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A Comparative Study Between Conventional and Modified Leishman Stain International Journal of Research and Review Vol.8; Issue: 2; February 2021 Website: www.ijrrjournal.com Original Research Article E-ISSN: 2349-9788; P-ISSN: 2454-2237 A Comparative Study between Conventional and Modified Leishman Stain Rizwana Abdul Hye1, Bindiya Gisuthan2, Indira Kariveettil3 1Assistant Professor, Dept of MLT, Alshifa College of Paramedical Sciences, Perinthalmanna. 2Assistant Professor, Dept of Pathology, Govt Medical College, TVPM-11, Kerala. 3Assistant Professor, Dept of MLT, Govt Medical College, TVPM-11, Kerala. Corresponding Author: Bindiya Gisuthan ABSTRACT modified method was 5.39, and the total score possible is 6. Photomicrograph showed Introduction: Leishman stain has been used as excellent results with modified Leishman stain. the stain of choice for peripheral blood films Thus from these values we can interpret that since many decades .But it has a disadvantage of modified method gave much more acceptable consuming 15 minutes for the procedure alone results than that of conventional method. thereby increasing the turnaround time of Conclusion: Unlike the conventional method peripheral smear reporting. In this study which requires a total of 15 minutes, to modified Leishman stain was made by adding complete the staining process, modified phenol to conventional Leishman to reduce the Leishman staining techniques takes only 3 staining time to 3 minutes without interfering minutes. Blood films can be stained within a with the quality of stain. short period of time thus aiding in rapid Aim: To study the quality of modified diagnosis and treatment of patients. Leishman stain in comparison with conventional preparation on peripheral blood smears. Key words: Leishman stain, Modified Leishman Materials and Methods: The present cross- stain, Phenol sectional study was carried out in Central Haematology laboratory of a tertiary health care INTRODUCTION centre in Southern India. A pilot study was done Romanowsky stain which is a to determine the ideal ratio of phenol to combination of an acidic stain and basic Leishman powder in preparing modified stain stain is the most common stain used without compromising the staining quality. worldwide for staining blood films. [1,2] The After obtaining consent, the blood sample was collected in anticoagulated vials. Sample size staining technique is named after the Russian physician Dmitri Leonidovich was calculated to be 85.Two thin wedge smears [3] were prepared from each sample and one was Romanowsky (1861-1921). The special stained with conventional Leishman stain and property of Romanowsky stains to other with modified Leishman stain. The distinctively stain granules of each cell is staining characteristics were assessed and scored due to two components Azure B (trimethyl by an experienced pathologist in terms of 6 thionine) and eosin Y parameters- RBC pattern, nuclear pattern, (tetrabromoflourescein). [4,5] The original neutrophil granules, eosinophil granules, Romanowsky combination contained platelets and background staining. The grades polychrome methylene blue and eosin. The were 1, 2, 3 & 4 (poor, satisfied, good and azure B and eosin Y was advocated for the excellent). Smears each with grades 2, 3 and 4 International Committee for Standardization were selected for study. The results were [6] analysed by Statistical Package for the Social in Haematology (ICSH). Azure B is one Sciences (SPSS) software version 16.0. of the oxidative products of methylene blue Result: The mean value for the staining quality and is superior to other azures, the reason it using conventional method was 4.73 and for International Journal of Research and Review (ijrrjournal.com) 5 Vol.8; Issue: 2; February 2021 Rizwana Abdul Hye et.al. A comparative study between conventional and modified Leishman stain. is more commonly employed in duly signed consent form in both English Romanowsky dyes. [7] and Malayalam. Original Romanowsky method was modified by William Boog Leishman a Study population / study material British pathologist and was called as Included all patients(OP&IP) whose Leishman’s stain. [8] Many modifications blood samples are collected into clean , dry have been done in Romanowsky dyes and penicillin bottles containing proper Leishman stain occupies an intermediate concentration of EDTA anticoagulant position among the various modifications (1.5mg/ml of blood) available. Inclusion criteria-Properly collected Leishman stain is routinely used in and labelled blood smears from Central Hematology labs to stain peripheral blood hematology laboratory, Govt. Medical films. [2] But the procedure is time College Hospital. consuming and cannot meet emergency Exclusion criteria-Unlabelled and needs in very sick patients. A very