ROMANOWSKY STAINS-1.pptx

The various romanowsky stains explained

1. MBARARA UNIVERSITY OF SCIENCE & TECHNOLOGY DEPARTMENT OF MEDICAL LABORATORY SCIENCE MLS 2112: HAEMATOLOGY & BLOOD TRANSFUSION TOPIC: ROMANOWSKY STAINS BY Tumukunde Benjamin

2. Objectives By the end of 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 Stains are 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 Romanowsky stains 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 stains work 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 Romanowsky stains Field stains (A and B) Giemsa stain May- Grünwald Leishman stain Wright's

8. Uses of Romanowsky stains Staining of blood and bone marrow specimens for cytopathological findings. Eg Leukemias Detection of malaria and other haemoparasites. Eg Trypanosomes.

9. Preparation of Romanowsky stains 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 c ommercially   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 Preparation of 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 Giemsa stock 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 Stock solution 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 stain preparation 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 stain preparation 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 preparation cont’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 for Leishman 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 for Wright’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 for May-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 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 for Field 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 procedures for 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 best in your endeavours