Practical Physiology Lab (3) - Manual White Blood Cells Count 2023-2024 PDF

Jaber bin Hayyan for Medical and Pharmaceutical Sciences Practical physiology 2nd year 2023-2024 Lab (3) Manual White blood cells count Lecturer: Karrar Abd...

Jaber bin Hayyan for Medical and Pharmaceutical Sciences Practical physiology 2nd year 2023-2024 Lab (3) Manual White blood cells count Lecturer: Karrar Abduljabar Alqershi M.Lab.Med Practical physiology 2nd year White blood cells count Learning objectives: After this practical session, the student should be able to - Understand the purpose, idea and the principle of the manual WBCs counting. - Count and interpret the result. - Understand the calculation procedure - Know the sources of errors. - Know the normal reference ranges for adult males and females. - Know when WBCs increase or decrease and why? Jaber bin Hayyan for Medical and Pharmaceutical Sciences/ Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year White blood cells count Manual White blood cells count The purpose of the test: The student should be able to perform and count the number of the white blood cells using Neubauer’s counting chamber (Haemocytometer). The idea of the test: - Blood is diluted in buffered ammonium oxalate or glacial acetic acid which haemolyses mature RBC and preserves the platelets, leukocytes and reticulocytes in their native state. - When the RBCs have been completely lysed, the solution will be clear (not turbid) and counting can proceed. The principle of the test: The blood specimen is diluted 1:20 with the WBCs diluting fluid (1% acetic acid) and cells are counted under low power (10x objective) or high power (40x objective) by using a counting chamber. The number of cells in undiluted blood are calculated and reported as the number of white cells per cubic mm of whole blood. Materials and method: Reagents and equipment: 1- White cell diluting fluid (1% Acetic acid). 2- Pipettes and tips. 3- 20µL Sahli pipette. 4- Kahn tubes. 5- EDTA anticoagulated or capillary blood. 6- Microscope. 7- Improved Neubauer haemocytometer. 8- Ethanol 70%. 9- Lancets. Jaber bin Hayyan for Medical and Pharmaceutical Sciences/ Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year White blood cells count Method: 1- Transfer 380µL of 1% acetic acid into a small Kahn tube. 2- Wipe finger with cotton soaked with alcohol, with a sterile lancet do small prick on the finger tip. Use pipette. Aspirate 20µL of blood by 20µL Sahli pipette. 3- Wipe the outside of the pipette, clean with a piece of dry gauge without touching the opening of the capillary. 4- Pour the 20µL capillary blood into 380µL acetic acid. Withdraw and pour several times to ensure that all the capillary blood has been mixed thoroughly. That gives a dilution of 1:20. Leave it for 2-3 minutes for complete lysis. 5- Clean the haemocytometer with ethanol and ensure chamber is dry and dust free. 6- Put the glass cover on the Neubauer chamber central area. Use a flat surface to place the chamber, like a table or a workbench. 7- Touch the tip of the pipette on the surface of the counting chamber 45 degree. (at the edge of the cover slip). 8- Remove a small sample of the 1/20 dilution using a capillary tube or transfer pipette and carefully fill both sides of the haemocytometer. Do not overfill the chamber. 9- Allow approximately 1 minute for the cells to settle in the chamber. Place the chamber on the microscope stage. Using the low power objective (x10) (or 40x) with the light correctly adjusted; count the number of leukocytes in the four large corner squares. 10- Count the cells that overlap two of the outer sides of the large square but disregard the cells that overlap the other two (it does not matter which sides are counted but one should try to be consistent and use the same two sides each time a count is performed). For a reasonable distribution there should be no more than 10% difference in each square. 11- Count the number of cells on the other side of the chamber -if there is >10% difference between the counts, the test should be repeated by reloading the chamber and performing a new count. Counting the cells The chamber slide: - Neubauer chamber’s counting grid is 3 mm x 3 mm in size. the grid has 9 squares each measuring 1mmx1mm - The central large square is subdivided into 25 squares - Each of this 25 squares is further subdivided into 16 squares. Jaber bin Hayyan for Medical and Pharmaceutical Sciences/ Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year White blood cells count - Each group of 16 small squares is - separated by closely ruled triple lines. - The 4 corner squares are further - divided into 16 small squares - The depth of the chamber is 0.1 mm. -. Jaber bin Hayyan for Medical and Pharmaceutical Sciences/ Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year White blood cells count Jaber bin Hayyan for Medical and Pharmaceutical Sciences/ Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year White blood cells count Counting: - Count the cells in the four large squares at the corners (pointed by large blue circles). - Cells touching the upper and left limits should be counted, unlike cells touching the lower and right limits which should not be taken into account” https://www.youtube.com/watch?v=NHy-7XWxE5E Calculations: Jaber bin Hayyan for Medical and Pharmaceutical Sciences/ Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year White blood cells count Notes:  When the white cell count is very high (eg: Leukemia), the count should be repeated with a larger dilution (1:100 or even 1:200). And adjust the calculation factor.  When the count is very low, it should be repeated by using a smaller dilution (1:10 or 1:5).  The diluent fluid must not be contaminated with dust, cells or bacteria.  The haemocytometer and coverslip must be kept clean.  Once the chamber has been filled, the count should be performed immediately. Delaying your count or leaving the chamber in a direct beam of light on the microscope stage may lead to evaporation and inaccurate count. Sources of errors: Manual cells count are subject to a number of errors and most of them are technical errors in counting, may be due to any of the following factors: Low counts:  Insufficient blood being drawn into the pipette.  Insufficient mixing.  Insufficiently filling the counting camber.  Undue delay in performing the count after filling the chamber.  Uneven distribution of the cells in the counting chamber.  Inaccurate calibrated pipettes.  Faulty counting technique and/or errors in calculations. High counts:  Too much blood being drawn into the pipette  Insufficient mixing of the blood/ diluent suspension  Overfilling the counting chamber  Uneven distribution of the cells in the counting chamber  The inclusion of the yeasts and particles of duct in the count.  Inaccurately calibrated pipettes  Faulty counting technique and/or errors in calculations. Reference ranges:* For adult males: 4-11 109 /L* *(Hoffbrand’s essential haematology, 7th edition, 2016) For adult females: 4-11 109 /L* Jaber bin Hayyan for Medical and Pharmaceutical Sciences/ Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year White blood cells count Clinical significance: - Increase: Known as Leukocytosis. Usually seen in infection, stress, inflammatory disorder or leukemia. - Decreased: Known as Leukocytopenia. Usually seen in treatment (chemotherapy or radiation) or long term use of broad spectrum antibiotics References: 1- Hematology practical manual, RMIT University, 2014. 2- https://emedicine.medscape.com/article/956278-overview Jaber bin Hayyan for Medical and Pharmaceutical Sciences/ Faculty of Medicine lecturer: Karrar Alqershi