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- 1. MBARARA UNIVERSITY OFSCIENCE & TECHNOLOGY DEPARTMENT OF MEDICAL LABORATORY SCIENCE MLS 2112: HAEMATOLOGY & BLOOD TRANSFUSION TOPIC: ROMANOWSKY STAINS BY Tumukunde Benjamin
- 2. Objectives By the endof the topic, we should be able to;- • Define Romanowsky stains, • State the principle of Romanowsky stains, • Know the types of Romanowsky stains, • Know how different Romanowsky stains are prepared, • Know how different Romanowsky stains are used, • Know QC procedures in the use of Romanowsky stains.
- 3. Introduction • Romanowsky Stainsare named after a man called Dmitri Leonovich Romanowsky who invented it in 1891 • Defined as; stains that are used in haematology and cytological studies to differentiate cells in microscopic examinations of blood and bone marrow samples. • They are also applied to detect the presence of haemoparasites such as malaria, trypanosomes, leishmania and others parasites.
- 4. Introduction cont’d • Romanowskystains are neutral in a way that they are composed of oxidized methylene blue (azure) dyes and Eosin Y. • The azures are purplish- blue and are basic while the eosin is pinkish red and is acidic. • They stain the cell components differently depending on their PH. • This ability to produce a wide range of hues (colours) allowing cellular components to be easily differentiated is called Romanowsky effect/ metachromasia
- 5. Principle • Romanowsky stainswork on the principle that “the acidic components of the cell are stained by the basic dye (azure) forming a blue-purple colour while the basic components of the cell are stained by the acidic dye (eosin Y) forming a pin- red colouration.”
- 6. Staining characteristics Azure B(Methylene blue) staining Eosin Y staining
- 7. Types of Romanowskystains • Field stains (A and B) • Giemsa stain • May-Grünwald • Leishman stain • Wright's
- 8. Uses of Romanowskystains • Staining of blood and bone marrow specimens for cytopathological findings. Eg Leukemias • Detection of malaria and other haemoparasites. Eg Trypanosomes.
- 9. Preparation of Romanowskystains 1. Field stains • Are water based stains. • Field stain A is basic containing methylene blue derivatives and • Field stain B is acidic containing eosin Y Preparation of Field stain A from commercially available Powder. • Heat distilled water up to 60 °C • Measure 600 ml heated distilled water in a conical flask • Add 5 grams of commercially available Field stain A powder • Mix the powder until it dissolves completely • Filter the solution • Label with date of preparation.
- 10. Field stains cont’d Preparationof Field stain B from commercially available Powder. • Heat distilled water up to 60°C • Measure 600 ml heated distilled water in a conical flask • Add 4.8 grams of commercially available Field stain B powder • Mix the powder until it dissolves completely • Filter the solution • Label with date of preparation.
- 11. 2. Giemsa preparation Giemsastock solution • Dissolve 7.6 g of Giemsa powder with 500 ml of methanol • Heat the solution up to 60°C • Add 500 ml of glycerin • Filter the solution • Label the container with preparation date • Store the solution for 1 – 2 months before use in a cool dark place Note: Ensure that the glass ware is dry while weighing Giemsa powder because any contact with water will spoil the remaining powder Giemsa working solution (10%) • Measure 10 ml of Giemsa stock solution in a measuring cylinder • Add 10 ml of methanol • Add 80 ml of distilled water • Mix thoroughly • The reagent is ready for use
- 12. 3. May-Grunwald preparation Stocksolution • Weigh 0.3 g of May Grunwald dye • Dissolve it in 100 mL absolute methanol • Warm the mixture to 50°C in a water bath for a few hours and allow it to cool to room temperature. • Gently mix on an automatic rotator for 24 hours. • Filter the mixture and stain is ready for use. Working reagent (15%) • Measure 30 ml of May-Grunwald solution • Add 20 ml of buffered water • Add 150 ml of distilled water and mix. The stain is ready for use N.B: Some Labs use 10%
- 13. 4. Wright’s stainpreparation • Weigh 1.0 g of Wright’s powder • Mix with 400 ml of water free methanol • Label the container with preparation date
- 14. 5. Leishman stainpreparation Leishman stock solution • Weigh 0.15g of Leishman stain powder and grind it in a glass • Put the powder into a bottle through a funnel and gently add 20 ml of methanol through the same funnel to ensure that all the dry stain is washed into the bottle • Shake the mixture in a circular motion for 2-3 minutes • Add the remaining amount of methanol to the mixture through the same funnel up to 100ml mark • Cap the bottle and shake for more 2 minutes • Label the bottle and keep the mixture for a week. There after, the stain is ready for use.
