RBC COUNT USING HEMOCYTOMETER.pptx presenting by dr Sangeeta swat

Rbc count using unbars chamber

1. RBC COUNT USING HEMOCYTOMETER Dr. Rashmi Prakash Gurao Department of Kriya Sharir National Institute Ayurveda Deemed University. Jaipur

2. PRINCIPLE OF TOTAL RBC COUNT USING HEMOCYTOMETER Very large numbers of Red Blood Cells are present in the Blood Specimen. Practically, counting this amount of Red cells directly under the microscope is highly impossible. So, the Red Blood cells are counted by using a special type of chamber, designed for the counting of blood cells in the specimen, known as Hemocytometer or Neubauer’s chamber. For this, the blood specimen is diluted (usually in 1:200 ratio) with the help of RBC diluting fluid (commonly the Hayem’s Fluid) which preserve and fix the Red blood cells. The Hayem’s fluid is isotonic to the Red blood cells and does not cause any damage to it.

3. The Normal Saline solutions can also be used for this but it causes the slight creation of red blood cells and allows rouleaux formation which may cause the errors in results. After diluting the specimen, the content is charged on Hemocytometer / Neubauer’s chamber and the cells are counted in the areas specific for RBC count. Nowadays, two types of RBC Diluting fluid are commonly used in Laboratories – Hayem’s RBC Diluting fluid Formalin Citrate diluting fluid

4. The composition of Hayem’s diluting Fluid-Mercuric Chloride ,Sodium sulfate Sodium chloride ,Distilled water

5. Introduction of H emocytometer : A hemocytometer (also known as a haemocytometer or a cell counting chamber ) is a tool used for manual cell counting. As the name implies, the hemocytometer was originally invented for quantifying blood cells.

6. Counting Chamber: Improved Neubauer Chamber The counting chamber was introduced by Crammer in 1805. Its modification by Thoma, and later by Neubauer remained in use for a long time. Improved Neubauer chamber is in current use. The counting chamber is a single, solid, and heavy glass slide . Extending across its middle third are three parallel platforms (pillars or flanges) separated from each other by shallow trenches (moats, gutters, or troughs).

7. The central platform or "floorpiece" (sometimes also called the plateau) is wider, and exactly 0.1 mm (one-tenth of a mm) lower than the two lateral pillars. The floorpiece is divided into two equal parts by a short transverse trench in its middle. Thus, there is an H-shaped trench or trough enclosing the two floorpieces.

8. The two lateral platforms can support a coverslip which, when in position, will span the trenches and provide a capillary space 0.1 mm deep between the undersurface of the coverslip and the upper surface of the floor pieces. Identically ruled areas, called "counting grids , consisting of squares of different sizes, are etched on each floorpiece. The two counting grids allow RBC and WBC counts to be made simultaneously if needed, or duplicate samples can be run.

9. Thoma's Chamber In this counting chamber, not used now, the central depressed platform is circular. The grid is only 1 mm² , consisting of 25 groups of 16 smallest squares each. The dimensions of the smallest squares are the same, i.e., 1/20 mm x 1/20 mm. The 1 mm squares at the corners are absent.

10. Old Neubauer Chamber There are nine 1 mm squares. The four corner groups of 16 squares each are for WBC counting, while the central 1 mm² area has 16 groups of 16 squares each for RBC counting (rather than 25 groups of 16 smallest squares as in improved Neubauer chamber). The medium squares are separated by triple lines.

11. The Counting Grid The ruled area on each floorpiece, the counting grid, has the following dimensions: Each counting grid measures 9 mm² (3 mm x 3mm). It is divided into nine large squares , each 1 mm²(1 mm × 1 mm). Of these nine squares, the four large corner squares are lightly etched, and each is divided by single lines into 16 medium-sized squares each of which has a side of 1/4 mm, and an area of 1/16 mm (1/4 mm x 1/4 mm). These four large corner squares are employed for counting leukocytes and are, therefore, called WBC squares.

