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Tuberculosis (TB) is a global public health problem. Despite recent success in improving TB cure rates, diagnosis and case detection remains a major obstacle to its control. In this study the usefulness of four fixatives in improving Acid Fast Bacilli (AFB) microscopy diagnosis of TB was assessed. The study was conducted at Muhimbili National Hospital(MNH) in Dar es Salaam, Tanzania. A total of 560 cases of peripheral lymphadenopathies had fine needle aspiration cytology (FNAC) done. Out of these cases, 110 (19.6%) were diagnosed by ZN stain to be TB lymphadenitis. Different types of fixatives in diagnosis of TB were compared. Each specimen was smeared and fixed on to four glass slides using four fixatives (95% ethyl alcohol, Acetone, Carnoy’s fluid and 10% neutral buffered formalin), and one slide fixed using air dried method. Subsequently, Zeelh-Neelsen (ZN) stained slides were examined under a microscope for AFB. Of the 101 cases, 53 were TB adenitis, as diagnosed by AFB smear microscopy on ZN stained slides, and 48 were adenitis due to other causes. The diagnostic yield of AFB was highest when using 95% ethyl alcohol which was observed to be 30.2% (16/53) when AFB was 1+, 58.5% (31/53) when AFB was 2+ and 11.3% (6/53) when AFB was 3+, and making a total yield of 100% (53/53). Air dried smears also gave a relatively high yield with total yield of 94.3% (50/53). When 95% ethyl alcohol was used as a gold standard fixative, air dried slides had highest sensitivity (94.3%) and all fixatives had high specificity. The study has demonstrated that 95% ethyl alcohol is an excellent fixative. It is therefore recommended that 95% ethyl alcohol be used to form part of the routine fixation of FNAC smears.