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Year : 2004  |  Volume : 22  |  Issue : 1  |  Page : 72

Shortcomings require internal quality control

Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029, India

Correspondence Address:
Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029, India

How to cite this article:
Mirdha B R. Shortcomings require internal quality control. Indian J Med Microbiol 2004;22:72

How to cite this URL:
Mirdha B R. Shortcomings require internal quality control. Indian J Med Microbiol [serial online] 2004 [cited 2021 Feb 26];22:72. Available from:

Dear Editor,
This is with reference to the correspondence published in January issues of 2003,[1] which certainly needs clarifications for some scientifically unacceptable remarks that have been communicated.
Flurochrome dyes / stains which were used initially for detection of Mycobacterium tuberculosis, were reported to give good visualisation of malaria parasite. In parasitology, the use of flurochromes mainly Acridine Orange (AO) for diagnosis was first applied to blood parasites by Ambriose et al[2] and Sodeman described for the first time the AO staining of thick blood film for malaria.[3] Thereafter, World Health Organisation (WHO) Malaria Working Group proposed AO staining for the identification of malaria parasite. The technique has been extensively evaluated and validated under both experimental and field conditions. AO staining also has potential value and is being used for diagnosis of other microbial infections with blood samples (eg. microfilaria, trypanosomes) as well as with other biological specimens such as bone marrow (Leishmania spp.) and cerebrospinal fluid).
Established knowledge goes with the fact that the flurochrome acridine orange is capable of multicoloured fluorescence. This metachromasia depends upon its binding with nucleic acids. AO fluoresces yellow with double stranded (DS) nucleic acids and red with single stranded (SS) nucleic acids. Malaria parasite contains both nuclear deoxyribonucleic acid (DNA) and cytoplasmic ribonucleic acid (RNA), thus the parasite inside the red cell is differentially stained by AO and can be easily detected by observing under ultraviolet light in fluorescent microscope or by use of daylight illuminated light microscope fitted with interference filter.[4]
Author's view of not being able to differentiate leucocytes [polymorphonuclear cells (PMN cells) and lymphocytes with a homogenous large nucleus] from trophozoites of malaria parasite within red blood cells (RBCs) in AO stained peripheral blood film is not at all an acceptable fact. This remark puts a serious doubt about the quality of reagent that has been used by the author in their study. Such problem has never been encountered or reported by any of the workers worldwide. In fact, this is a remotest possibility. “Reversal of staining” (DNA getting stained red and RNA becoming yellow) does occur due to use of inappropriately made and stored reagents. In practice, this is the stage where the reagent needs to be changed or discarded immediately. Stability of AO stain is paramount and is improved by storage for a month or more at 4°C in the dark before use. In addition, clarity and pH stability are essential to obtain desired differential staining which permits rapid and reliable recognition of parasites.
Secondly, the conclusions made by author with only one hundred specimens in an malaria endemic country with probable use of inappropriate quality stain, needs further evaluation to disprove the efficacy of the technique. Our results and experience over a period of half a decade, have demonstrated higher sensitivity, precise morphological characterisation of both uninfected and parasitised red blood cells and greater rapidity of AO compared to Romanowsky's stained blood film.[5] Although, AO technique is not very satisfactory at times in species differentiation especially during synchronous multiplication of malaria parasite (trophozoite stage), but once the classical variations of trophozoites' position (accole), size (in relation to RBCs) and numbers within RBC(multiple infections) are taken into account, it is never ever a problem in species identification in malaria diagnosis. Quality control is one of the priorities in today's laboratory practice. Problems experienced by the author mostly pertain to the reagent used than the technique per se. 

 ~ References Top

1.Das S. Shortcomings of Acridine orange staining for malarial parasite. Indian J Med Microbiol 2003;21(1): 66.  Back to cited text no. 1    
2.Ambroise TP, Michel BJ, Despeignes J. Identification des parasites sanguicoles par coloration a l'acridine orange et microscopic de fluorescence. Bulletin de la socie'te' de pathologie exotique 1965;58:630-639.   Back to cited text no. 2    
3.Sodeman TM. The use of flurochromes for detection of malaria parasites. American J Trop Med Hyg 1970;9: 40-42.  Back to cited text no. 3    
4.Kawamato F. Rapid diagnosis of malaria by fluorescence microscopy with light microscope and interference. Lancet 1991;337:200-202.  Back to cited text no. 4    
5.Mirdha BR, Samantray JC, Burman D, Mishra B, Ghimire P. Quantitative buffy coat: a special adjunct for diagnosis of malaria. J Com Dis 1999;31(1):19-22.  Back to cited text no. 5    
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