supravital stains.p ptx

hhhhh

1. TECHNICAL UNIVERSITY OF MOMBASA HAEMATOLOGY 1 AML 4206 ZINDY CHEROTICH KIRUI-BMLS/006J/2014 HEZRON KAILANYA KAMIRI-BMLS/023J/2014 SAMUEL ODUOR OKOTH-BMLS/033J/2014 DR.WIGINA 16/3/2016.

2. Introduction Supravital stains. Used in microscopy to examine living cells that have been removed from an organism. Most commonly supravital stain is performed on reticulocytes using new methylene blue or brilliant cresyl blue. Other supravital dyes for red blood cells staining are; Crystal violet , methyl violet , Nile blue and Hoechst stain New methylene blue. Principle- non-nucleated immature erythrocytes contain nuclear remnants of RNA and the cell is known as a reticulocyte. To detect the presence of this RNA, the red cells must be stained while they are still living, RNA appears as a reticulum with the red cells.

3. Method Deliver 2 or 3 drops of the dye solution into a 75-X10- mm plastic tube by means of a plastic Pasteur pipette. Add 2-4 volumes off the patients EDTA-anti-coagulated blood to the dye solution and mix. Keep the mixture at 37 degrees Celsius for 15-20 minutes. Re-suspend the red cells by gentle mixing and make films on glass slides in the usual way. When dry, examine the films without fixing or counter staining. The exact volume of blood to be added to the dye solution for optimal staining depends on the red blood cells.

4. A larger proportion of anemic blood and a smaller proportion of polycythemia blood ,should be added than of normal blood. In successful preparation, the reticulofilamentous material should be stained deep blue and the non-reticulated cells should be stained diffuse shades of pale greenish blue .Films should not be counterstained. The reticulofilamentous material is not better defined after counterstaining and precipitated stain overlying cells may cause confusion.Moreover,Heinz bodies will not be visible In fixed and counterstained preparations.

5. If the stained preparation is examined under phase contrast , both the mature red cells and reticulocytes are well defined. By this technique, late reticulocytes characterized by the presence of remnants of filaments or threads are readily distinguished from cells containing inclusion bodies. Satisfactory counts may be made on blood that has been allowed to stand (unstained) for as long as 24hours although, the count will tend to decrease after 6-8hours unless the blood is kept at 4 degrees Celsius.

6. Counting reticulocytes An area of film should be chosen for the count where the cells are undistorted and where the staining is good. A common fault is to make the film too thin, however the cells should not overlap . To count the cells use power 100 oil immersion objective and if possible, eyepieces provided with an adjustable diaphragm. If eyepiece with an adjustable diaphragm are not available, a paper or cardboard diaphragm, in the center of which has been cut a small square with sides about 4mm in length, can be inserted into an eyepiece and used as less convenient substitute.

7. The counting procedure should be appropriate to the number of the reticulocytes present. Very large numbers of cells have to be surveyed if a reasonably accurate count is to be obtained when only small numbers of reticulocytes are present. When the count is less than 10%, a convenient method is to survey successive fields to determine the average number of red cells per field.

8. Calculation number of the reticulocytes in N field = X Average number of red cells per field=Y Total number of red cells in N field =XY Reticulocytes %=(N/NY)100% Absolute reticulocyte count =% x red blood cell Thus, when the reticulocyte % is 3.3 and the red blood is 5x10^2 per liter, the absolute reticulocyte count per liter is as follows:(3.3/100 )by 5x10^12=165x10^9 It is essential at the reticulocytes count exceeds 10%,only a relative small number of cells will have to be surveyed to obtain a standard error of 10%

9. An alternative method is based on the principle of a balanced sampling, using a miller ocular. This an eyepiece giving a square field, in the corner of which is a smaller ruled square, one-ninth the area of the total square. Reticulocytes are counted in the large square and the total number of red cells are counted in the small square.

10. Fluorescent method used to distinguish various components of a sample of blood . Red blood cells fluorescent stain. Red blood cells-fluorescein Isothiocyanate(FIT-C) dye . It is a synthetic organic molecule that binds to membrane proteins and fluorescence under fluorescent light. Tris - Buffered Ringers(TR) is use to wash blood samples via a centrifuge during staining process. Tri-Buffered Ringer albumin solution(TRA) IS Used to maintain healthiness of cells for overnight storage. The use of albumin is to maintain biconcave shape of normal erythrocytes.

11. Reticulocytes count. Reticulocytes can be counted manually by fluorescence microscopy on appropriately stained films method. Add 1 volume of alcidine orange solution (50mg/100ml of 9g/1NaCl) to 1 volume of blood. Mix gently for 2 minutes, make films on glass slides ,dry rapidly and examine by a fluorescent microscope.

12. RNA gives an orange red fluorescence, the brightness and color of the fluorescence fluctuates and the preparation quickly fades when exposed to light, also it requires a special fluorescence microscope . It is thus not suitable for thin use of reticulocytes counting.

13. white blood cells florescence staining. White blood cells - a dye composition for differential analysis of white blood cells comprising a hypotonic nuclear solution of a metachromatic flourochrome dye e.g. alcidine orange. Alcidine orange stained white blood cells are clearly visible in the acquired fluorescent image. The software indicates counted cells with a green circle.

14. References Hiller R . Glass , Hematology 1Human press second edition , ISBN 158829575. Provan D & Gibbean J.G(2005)Molecular Haematology , second edition blackwell publishing Hd,ISBN10:140511255 Wikipedia(google.com)