Practical Blood Physiology Lab 2: Manual Red Blood Cells Count (2023-2024) PDF

Jabir Ibn Hayyan university for Medical and pharmaceutical sciences Faculty of Medicine Practical blood physiology 2nd year 2023-2024 Lab 2 Manual red blood cells count...

Jabir Ibn Hayyan university for Medical and pharmaceutical sciences Faculty of Medicine Practical blood physiology 2nd year 2023-2024 Lab 2 Manual red blood cells count Lecturer: Karrar Abduljabar Alqershi M.Lab.Med  Practical physiology 2nd year Red blood cells count Learning objectives: After this practical session, the student should be able to - Know the definition of blood, what is made from and the main functions? - Understand the purpose, idea and the principle of the manual RBCs counting. - Count and interpret the result. - Understand the calculation procedure - Know the normal reference ranges for adult male and females. - Know when RBCs increase or decrease and why? Introduction: 1- What is the blood? Blood is a combination of plasma and cells that circulates through the entire body. It is a specialized bodily fluid that supplies essential substances around the body, such as sugars, oxygen, and hormones. It also removes waste from the cells in the body. 2- What is blood made from? Blood consists of plasma, red and white blood cells, and platelets. Plasma: This constitutes approximately 55% of blood fluid in humans. Plasma is 92 percent water, and the contents of the remaining 8 percent include:  carbon dioxide  glucose  hormones  proteins  mineral salts  fats  vitamins The remaining 45 % of the blood mainly consists of red and white blood cells and platelets. Jabir Ibn Hayyan university for Medical and pharmaceutical sciences / Faculty of Medicine lecturer: Karrar Alqershi  Practical physiology 2nd year Red blood cells count 3- Functions of blood Blood has a number of functions that are central to survival, including:  Supplying oxygen to cells and tissues  Providing essential nutrients to cells, such as amino acids, fatty acids, and glucose  Removing waste materials, such as carbon dioxide, urea, and lactic acid  Protecting the body from infection and foreign bodies through the white blood cells  Transporting hormones from one part of the body to another, transmitting messages, and completing important processes  Regulating acidity (pH) levels and body temperature -  The platelets in blood enable the clotting, or coagulation, of blood. When bleeding occurs, the platelets group together to create a clot. The clot becomes a scab and stops the bleeding, as well as helping to protect the wound from infection. Jabir Ibn Hayyan university for Medical and pharmaceutical sciences / Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year Red blood cells count Manual Red blood cells count The purpose of the test: The student should be able to perform and count the number of the red blood cells using Neubauer’s counting chamber (Haemocytometer). The idea of the test: - Red blood cells contain a substance called haemoglobin which transports oxygen around the body. The amount of oxygen that's delivered to your body's tissues will depend on the number of red blood cells you have and how well they work. - Approximates the number of the circulating red blood cells and thus gives an estimate of the blood’s oxygen carrying capacity. - Helpful in diagnosis of certain pathological cases such as anaemia. The principle of the test: RBC diluting fluid is isotonic with blood, hence hemolysis does not take place. Normal Saline also can be used. But it causes slight creation of red blood cells and allows rouleaux formation. The blood specimen is diluted 1:200 with the RBC diluting fluid and cells are counted under high power (40 x objective) by using a counting chamber. The number of cells in undiluted blood are calculated and reported as the number of Red cells per cu mm of whole blood. Materials and method: Materials: 1- Whole blood using EDTA or a direct capillary blood. 2- RBCs diluting fluid or normal saline (NaCl 0.9) 3- Red blood cells pipette. 4- Neubauer’s counting chamber. 5- Lancets, cotton, alcohol70%, 6- Microscope. Note: The mostly used RBCs diluting fluid is called (Hayem’s) solution and it is composed of the following ingredients: Mercuric chloride 0.25 gm Sodium sulphate 2.50 gm Sodium chloride 0.50 gm Distilled water 100.0ml Final pH ( at 25°C) 5.9±0.1 Jabir Ibn Hayyan university for Medical and pharmaceutical sciences / Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year Red blood cells count Method: 1- Wipe finger with cotton soaked with alcohol, with a sterile lancet do small prick on the finger tip. Use pipette. Aspirate blood to 0.5. 2- Wipe the outside of the pipette,clean with a piece of dry gauge without touching the opening of the capillary. 3- Immediately draw diluting fluid to the 101 mark past the bulb. This gives a dilution of 1:200. Note: you can make that dilution by adding 10µL EDTA blood to 1.99mL (1990 µL) diluting fluid to get 1:200 D.F. 4- Rotate the pipette for 3 minutes immediately before filling the haemocytometer. To allow the blood to be mixed thoroughly. 5- Expel, first 4 - 6 drops from the pipette and fill one side of counting chamber. 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- Place the chamber on the stage of the microscope and allow 2 minutes for the cell to settle 9- Scan the counting area with 10x objective lens. 10- Use 40x objective, include all cells lying on the upper and left lines of any sequare, ignore the cells on the lower and right- hand lines. 11- Count the cells in five groups of 16 small sequares i.e 80 small sequares. Counting the cells The chamber slide: Thick glass slide with inscribed platforms of well-known area and precisely controlled depth under coverslip. - 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. Jabir Ibn Hayyan university for Medical and pharmaceutical sciences / Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year Red 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 0.1mm. Jabir Ibn Hayyan university for Medical and pharmaceutical sciences / Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year Red blood cells count Counting: - Count the cells in the four squares at the corners and the middle one (pointed by small 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” - Jabir Ibn Hayyan university for Medical and pharmaceutical sciences / Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year Red blood cells count Calculations: Reference ranges:* *(Hoffbrand’s essential haematology, 7th edition, 2016) For adult males: 4.5-6.5 1012/L For adult females: 3.9-5.6 1012/L Clinical significance: DECREASE ↓: (Erythrocytopenia) is usually seen in anaemias which result from either: - Decreased red cell production: e.g Iron deficiency anaemia (IDA), VB12 deficiency anaemia (Megaloplastic anaemia, erythropoietin deficiency, Bone marrow failure, Malnutrition, Leukemias. - Increased red cells loss or destruction: e.g: acute or chronic blood loss (Bleeding), Haemolysis e.g: sickle cell anaemia (hereditary), mismatched transfusion..etc INCREASE ↑ (Erythrocytosis): - Physiological: Low O2 level, dehydration, cigarette smoking - Pathological: e.g Polycythemia Vera (hereditary), congenital heart disease, lung (pulmonary) diseases, renal cell carcinoma. Jabir Ibn Hayyan university for Medical and pharmaceutical sciences / Faculty of Medicine lecturer: Karrar Alqershi Practical physiology 2nd year Red blood cells count References: 1- https://www.medicalnewstoday.com/articles/196001.php 2- http://www.celeromics.com/en/resources/Technical%20Notes/cells-chamber-concentration.php 3- http://www.nhs.uk/conditions/Red-blood-count/Pages/Introduction.aspx 4- Red blood cells count. DR.ANINDITA SAHA pdf. 5- HiMedia technical data. RBC diluting fluid,2015. 6- Hematology practical manual, RMIT university, 2014. 7- https://www.healthline.com/health/rbc-count. Accessed 21st of October,2019. Jabir Ibn Hayyan university for Medical and pharmaceutical sciences / Faculty of Medicine lecturer: Karrar Alqershi