12. The central densely etched large square (1 mm x 1 mm) , called the RBC square , is divided into 25 medium-sized squares , each of which has a side of 1/5 mm. Each of these medium squares is set off (separated) from its neighbors by very closely placed double lines (tram lines) or triple lines. These double or triple lines extend in all directions beyond the boundaries of the 9 mm² ruling, i.e., in between all the WBC squares around the central RBC square.

13. Each of the 25 medium squares (side = 1/5 mm), bounded by double lines (which are 0.01 mm apart) or triple lines, is further divided into 16 smallest squares by single lines . Thus, each smallest square has a side of 1/5 x 1/4 = 1/20 mm, and an area of 1/400 mm².

14. Procedure of the Total Red Blood Cell (RBC) Count Take 3.98 ml of RBC diluting fluid in a Clean, Dry and Grease free Test tube. If you don’t have variable pipette in the lab which can measure 3.98ml or 3980 μl of Diluting fluid then Take 4 ml of Diluting fluid with the help of 5ml Graduated pipette in the test tube and discard 20 μl of fluid using a micropipette or RBC pipette. Now add 0.02 ml or 20 μl of Blood Specimen to the tube containing diluting fluid with the help of micropipette or RBC pipette. Mix well for few minutes and ready your Hemocytometer / Neubauer’s Chamber. Take out the Neubauer’s chamber / Hemocytometer from its case and clean it using a swab or gauze piece. Similarly, clean out the cover glass and place it over the grooved area of Hemocytometer. Here a special type of cover glass is used which is 0.4 mm thick with very smooth surface and even thickness so that the space between the grooved area of the chamber and cover glass is exactly 0.1 mm.

15. Now, take out the RBC pipette and fill it with the Diluted Specimen, mix the solution well and then discard 1-2 drops from the pipette before charging the chamber. Gently press the rubber tube of the RBC pipette, so that the next drop of fluid is in hanging position. Touch the Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45° approximately. Allow a small amount of fluid from the pipette to fill into the chamber which occurs by the Capillary action. Do not overcharge the chamber and there should be no air bubble in the Chamber.

16. COUNTING THE RED BLOOD CELLS UNDER MICROSCOPE Focus the ruling using the 10x Objective lens and then Count the RBCs in 5 small squares of the central square as described above, using the 40x Objective lens. Count the cells which are lying on the right and lower lines of the 5 small squares but not the opposite line. In case of marginal cells, count the cells on ‘L’ line that is either on Right and Lower lines or Left and Upper lines.

17. CALCULATIONS FOR THE TOTAL RBC COUNT USING HEMOCYTOMETER After counting the cells under the microscope, we know the No. of RBC in 5 squares of the central square. Let’s consider it as ‘N’ no. of cells. Now, the volume of the fluid inside the chamber is the product of Area and depth of the Hemocytometer / Neubauer’s chamber

18. The central area is the 1 sq. mm which is divided into 25 parts so the area is 25 squares = 1 sq. mm Out of these 25 squares, the RBCs are counted in 5 squares. So the Area of 5 small squares is 5/25 i.e. 1/5 The depth of the Hemocytometer is 0.1 mm as described above in a short description of Hemocytometer. Now Apply the Following formula to get the Total Red Blood Cell Count – Total RBC Count = N × Dilution / Area × Depth N × 200 (or 100 as the dilution is made) / (1/5 × 0.1) tToal RBC count = N × 10,000 / mm3

19. NORMAL VALUES OF RED BLOOD CELLS In Males – 4.8-5.5 million/mm3 In Females – 4.5-5 million/mm3

20. PURPOSE OF PERFORMING TOTAL RBC COUNT The purpose of performing Total Red Blood cell count is to know whether or not you are suffering from Erythrocytosis or Polycythemia (i.e. the increase in the no. of Red Blood Cells to more than 6.5 million/mm3) or Erythrocytopenia or Erythropenia (i.e. the Decrease in the no. of Red Blood Cells to less than 3.5 million/mm3).

21. THANK YOU