White Blood Cells Count Using Hemocytometer Salman bin Abdulaziz University College of Pharmacy Clinical Pharmacy Department Physiology-I (Practical Part) White Blood Cells Count Using Hemocytometer Abdulaziz.bin.mohammed2@gmail.com 9/17/2018

White Blood Cells - WBCs white blood cell, also called leukocyte ,  a cellular component of the blood that lacks hemoglobin, has a nucleus, is capable of defends the body against infection and disease by ingesting foreign materials and cellular debris, by destroying infectious agents and cancer cells, or by producing antibodies. The normal range of leukocyte is 4,800 - 10,000/cubic millimeter. Red blood cells are confined to the bloodstream and functions in the blood. White blood cells, by contrast, are able to slip into and out of the blood vessels and go to tissues (by process called diapedesis). WBCs Disorders: 1. Leukocytosis: is a high white blood cell count Microbes, anesthesia or surgery 2. Leukopenia is low white blood cell count radiation, shock or chemotherapy. 9/17/2018

9/17/2018

WBCs Disorders: 1. Leukocytosis is a high white blood cell count Microbes, anesthesia or surgery. 2. Leukopenia is low white blood cell count radiation, shock or chemotherapy. 9/17/2018

White Blood Cells Types WBC’s are divided into two categories: Granulocytes, and Agranulocytes. Granulocytes contain granules within the cytoplasm and include: Neutrophils, Eosinophils, and Basophils. Agranulocytes contain very little, if any, granules and include: Lymphocytes and Monocytes. 9/17/2018

White Blood Cells Types Neutrophils (Granulocyte) Neutrophils have a multilobed nucleus (3-5lobes) and very fine granules Function: Release lysozymes which destroy/digest bacteria Neutrophils are produced by bone marrow, they fight disease by migrating to the point of infection, absorbing bacteria, and destroying them (phagocytosis) Neutrophil (purple) migrating through tissue to engulf bacteria through phagocytosis. 9/17/2018

Eosinophils (Granulocyte) Nucleus with 2 or 3 lobes connected by a thin strand Function: Attack parasitic worms and play an important role during allergies and asthma. albendazole 9/17/2018

Basophils (Granulocyte) The rarest of the WBCs, have S shaped nucleus contain large histamine-containing granules that stain dark blue. Function: Basophils are involved in inflammatory and allergy by releasing heparin, histamine & serotonin. the rarest of the WBCs, have S shaped nucleus contain large histamine-containing granules that stain dark blue. 9/17/2018

Lymphocyte (Agranulocyte) Dark, oval to round nucleus Cytoplasm sky blue in color Function: B cells: Destroy bacteria and their toxins Turn into plasma cells that produces antibodies T cells Attack viruses, fungi, transplanted organs, cancer cells & some bacteria. 9/17/2018

Monocyte (Agranulocyte) Monocytes are the largest of the WBCs. Except for their more abundant cytoplasm and indented (kidney like) nucleus, they resemble large lymphocytes. When they migrate into the tissues, they change into macrophages. Macrophages are very important in fighting chronic infections, such as tuberculosis. Function: Macrophages absorb disease-producing materials, such as bacteria through phagocytosis. 9/17/2018

9/17/2018

White Blood Cell (WBC) Count Expressed as: Number of white blood cells per a unit volume (x 103 / cu.mm.) (x 103 / mm3.) (x 103 / µL.) (x 109 / L.)

WBC Count (Cont.) Reagent WBC diluting fluids 1. Turk’s solution Specific Equipment White pipette (Thoma type) 2. Rubber pipe 3. Plastic syringe 4. Pipette vibrator 5. Hemocytometer & cover slip (Counting chamber) 6. Microscope 7. Counter (hand tally) 8. Report form Reagent WBC diluting fluids 1. Turk’s solution Glacial acetic acid Gential violet Distilled water 2. Acetic acid (2%) 3. Hydrochloric acid (1% )

WBC Count (Cont.) Procedure 1. Mix the blood sample thoroughly. 2. Fill blood into white pipette at 0.5 mark. 3. Fill reagent add up into the pipette to 11 mark. 4. Shake the pipette on the vibrator for 1 min. 5. Discarded the first 3-4 drops. 6. Fill in the hemacytometer nicely. 7. Allow WBC to set down for 2-3 min. 8. Count 4 white squares under microscope (x100 lens). 9. Calculate the WBC concentration.

WBC Count (Cont.) High 0.1 mm. 1 mm. W W 1 mm. R R 1 mm. R R R W W

Hemacytometer counting chamber (WBCs) Hemacytometer counting chamber (WBCs). Areas marked A, B, C, and D are used to count white blood cells.

WBC Count (Cont.) W 1 mm. High 0.1 mm. 1 mm.

WBC Count (Contd) Top Left

WBC Count Calculation 1. Volume of 1 white square = 1 x 1 x 0.1 cu.mm. 2. Volume of 4 white square = 0.1 x 4 cu.mm. = 0.4 cu.mm. 3. In 0.4 cu.mm. the WBC count = N (counted No.) 4. In 1 cu.mm. the WBC count = N x 1 / 0.4 = N x 2.5 5. The dilution for WBC = 10/0.5 = 20 6. The final WBC count = N x 2.5 x 20 = 50 x N (/cu.mm.)

WBC Count (Cont.) Calculation Normal range = 4,800 - 10,000/ cu.mm. White cell count = number of cells counted (N) x volume factor (=2.5) x dilution factor (=20) = N x 50 Normal range = 4,800 - 10,000/ cu.mm. 5 - 10 x 103 / cu.mm.

Do You Have Any Questions? Thank You For Your Attention Do You Have Any Questions? 9/17/2018