few improperly collected smears are excluded. modifications have been tried in Leishman stain to reduce the time taken for staining. Study period Since it is an era of rapid diagnostic A period of six months after the techniques and automated machines are approval of ethics committee. used for getting quick blood count results modifications are being tried in Leishmans Sample size calculation stain to reduce staining time without Formula, compromising the quality. In modified N = Leishman stain, phenol an accentuating agent is used to optimize staining and to reduce the time taken. [9] Phenol which is Where; P = used in Zeil Neelson staining for optimizing staining is used in modified Leishman stain P1: Proportion in the first group= 80%, P2: expecting the same result. Phenol act by Proportion in the second group= 60%, α: changing the pH of the staining solution Significance level = 5% thereby increasing the rate of stain uptake [10] 1-β: Power =80% by the tissue. Phenol also helps in N = 85 in each group ripening of the modified Leishman staining A pilot study was conducted to get solution and thus shortens fixing and optimum concentration of phenol by staining time. This will help in overall preparing stains using various ratios of reduction in turn-around time (TAT) of phenol and Leishman powder in acetone peripheral smear reporting. This study was free methanol. undertaken to compare the quality of modified Leishman stain with conventional Components of Modified Leishman Stain; preparation on peripheral blood smears. Component ingredients of modified Leishman stain are Leishman powder, MATERIALS AND METHODS absolute methanol, and phenol crystals. This cross sectional study was carried out in the Central hematology Determination of Phenol: Leishman laboratory and Department of MLT at a Powder Ratio/100ml of Leishman tertiary care hospital in south India. It was Weighed 30mg, 37∙5mg, 50mg, approved by the Institutional Ethics 75mg and 150mg of phenol crystals with Committee. Total two smears were collected 150mg of commercially-prepared Leishman from each patient and all patients were powder into different brown screw- capped properly informed about this study with a bottles to get phenol: Leishman powder in International Journal of Research and Review (ijrrjournal.com) 6 Vol.8; Issue: 2; February 2021 Rizwana Abdul Hye et.al. A comparative study between conventional and modified Leishman stain. the ratios 1:5, 1:4, 1:3, 1:2 and 1:1 Took an air dried smear prepared by respectively and this was dissolved in 100ml manual wedge method and covered the of absolute methanol to get 100ml of smear with undiluted stain, kept for 1-2 modified Leishman solution. The minute. preparation was done at room temperature Added twice volume of buffered water and the stain was kept protected from direct (pH 6.8) to dilute the stain. sunlight. The blood films were assessed after Mixed the water with stain by gently staining with modified stains in the ratio 1:3 blowing with a plastic bulb pipette or and became the reference phenol: Leishman with a straw. Allowed to stain for 10-12 powder ratios with optimal staining min performance. Inadequate fixing and Washed off stain with tap water. improper staining times of staining process Flooded the smear with buffered water, resulted in under-staining or over-staining pH 6.8 for 2 minutes and rinsed with of blood films. This caused wrong water and wiped the back of the slide differential count results. The fixing and and kept for drying. staining times of staining process were determined by experimenting the procedure For modified method time thus leading to development of new Covered the smear with undiluted staining procedures. modified Leishman stain for 25 seconds Collection of blood - Blood samples were Added twice volume of buffered water collected by venipuncture from the median to dilute the stain. cubital vein. Allowed to stain for 50 seconds. Blood smear preparation Washed off the stain with water. Two separate smears were prepared Flooded the smear with buffered water from each patient; those smears were with a pH6.8 for 2 minutes and rinsed allowed to air dry. One smear used for with water. conventional Leishman staining and the Wiped the back of the slide and kept for other smear was used for modified drying. Leishman staining. The morphological features of RBC, Neutrophil, Eosinophil and Platelets STAINING PROTOCOL stained with conventional and modified For conventional method Leishman stain in oil immersion(100X) is depicted in fig 1, 2, 3 & 4 Fig 1 RBC pattern stained with conventional preparation, 100x. Fig 1 RBC pattern stained with modified Leishman stain, 100x. International Journal of Research and Review (ijrrjournal.com) 7 Vol.8; Issue: 2; February 2021 Rizwana Abdul Hye et.al. A comparative
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