.
Optimal preservation of morphology.
Optimal density.
High yield cells.

Cerebrospinal Fluid (CSF)
CSF is the fluid found within the cavities of the brain and the spinal marrow. Healthy CSF
contains a maximum of 5 cells/mm3. Depending on where the sample is taken, the fluid
consists mainly of lymphocytes and monocytes. In addition, CSF contains albumin and
sugar. Frequently pathological CSF is characterized by a general increase of
mononuclear cells as well as by the presence of erythrocytes and granulocytes. The
concentration of sugar and albumin may also be increased.
In a cytological slide preparation the cells contained in the CSF are identified. In special
cases the cell types mentioned above are joined by tumor cells and pathogenic agents
such as bacteria and parasites. The biochemical analysis of the CSF provides information
on wether the concentration of albumin or sugar has changed.

The examination of CSF is a useful instrument for the diagnosis of:

Vascular lesions such as meningeal hemorrhaging.
Infectious diseases such as meningitis and AIDS.
Parasitosis such as toxoplasmosis.
Primary and secondary tumors.
Neurologic diseases such as multiple sclerosis.
Damage caused by environmental influences such as lead poisoning.
Speciments of CSF are never alike, therefore, a versatile technique of preparation is
required.

A good preparation of the cells present in the CSF has the following characteristics:

High yield.
No selective loss.
Arrangement in a constant layer of single cells, concentrated on a small area, with a clean
background.
Easy identification of the various populations.
The HETTICH cyto-technique meets all requirements demanded of an optimal preparation.

How to assemble the cyto accessories can be learnt from our leaflet "Perfect preparations
- with the new HETTICH cyto-system all it takes is a turn".



Assemble cyto-insert:
Slide preparations of CSF normally require a dry fixation. Assembly of the cyto-insert,
therefore, includes a filter card (compare point B 1 in our leaflet).
HETTICH offers chambers of 4 different sizes:

Areas of sediment (mm2):
30
60
120
240

Content approx. (ml):
1
2
4
8




Additionally, different kinds of chambers with 3-4 vials are available. This means even
more variability.

Fill with specimen:
In order to determine the amount of CSF necessary for a preparation some of the points
mentioned below must be considered.
The quality of the preparation depends on:

the number of cells processed: the more the better
the distance between the cells in the sediment: the smaller the better
This means: for low cell counts select small chambers, for high cell counts select large
ones.
The smallest chamber (30 mm2) can be filled with specimens containing up to 20,000
cells. For higher cell counts a correspondingly larger chamber has to be used.
The specimen`s concentration of albumin also influences the quality of the preparation.

When using cell-poor specimens (up to 1,000 cells per specimen) the quality of the
preparation is often insufficient. The reason is a small concentration of albumin
(approximately 0.2 mg/ml) usually present in healthy CSF. The danger that only burst
cells are found on the slide is very great. Therefore, we recommend to add a few drops of
human serum or albumin. The final concentration of albumin should range between 10
and 60 mg/ml. Obviously the supernatant of these specimens is not suitable for
biochemical analyses.

Which chamber for which specimen?

Content of cells: (cells/sample
up to 2.000
2.000-20.000
above 20.000

Addition of albumin
yes
no
no

Recommended size of chamber
30 mm2
30 mm2
above 60 mm2





Sedimentation
-Put the inserts into the cyto-suspensions.
In case of infectious material place on hygienic lid (compare point B 2 in our leaflet.
-Centrifugation: 3 minutes, RCF = 275
(i.e. 1,500 rpm with the 6-place rotor, 1,700 rpm with the 4-place rotor)

Now the cells of the specimen are spread on the slide. The hygienic lid warrants
aerosol-free centrifugation.

Removal of supernatant Remove the supernatant carefully with a Pasteur pipette except
for a small residue. When pipetting off follow the surface of the fluid from the top to the
bottom with the tip of the pipette. Do not stir and do not touch the slide. Leave a small
residue in the chamber. If not enriched by albumin the supernatant is then available for
further examinations (analysis of sugar and albumin).

Drying the sediment
In order to perform the Giemsa method of staining, the sediment has to be dried.
- Unfasten adaptor ring and take off the cyto-chamber after having removed the
supernatant (compare point B 4 in our leaflet).
- Centrifugation: 1 minute, RCF = 1,100
(i.e. 3,000 rpm with the 6-place rotor, 3,400 rpm with the 4-place rotor).

The residual fluid is pressed away by the centrifugal force and absorbed by the filter card.
The cells remain on the sediment and are optimally spread. Shrinkage of leukocytes and
formations of salt cristals are avoided.

Fixing and staining
Immediately after drying the sediment can be submitted to the fixing and staining
procedures.

Drying of the stained slides
When performing the Giemsa or May-Grünwald-Giemsa methods of staining, the slides
will be rinsed with buffer (Weise, Sörensen) at the end. Afterwards they have to be dried.

This can easily be performed with the HETTICH-labora-system-frame for 6 slides.

Place the slides rinsed with Weise buffer into the frames.
Put them into the centrifuge.
Centrifugation: 1 minute, RCF = 275.
(i.e. 1,500 rpm in the 6-place rotor, 1,700 rpm in the 4-place rotor).
Remove the frames with the dry slides.
Wipe the suspensions dry.
The preparations are ready for mounting or observation.
Up to 36 slides can be dried in the 6-place rotor.
CSF CYTOLOGIE
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