HETTICH-Methods
Preparation of BAL Specimens for Cytological Examination
(BAL:"Bronchio-Alveolar Lavage")
The bronchio-alveolar lavage (BAL)
BAL is a lavage of the lower respiratory tract with physiologic salt solution. It serves to collect endogenic
cell specimens and/or invaded foreign bodies for microscopic examination. The BAL specimen of a
healthy non-smoker contains about 90 % macrophages, maximally 15% lymphocytes, up to 3%
granulocytes, and up to 0.5% eosinophiles. BAL is cell-rich and in most cases mucus-containing.
In case of an illness the composition of the cell population is modified (e.g. in case of arcoidosis or
fibrosis) or other kind of cells are added (e.g. tumour cells). Inhaled foreign bodies (e.g. asbestos
particles) and pathogens (e.g. tubercle bacilli, pneumocystis carinii in case of AIDS) can be immediately
detected. For this reason cytopreparations of BAL specimens are referred to for diagnosing many
diseases of the respiratory tract and the lungs. The common staining method is that according to
Pappenheim; lately immunocytochemical staining methods are also used.
Advantages of the HETTICH-method:
easy preparation
slide preparation of optimal quality, i.e.
sediment of even cell distribution
well spread cells
all kinds of cells are presented reproducibly
no selective ioss of cells
easy examination, as the cells are concentrated on a small area only
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".
How to prepare BAL specimens with the HETTICH cyto-accessories
Removal of mucus
Filter specimen through two layers of gauze.
Assembly of cyto-insert
Either fasten the 2 ml cyto-chamber with a diameter of 60 mm² (1664) or the 4 ml cyto-chamber with a
diameter of 120 mm² (1665) on the slide resp. the slide holder (compare point A1 in our leaflet).
Filling with BAL specimen
Fill in so much specimen that the chamber will contain 1,000 to 2,000 cells each mm² of the sediment.
Consequently about 60,000 to 120,000 cells have to be filled into the 60 mm² chamber and 120,000 to
240,000 cells into the 120 mm² chamber (cells counted in the Fuchs-Rosenthal counting chamber).
Sedimentation
Centrifuge for 20 minutes at an RCF of approx. 275 (1,500 rpm with 6-place rotor, 1,700 rpm with the 4-
place rotor). In case of infectious material place on hygienic lid (compare point A2 in our leaflet).
Removal of supernatant
Pipette-off supernatant carefully, if possible, leaving no residue.
Drying the sediment
Remove cyto-chamber (compare point A4 in our leaflet), air-dry the sediment.
Fixing and staining
Drying the stained slides
This is fast and easily done with the HETTICH-labora-system-frame for 6 slides:
Place the slides that have been rinsed with Weise buffer into the frames.
Put the frames into the centrifuge.
Centrifugation: 1 minute, RCF = 275
(1,500 rpm with 6-place rotor, 1,700 rpm with the 4-place rotor).
Remove the frames containing the dry slides.
Wipe the suspensions dry.
The preparations are ready for mounting or examination now.
Up to 36 slides can be dried in the 6-place rotor.
Though Dr. Barkhofen`s method (Dr. Barkhofen, Schillerhöhe, Stuttgart/Gerlingen) proceeds from more
time-consuming procedure, it provides preparations of rather better quality (the cells are further spread).
Dr. Barkhofen`s method differs in following points from the above described:
Removal of supernatant
Tilt the entire cyto-insert sideways and let the liquid drain off. Allow the cyto-insert to dry in this position
for at least 1 hour – or longer, if possible (over night!)
Drying the sediment
Remove cyto-chamber, let possible ring-shaped residue air-dry.
Ordering-Informations
Order.-No.
Rotofix 32
1205
4-place rotor
1624
6-place rotor
1626
8-place rotor
1648
cyto-suspension
1660
hygienic lid
1661
slide carrier with adaptor ring
1662
cyto-chambers:
single, 6,2/30 mm²
1663
single, 8,7/60 mm²
1664
single, 12,4/120 mm²
1665
single, 17,5/240 mm²
1666
labora-system-frame
for 6 slides
1285
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