Nitroblue-Tetrazolium (NBT) Reduction Test in Cultured Macrophages from Bone Marrow and Peripheral Blood: A Phagocytic Activity Assay from Leishmaniasic Dogs
*Pablo Gomez Ochoa, Juan Antonio Castillo, Manuel Gascon Perez, Javier Lucientes Curdi, Cristina Rodriguez Serrano, Maria Jesus Ramiro Ibañez
NBT was firstly used in the sixties in human medicine as a diagnostic tool for chronic granulomatous disease. Coloration change from yellow to blue -formazan-that NBT exhibits when it becomes insoluble because of fagolisosome enzyme action, is the morphological base of the test. From then to now it has been performed to value germ-killing ability of neutrophils. This work's main objective is to evaluate the NBT reduction test in macrophages cultured from peripheral blood and bone marrow from dogs with active leishmaniasis.
Samples from jugular vein, and rib marrow aspirations were taken, separating later mononuclear cells by centrifugation in a 1077 gradient, sowing 1000000 cells per plate with McCoy´s 5 A medium adding autologous serum. Incubation was made at 37° C in a environment of 5% CO2 and 100% relative humidity. At 10th day culture supernatant was removed, following Park et al. (1968) technique, covering after with NBT, 0.2% solution tampon phosphate M/15 pH7,2, and were incubated during 15 minutes in a wet chamber at 37° C and afterwards other 15 minutes at environmental temperature. May-Grünwald-Giemsa contrast staining was made. The optical microscopy reading valued as NBT positive cells all those that showed dark blue cytoplasm granules.
Among 5 to 10 % of healthy dogs macrophages were NBT test positives, noticing a fine disseminated spotted granulation into cytoplasm. In samples from leishmaniasic dogs a NBT test positivity around 50 to 80% was detected, consisting on coarse sole granules localized into big vacuoles. The bone marrow culture proved much more growth of fibroblasts- relationship macrophages/fibroblasts 2:1- with confluence areas( CFU-F) at 10th day. 5% of fibroblasts were positives to NBT test with a coarse granular pattern. Differences between total growth of both series weren't recorded.
The NBT reduction test is reliable for cells obtained from liquid medium culture, allowing us to a right macrophagic valoration. In dogs with active leishmaniasis, a raising in NBT test positivity was recorded- even a 80% of the macrophages- and moreover its showed a specific cytological pattern. The NBT test positivity inside marrow fibroblasts gives clear proofs of underestimated role that plays these cells during disease. The practice utility of NBT reduction test in the hematopoietic cultures allow us to identify macrophagic elements and to value germ killing against infection, giving a good prognosis factor in leishmaniasis.
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