immunology of infectious and parasitic diseases - XXXVII Congress ...

STANDARDIZATION OF LEUKOCYTE ANALYSIS FROM SPUTUM BY
FLOW CYTOMETRY TECHNIQUE
PÂMELA CRISTINA GASPAR 1 ; RENATA CRISTINA MESSORES RUDOLF-
OLIVEIRA 1 ; LETÍCIA MURARO WILDNER 1 ; ANA CAROLINA RABELLO DE
MORAES 1 ; VANESSA MENGATTO 1 ; MANOELA LIRA REIS 1 ; CHANDRA
CHIAPPIN CARDOSO 1 ; MARIA LUIZA BAZZO 1 ; MARIA CLÁUDIA SANTOS-
SILVA 1 .
1 Federal University of Santa Catarina, Florianópolis, Brazil.
Introduction: Tuberculosis (TB) is an infectious disease caused by
Mycobacterium tuberculosis. Information related to sputum cytology found in
pulmonary tuberculosis is scarce, mainly it is unknown if there are differences in
pulmonary immune cellular response between patients HIV-positive and HIVnegative
and if there is a correlation between cytology and pulmonary response
to TB treatment. Thus, the aim of this study was to standardize a technique to
analyze leukocytes phenotype from sputum by flow cytometry. Methods and
Results: To obtain the cells, sputum was fluidized with dithiothreitol (DTT).
Approximately 1 g of sputum was incubated with 4 ml of DTT (0.1% - ratio 1:4)
for 15 min on a rolling mixer. The same volume of phosphate buffered saline
(PBS) (4 ml to 1 g sputum) was added to the mixture homogenized and
incubated for another 10 min. The suspension was filtered through a 50 mm cell
strainer and the filtrate was centrifuged (200 x g, 10 min). After centrifugation,
cells were washed with PBS and the pellet was resuspended in 1 ml of PBS.
Cell viability was determined using the tryptan blue exclusion method. To
prevent aerosol damages caused by sputum contaminated with M. tuberculosis
the tubes were left to stand for 10 min after each agitation and after sputum
fluidization, mycobacterium bacilli were killed by treatment with
paraformaldehyde (0.5% v/v for 40 min or 1.0% v/v for 30 min). In order to
evaluate DTT and paraformaldehyde influence on surface markers expression,
leukocytes from peripheral blood were submitted to the same fluidization and
paraformaldehyde protocol. To asses surface marker expression, samples were
staining with anti-CD45, -CD3, -CD4, -CD8 according to the manufacturer‟s
instructions. The results showed that surface markers were preserved. After
verify that all described procedures did not affect peripheral blood cells surface
markers, the sputum cells obtained and treated with paraformaldehyde were
also staining with cited monoclonal antibodies. Monocyte/macrophage,
lymphocyte and neutrophils cells were observed. In this way, it was verified that
surface markers of sputum cells were also preserved after all procedures and
could be detect by flow cytometry technique. Conclusion: The standardized
technique proved to be efficient in evaluation of cellularity from sputum cells by
flow cytometry.