Differential Leucocyte Count PDF

DIFFERENTIAL LEUCOCYTE COUNT Dr Iram Jaan Lecturer,Physiology.  Learning objectives On completion of the practical class, the student will be able to: Identify the different WBCs and know the percentage of different white blood cells present in the blood....

DIFFERENTIAL LEUCOCYTE COUNT Dr Iram Jaan Lecturer,Physiology.  Learning objectives On completion of the practical class, the student will be able to: Identify the different WBCs and know the percentage of different white blood cells present in the blood. Understand the clinical significance of doing a differential leucocyte count. DIFFERENTIAL LEUCOCYTE COUNT (DLC) REQUIREMENTS Microscope Glass slides (One of the glass slide is used as a spreader which has smooth edges to spread the blood onto the slides) Leishman stain or Field’s stain Distilled water Cedar wood oil Lancet Alcohol swab DIFFERENTIAL LEUCOCYTE COUNT (DLC) COMPOSITION OF FIELD’S STAIN Alcohol (Methanol) – Used to fix the blood that is spread on the slide. Eosin (Acidic dye) – stains the basic particles (basic granule) of the WBC Mehtylene blue (Basic dye) – stains the acidic particles (nucleus and acidic granules of the WBC  STEPS FOR THE DLC 1. Preparation of blood film. 2. Staining of the blood film with Field stain. 3. Examination of film. 4. Identification of white blood cells. 5. Counting.  1. PREPARATION OF BLOOD FILM Using aseptic precautions a skin puncture is made on the left middle finger and a small drop of blood is obtained. Then a clean grease free glass slide is taken and one end of the slide surface the drop of blood is touched.  1. PREPARATION OF BLOOD FILM i. Then a spreader is taken and grasped with the fingers and thumb of the other hand and held at an angle of 45 degrees to the first slide (which contains the drop of blood) ii. The edge of the spreader is placed on the first slide just in front of the drop of the blood and allowed to spread. iii. The spreader is moved on the first slide with a single motion applying gentle pressure. iv. The smear should neither be too thick or too thin, without any ridges or holes and should cover 3/4th of the slide. v. The blood film is dried by waving the slide in air. 1. PREPARATION OF BLOOD FILM  2. STAINING THE BLOOD FILM The dried smear is dipped into a bottle of Leishman stain (such that the whole film is covered by stain and left for 2 - 3 minutes)  2. STAINING THE BLOOD FILM After 2- 3 minutes the stain is diluted with distilled water for 6 - 8 minutes. After 6 - 8 minutes the smear is dried in air.  3. EXAMINATION OF BLOOD FILM A good smear is light purple in color and rectangular with a dense head end and a tapering tail end. A drop of cedar wood oil is then placed on the slide and the objective is turned to oil immersion and observe the different types of white blood cells. [Cedar wood oil has the same refractive index of glass (1.5) thus it reduces the diffraction of light and increase the clarity] 3. EXAMINATION OF BLOOD FILM Place the slide on the stage of the lens. Add a drop of cedar wood oil. Switch the lens objective to 100x. Carefully raise the stage until the oil touches the lens objective. 4. IDENTIFICATION OF WHITE BLOOD CELLS Identification of the cells is done by noting: a) Size of the cell b) Shape of the nucleus and the number of lobes c) Staining of the nucleus, its intensity and uniformity d) Presence of granules, their size, number and staining reaction. IDENTIFICATION OF WHITE BLOOD CELLS White blood cells are classified as granulocytes and agranulocytes. Granulocytes: are white blood cells that have granules inside their cytoplasm (neutrophils, eosinoplils and basophils). Agranulocytes: are white blood cells that do not have granules inside their cytoplasm (monocytes and lymphocytes). IDENTIFICATION OF WHITE BLOOD CELLS IDENTIFICATION OF WHITE BLOOD CELLS Neutrophils Diameter: 10-14 microns Nucleus: 2-5 lobes Granules: Fine and violet in color, they take up both, basic and acidic stains Eosinophils Diameter: 10 - 14 microns. Nucleus is bilobed. Granules in cytoplasm are coarse and pink in color as they take the acid stain - Eosin.  IDENTIFICATION OF WHITE BLOOD CELLS Basophils Diameter: 8-10 microns. The nucleus is bilobed. The granules are coarse and blue in color as they stain with methylene blue the basic stain  IDENTIFICATION OF WHITE BLOOD CELLS Lymphocytes Diameter: large lymphocyte is 10 - 14 microns smaller ones is 7 - 9 microns. Nucleus: round or slightly bean shaped, it is comparatively larger,and the cytoplasm is less. The cytoplasm is clear without granules. Monocytes Largest of all the leucocytes with a diameter of 16 - 22 microns. Nucleus: kidney or horse shoe shaped and it is mostly pushed to one side The cytoplasm is clear without granules.  5. COUNTING OF WHITE BLOOD CELLS 100 small squares are drawn on a paper. After identifying every cell it is entered by writing the letter ❖ N for Neutrophil (50 -70%) ❖ E for Eosinophil (1 - 4%) ❖ B for Basophil (0-1%) ❖ L for Lymphocyte (20 -40%) ❖ M for Monocyte (2 -8%) 5. COUNTING OF WHITE BLOOD CELLS  FUNCTIONS OF WHITE BLOOD CELLS Neutrophil – Neutrophils defend our body against bacteria and fungi by releasing hydrolytic enzymes and antimicrobial proteins and phagocytize them. Eosinophil – Eosinophils specifically attack and kill parasitic worms and allergens (antiparasitic and antiallergic function) Basophils – Basophils release histamine and other mediators of inflammation. It also contains heparin Lymphocytes – They have a crucial role in immunity by direct cell attack (T lymphocytes) or via antibodies (B lymphocytes) Monocytes- Monocytes leave the circulation and enter the tissues and become macrophages. They are actively phagocytic and defend the body against viruses and chronic infections Learning resources Textbook: CL Ghai The Textbook of Practical Physiology 8th edition. Jaypee; 2013. ISBN-978-93-5025-932-0 , Chapter 1-12, Page no. 69 to 84