4. Hrvatski kongres kliniËke citologije 4th Croatian Congress ... - Penta

4. Hrvatski kongres kliniËke citologije / 1. Hrvatski simpozij analitiËke citologije / 2. Hrvatski simpozij citotehnologije
IMMUNODETECTION OF ANTIGENES IN CYTOLOGIC SAMPLES
Rešetar R
University Hospital Center Rijeka, Croatia
Objective: Presentation of the procedures of antigene detection in cytologic smears
depending on their cell location. Methods: Basic rules of immunodetection methods
have been used in the research. Cell antigens can be divided into three basic groups:
membrane, cytoplasmic and nuclear antigens. Since membrane and cytoplasmic antigens
are more accessible to the reaction with specific antibody due their cell location,
there were no difficulties in their detection. However, nuclear antigens, especially Ki-67
placed deep in the cell nucleus, can not be detected just by using classic procedures
of immunocytochemistry. Due to its specific location in the cell and in order to prove
nuclear antigens, an additional special technique called ‘antigen demasking’ is needed.
This technique is performed with the help of special fluid for ‘antigen demasking’ and
thermic treatment in the water bath. In immunocytochemical methods there are three
key steps important for the validity and positive result of reactions: 1. preparation fixation
time; 2. precisely performed ‘antigen demasking’; 3. incubation time for primary
monoclonal antibody. In immunocytochemical methods we have combined the following
procedures:1.fixation time: 2-10 min, 2.’antigen demasking’ time: 5-20 min, 3. fluid for
‘antigen demasking’: citrate buffer (ph=6) or Tris/EDTA (ph=9),4. incubation time for primary
monoclonal antibody: 30-90 min. For all types of antigens we used sylanized glass
impregnated by special adhesion matters. Results: Membrane and cytoplasmic antigens
are detected by classic procedures of immunocytochemistry with 10 minute fixation and
30-60 minute incubation with antibodies. The following procedure showed the best results
for the detection of nuclear antigens: 10 minute fixation in cold acetone, 5 minute
incubation in water bath on 95 °C in citrate buffer (ph=6) and 90 minute incubation with
primary antibody. Conclusion: For proving specific nuclear antigens all four steps are
needed as well as the procedure of ‘antigene demasking’ which includes thermic treatment
in water bath with obligatory use of fluid for ‘antigene demasking’. Membrane and
cytoplasmic antigens require only the combination of fixation and adequate incubation
time.
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