ere are various types of flood cells preset in our body, having different shapes and izes rer ake upthe sain as pr their structures. Some of the Components ofthe blood ecls aeeAiTe Le they havea great ainiy for bsie dyes whereas some of the components ils uz they havea high affinity for acidic Dyes and then there are some wal and has the high ality for neutral stains. jog laboratory ae the combination of these three types hich acts as the mordant and are Aiden Components ofthe cells whieh arene Therefore the stains used in Haematol. of sane. Besides the dyes, a buffer is added to te stain w' Srhences the staining reaction, results inthe better morphology of the blood cells under the Mioroscope, Romanowsky stains ae such types of stains that are universally employed for the aining ofblood cells Almost ll the Romanowsky group of stains has two essential sosin or Azure dye. Methylene blu isa basie dye which componenis i. Methylene blue & E facie high affinity forthe acidic components of the celle. Nucleus and Eos Azure isthe Acidic dye which has the high affinity forthe basic components ofthe cells ie. the eytoplasm. and Granules in some cells. Most ofthe Romanowsky stains are prepared with Methyl Slcoho! (Methanol) so that they act as a fixative as well asthe cellular stain. There re 4 different types of Romanowsky stains commonly used in Hematology laboratory for staining the blood cells~ + Leishman Stain + Giemsa Stain + Field’s Stain + Wright's StainLeishman sain is amin urs of Methylene blue and Fosin de, grepared in Alcohol medium te reat em a tierce sain at wed o vray ac tease ees SSSI Ault adhere pao sn muna see ara Ste hun and hcl cel nd appa ar ue an pi aoe Dae od sca fr the mina fa! finer be msrp sn td eer Mood pans Gunes ann pes beer ea Principle of Leishman staining Leishman Stain is a neutral stain for blood smears which was devised by the British streon W. B. Leishman (1865-1926). Ie consists of mixture of eosin (an aciie stan), and 1 buffered during Methylene blue (abasic stan) in Meth alcohol and is usualy diluted an the staining procedure. It stains the different components of blood in arange of shades between red and blue, It is based on a methanolic mintue of "polychromed” Methy ene hve and cosin, The methanolic stock solution is stable and also serves the purpose of direoty Fixing the smear eliminating a prefixing step. Leishman stain is commonly used when there is need to examine the Blood smear forthe Various blood cells, Differential Leucosyts count, ‘and also used to differentiate nuclear Platelets, RBCs, WBCs as Type of Anemia, Toxic Granules & Platelet count e nd eytoplasmic morphology ofthe various cells of the blood like Well as forthe parasites, This stain is the mest dependable stain for Peripheral blood film Scamination. The working principle of the Leishman stan is same as described above, As it joa lye of Romanowsky stains, ft contains both the Acidic and Basic dyes which have the affinity for Basie and Acidic components ofthe Blood cells respectively. The acid dye, Fosin, variably stains the Basic components ofthe cells i.e. the cytoplasm, Granules ete. and the Basi dye, Methylene blue stains the Acidic components especially the Nucleus ofthe {ell The stain must be diluted for use with Phosphate buffer to pH 6.8 or 7.2, depending on vifc technique used, The pH 6.8 is preferred when the morphology of blood cells isto red and pH 7.2 is good for parasitic studies. scientific disciplines study the microscopic anatomy of eed 10 Histology, cytology and other related tissues and cells, In order to achieve a good tissve and cellular structure, the samples n inner. Leishman powder dye is intended for microscopy staining be stained in a correct mat tsing different methods. It is typical purple dye for cell nuclei because of its molecular interactions between Eosin ¥ and Azure B-DNA complex. Staining intensity depends on the amount of Azure B and Azure B/Eosin Y ratio. Leishman stain is clasified as & Romanowsky stan. Iti used for differentiating leukocytes because staining results in different hues ranging from red to blue.8 The en {hat it forms a 30° 45° angle om the side of the blood ds ™ Weal the sm ‘Afr spreading allow the smear te dry. 2. Leishman staining technique ‘Place the unfixed blood smcar on the staining rack * Now cover the thin layer of blood smear with the undiluted Sain of Leishman ‘sain solution by courting the drops of Leishman sai, Allow ito act for 2 mints, + Ad the double the volume of disiled water using the pipet After adding mix, the fluids, = Allow ieto act for 10 minutes + Wash the slide with distilled water thoroughly and gently until the slide appears bright pink in eolur. After washing the slid, blot dry the slid using bloting paper. Now observe the dried slide under the microscope 3. Steps for the Microscopie Examination ‘© Wipe and clean the mieroscope before use, ‘= Place the microscopic slide on the stage and move the slide to get used to the stage ‘moving knobs. ‘© Choose the low power objective (10X) on the microscope and place the area of interest on the glass slide in position ‘© Lower the condenser and keep theirs diaphragm partially closed, Now use the coarse adjustment knob to focus the slide and view through the eye piece until a sharp image is create. ‘The objective lens may now be moved to the 40X magnification, Life the condenser and open the iris diaphragm fully at this position Use only the fine adjustment knob to focus th slide. Use of coarse adjustment knob in this phase may result in breakage of slide and objective lens. ‘Once the image is in focus, do the reading. ‘Add a drop of immersion oil and move the objective to 100X (oil immersion) lens.* Red blood cells are stained pink with more staining witha central ates of pallor Platelets sain as blush purple cel fragments. ident on the periphery along Neutrophils havea blue eytoplasm, a3 or more lobed nucleus al eranules, + Eosinophils have a blue cytoplasm, abi-lobed nucleus along with orange red granules. ‘+ Basophils havea blue eytoplasm,airegular shaped mucus along with purple + Monocytes have plenty of blue cytoplasm with hershoe shaped nucleus and no granules, + Lymphocytes have scanty blue cytoplasm with around shaped nucleus which may have a den. Result All the micro cellular structural components present insie the blood were observed clearly using the Leishman staining technique which is type of differential staining technique successfully.