- 15. Leishman stain preparationcont’d Leishman working reagent • Measure 50 ml of Leishman stock solution into a measuring cylinder • Add 75 ml of phosphate buffer (PH btn 6.4 – 7.0) • Add distilled water up to 250 ml mark (175 ml) • Mix and let the solution stand for 10 minutes before use
- 16. Staining procedure forLeishman A. Flooding method • Place the slide on the staining rack and flood it with methanol for 1 minute • Flood the slide with 20 drops of Leishman stain solution and allow to stand for 1 minute. Do not rinse • Apply 30 drops of buffer diluent • Mix gently by rocking the slide and allow to stand for 3 minutes • Pour off the mixture and rinse with distilled water for 10 – 15 minutes • Air dry the smear and examine microscopically. B. Dipping method • For the same steps with reagents in the coplin jars
- 17. Staining procedure forWright’s stain • Cover the air dried smear with un diluted stain for 2- 3 minutes. This partially fixes the smear. • Add equal amount of buffered water until a green scum appears on top. • Leave it stand for 5 minutes. • Without disturbing the slide, flood with distilled water and wash until the thinner part of the smear turns pinkish red • Allow the smear to air dry and examine microscopically
- 18. Staining procedure forMay-Grunwald stain It is together used with Giemsa stain May-Grunwald Giemsa staining • Fix the smear with methanol of 2 minutes • Cover the smear with May- Grunwald stain (10%/ 15%) for 5 minutes and tip off the excess • Cover the smear with 50% Giemsa stain for 15 minutes. • Rinse the smear with distilled water and leave it to air dry. • Examine microscopically
- 19. Giemsa staining procedure A.Thick smear • Prepare the stain using 1:50 dilution ratio (1 ml of the stain + 49 ml of buffered water) • Dip the smear in diluted Giemsa stain for 30 sec- 1 minute • Rinse the smear in distilled water 3- 5 times • Allow the smear to air dry. B. Thin smear • Prepare the stain using 1:20 dilution ratio (2 ml of the stain + 38 ml of buffered water) • Dip the smear in diluted Giemsa stain for 20 - 30 minutes • Rinse the smear in distilled water 2- 3 times • Allow the smear to air dry.
- 20. Staining procedure forField stains A. Thick smear • Dip an air dried smear in Field stain A for 4 seconds • Rinse with tap water • Dip the smear in Field stain B for 3 seconds • Rinse with tap water • Clean the back of the slide and leave to air dry. B. Thin smear • Fix the air dried smear with methanol for 1 minute • Dry in the air. • Dip fixed smear to Field Stain B (Red Stain) for 5 to 6 seconds. • Wash in running tap water. • Dip smear into Field Stain A (Blue Stain) for 10 to 30 seconds. • Wash in running tap water. • Leave to air dry
- 21. Quality control proceduresfor preparing & use of Romanowsky stains • Use of SOPs while preparing and using the stains • Appropriate weighing of powder stains while preparing stock solutions • Use of water free methanol while preparing the methanol based stains • Ensure that all solute particles of the powder are dissolved • Filtering of the stains before use • Use of smears from normal and abnormal samples to control stains before use • For stored samples, bring them to room temperature before processing them
- 22. All the bestin your endeavours