HETTICH-Methods*
Slide Preparation According to the One-step Method
with the Cyto Angle Chamber
Differential Cell Pictures of CSF (Cerebrospinal Fluid), Pleural and Ascitic Effusions and Dialysates*
* From the CSF Laboratory of the Institute of Clinical Chemistry and Laboratory
Diagnostics (Prof. Dr. Kluge and Dr. Roskos) at the University Hospital of Jena.
Lack of time, the 24-hour-availability of cytodiagnosis, a significant increase of requested
diagnostic parameters with, at best, the same amount of available sample material as well as
economic, organizational and concomitant personnel aspects (incorporation of specialized labo-ratories
in laboratory centres) demand the development and implementation of simple centrifuging techniques
that work carefully, reliably and guarantee reproducible cytodiagnoses from the collected body fluids.
Below described one-step method for the preparation of slides for the examination of cell suspensions
using cyto angle chambers in Hettich centrifuges meets these demands.
1. Selecting the suitable accessories
Angle chambers No. 1672 with a diameter of 8,7 mm and a sedimentation area of 60 mm² with filter
cards No. 1697 are best suited for 400 – 800 µl cell suspensions.
For routine examinations and traditional staining according to Pappenheim we recommend
the use of Polysine ™#-coated slides, for the immunocytological detection of cellular surface
markers, particularly tumour markers it is advisable to use Super Frost®# Color slides.
(# Registered trademark of Gerhard Menzel Glasverarbeitungswerk GmbH & Co. KG).
2. Preparing the samples
For buffering the sample and stabilizing the cells suspensions with a cell count below 10 / µl and
a protein content below 3000 mg / l (majority of CSFs) require the addition of protein, for which no upper
limit is to be observed. Fresh normal human serum available in laboratories serves the purpose (foetal
calf serum or albumin solution may also be used, but is not requisite).
To attain a protein content of more than 3000 mg / l with non-bloody, undiluted CSFs 50 µl serum is to
be added to 400 µl sample volume. The same applies to dialysates. If cell-rich, non-bloody or bloody
CSFs require dilution, either freshly prepared, refrigerated physiological NaCl solution / serum
consisting of 9 parts solution and 1 part serum (following called „9/1“ mixture) and allowed to warm up
to room temperature before use (compare 2.1.) or
cell-free supernatant gained by centrifugation from homologous CSFs if sufficiently available should be
employed. The centrifuging parameters are the same as for separating
serum from blood.
Body fluids with a normally high content of protein such as pleural or ascitic effusions only necessitate
the addition of protein in case of excessive dilution due to high cell counts (compare 2.3.).
2.1. Cerebrospinal fluid
Required is a minimum of 200 cells detectable on the sedimentation area to permit a satisfactory
proportional differentiation. Consequently, the cell count of the specimens should be determined with a
counting chamber (e.g. Fuchs-Rosenthal) before centrifuging. The centrifuging parameters
recommended under 3. allow a recovery rate of at least 25 %. CSFs with a cell content of e.g.
2 cells / µl, therefore, require specimen volumes of at least 400 µl. If the cell count is 1 cell / µl
(1/3 to 3/3 in the Fuchs-Rosenthal counting chamber), specimen volumes of at least 600 µl, better
800 µl, are obligatory.
Due to their otherwise reduced homogeneity specimen volumes should not fall short of 200 µl.
2.1.1. Non-bloody CSFs:
Dilution guidelines for specimen volumes of 400 µl per preparation with cell
counts of < 50 cells / µl : 400 µl CSF plus 50 µl serum
50 –150 cells / µl : 100 µl CSF plus 300 µl „9/1“ mixture or homologous supernatant
150 – 500 cells / µl : 50 µl CSF plus 350 µl „9/1“ mixture or homologous supernatant
> 500 cells / µl : 50 µl CSF plus 1000 µl „9/1“ mixture. Only fill 200 µl of it into the
angle chamber. Specimens with extremely high cell counts require
a still higher degree of dilution.
2.1.2. Bloody CSFs:
The degrees of dilution recommended for non-bloody CSFs do not entirely apply to bloody
CSFs, as aside from the count of white blood cells the share of erythrocytes also needs to be
considered. By choosing the right dilution ratio and the volume of the diluted cell suspensions not only
the overlapping by erythrocytes, but also the „diluting out“ of erythrophages and hemosiderophages
indispensable for correct diagnosis must be avoided. In order to get clearly evaluable slides it may
sometimes be necessary to make several preparations from one specimen diluted in various degrees.
2.2. Dialysates
In general dialysates do not need to be diluted. 50 µl serum is to be added to every 400 µl
sample volume.
2.3. Pleural and ascitic effusions
With pleural fluid and ascites the cell count typically varies between 50 and 5000 cells / µl.
So when processing 400 µl per preparations the same dilution ratio as for non-bloody CSFs
can be applied.
Because the protein content of 20 to 40 g / l of both effusions is quite high, dilution can be
effected with a 1:10 physiological saline solution without adding protein. Generally both fluids
can be collected in a sufficient quantity, so that they can also yield a sufficient amount of cell-free
supernatant for dilution.
3. Slide preparation with the angle chamber
3.1. Assembling the cyto insert
Fasten the angle chamber, the slide and the filter card with the neck ring on the slide carrier
(Cat. No. 1660) by turning the ring to its limit stop. While still retaining the tension turn the ring slightly
back so as to allow the filter card to absorb practically all supernatant during centrifugation
and leave no residue on the sediment.
3.2. Filling the specimen into the chamber
Place the cyto insert upright on the bench or into the cyto suspension (Cat. No. 1680) and pipette the pre-
processed specimen (as under 2. described) into the funnel of the chamber.
3.3. Centrifugation
There is a choice of two programmes:
Programme 1: 3 minutes at 100 x g
Programme 2: 4 minutes at 50 x g
For routine operation programme 1 is to be favoured.
3.4. Disassembling the cyto insert
Make sure that the entire liquid has been absorbed by the filter card before disassembling the
cyto insert. Take the insert out of the centrifuge, loosen the neck ring, take off the chamber and pull the
slide with the filter card out of the carrier. Remove the filter card carefully without obliterating the
sediment. Let the still moist sediment air-dry. (As a rule this takes 15 minutes, longest. A longer drying
period may damage the cells!)
Should there still be liquid in the angle chamber after centrifugation it has to be pipetted out of
the funnel while the chamber is still in vertical position. Due to the previous addition of serum the
supernatant must be discarded. You may now take the insert out of the centrifuge. Turn off the neck ring
with the insert in horizontal position. Allow the residual liquid on the sediment to disperse slowly and
evenly into the filter card to preserve the homogeneity of the sediment.
Marking the sedimentation area before staining proved helpful.
4. Staining the preparations according to Pappenheim
Stain in May-Grünwald solution (Merck) for 3 to 5 minutes, then rinse in distilled water.
Continue staining in diluted Giemsa solution for 10 to 15 minutes (1 ml stock solution from Merck with
10 ml distilled water).
After 3 minutes stains with May-Grünwald solution have attained sufficient intensity. In the majority of
cases they are still evaluable after 5 minutes. With ventricular cerebrospinal fluid or cells already altered
by therapy, however, the stains may grow too intensive for evaluation after 5 minutes.
Special stains and immunocytological markings of cell surfaces (e.g. lymphocyte and tumour markings)
can be carried out with the dried and unfixed cell sediments by observing special instructions.
The Angle Chamber
Preparation of a Dry Sediment in a Single Centrifuging Operation
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