1. RBC, WBC, & PLT Counts Nada Mohamed Ahmed, MD, MT (ASCP)i

2. Hemacytometers Hemacytometers  a precision-made slide for performing manual cell counts with the aid of a microscope. Hemacytometers are used when…… — automated cell counters and hematology analyzers are unavailable — blood cell counts are extremely low — to get a cell count for body fluids (spinal fluid, joint fluid, semen counts, and other bodily fluids)

3. Hemacytometers The hemacytometer is a counting chamber that contains two microscopically ruled areas marked off by lines. The most commonly used hemacytometer is the Neubauer based design. Glass hemacytometers are made of heavy glass with two counting areas. The chamber areas are covered with a coverglass and placed under the microscope for visual examination. The chamber depth in the Neubauer-type hemacytometer is 0.1 mm. A glass hemacytometer is reusable if disinfected between each use. Disposable hemacytometers can also be purchased. Instead of reusing these the coverglass is plastic and can only be used once.

4. Hemacytometers The hemacytometer contains two identical ruled areas that each have etched lines with squares of specific dimensions. In the Neubauer-type hemacytometer, the total lined area on each side is made of a large square (3 X 3 mm). This large square is divided into nine equal squares each 1 mm 2. The total area of all the squares is 9 mm 2. When the cover glass is put in place and fluid is added, the fluid volume in the area can be calculated.

5. Total WBCs count Value: 1-diagnosis of infection 2-help in diagnosis of leukemia 3-flow up treatment which cause leucopenia.

6. Principle 1-whole blood dilluted (1-20) times in 20% acitic acid 2-count wbcs in large corner squire 3-Calculate according to general formula to get TWBCs /cu mm # of cells X dilution = # cells / microliter 4 X 0.1

7. Equipments 1-Hemacytometers 2-Coverslip 3-Microscope 4-White blood cells count diluting fluid 20% acitic acid 5-Small test tub 6- 0.02 ml pipette 2ml pipette pasture pipette Specimen: – EDTA- anticoagulated blood or capillary blood is preferred.

8. haemocytometer chamber Thoma white pipette Rubber sucking tube

9. Steps 1-in clean dry test tube place 0.38 ml of 20% acitic acid 2- add 0.02 blood sample 3-remix well, wait for (2-3) min (lysis for RBCs) 4-Prepare the chamber 5-Remix the solution and place one drop from mixture in one 6-counting area in chamber (between glass &chamber) Avoid over filling Avoid air bubble 7-Place the chamber in flat position for 1-2 min (cell settelment) 8-Count WBCs in 4 large corner squire (count in L shape) Calculate to get the final result cell/cu mm

10. Performing a Manual WBC Count First determine if the liquid needs to be diluted. If it does follow the instructions for each diluting kit. Load the hemacytometer. Leave the hemacytometer for a few minutes to allow the cells to settle. Use the microscope (10 X) to locate the WBC counting area. You may have to lower the light on the microscope to visualize the WBCS. They will be refractile and round with a definite outline.

11. Performing a Manual WBC Count

12. Calculation: T.WBCs = No. of cells counted X dilution factor Volume counted Sum x 50 12 Sum X dilution factor (DF) Depth X Area of count Sum X dilution factor (DF) Depth X Area of count

13. Result: Reference range of T.WBCs: 4 - 11 X 10 3 /µL 4 - 11 X 10 9 / L 13

14. RBC Count & PLT Count The large center square is used for RBC and PLT counts. The center square is divided into 25 smaller squares, which are each subdivided into 16 squares. Only 5 of the 25 squares are used to count red blood cells. These 5 are usually the 4 outer squares and the inner most center square. The entire large center square is used to count platelets.

15. RBC Count & PLT Count First determine diluted fluids. If it does follow the instructions for each diluting kit. Load the hemacytometer. Leave the hemacytometer for a few minutes to allow the cells to settle. Use the microscope (10 X) to locate the RBC counting areas. The higher power (40 X) objective needs to be rotated into place to visualize the RBCs.

16. RBC Count & PLT Count RBCs are much smaller than the before mentioned WBCs. The cells touching either the top or left boundaries of the squares are included but the lower or right boundaries are not counted.