Chemistry of Blood Practical PDF

Chemistry of blood practical Complete Blood Count (CBC) Complete Blood Count (CBC) It provides information about the white blood cell (WBC), red blood cell (RBC), and platelet population present. This information includes the number, type, size, shape and some of the physical characteristics o...

Chemistry of blood practical Complete Blood Count (CBC) Complete Blood Count (CBC) It provides information about the white blood cell (WBC), red blood cell (RBC), and platelet population present. This information includes the number, type, size, shape and some of the physical characteristics of the cells It is actually a panel of tests that examines different parts of the blood and includes the following: 1- Red blood cell (RBC) count is a count of the actual number of red blood cells per volume of blood. 2 - Hemoglobin measures the amount of oxygen carrying protein in the blood. 3 - Hematocrit (HCT) measures the amount of space red blood cells take up in the blood. It is reported as a percentage. 5 - Mean corpuscular volume (MCV) is a measurement of the average size of your RBCs. 7- Mean corpuscular hemoglobin (MCH) is a calculation of the amount of oxygen carrying hemoglobin inside your RBCs. 8- Mean corpuscular hemoglobin concentration (MCHC) is a calculation of the concentration of hemoglobin inside the RBCs. 9- Red cell distribution width (RDW) is a calculation of the variation in the size of your RBCs. Reference range parameter unit range WBCs x 103/mL 4.5 - 11.0 RBCs x 106/mL m = 4.60 - 6.20 f = 4.20 - 5.40 HCT % m = 40.0 - 54.0 f = 38.0 - 47.0 HB g/dL m = 13.5 - 18.0 f = 12.0 - 16.0 MCV m3 80 - 96 MCH mmg 26 - 34 MCHC % 31 - 37 PLT x 103/mL 150 - 450 MPV FL 6.5 - 12.0 RDW % 11.6 - 14.6 CBC can applied by two way 1. Automated blood count 2. Manual blood count Automated blood count CBC is performed by an automated analyzer that counts the numbers and types of different cells within the blood. It aspirates a very small amount of the sample through the narrow tubing, within this tubing, there are sensors that count the number of cells going through it, and can identify the type of cell; this is called flow- cytometry. Manual Blood Count This measurement is made with a microscope and a specially ruled chamber (Hemocytometer) using diluted blood.  Hemoglobin Colorimetric determination of Hemoglobin Procedure: This method below is the manual method for spectrophotometer. Wavelength: 540 nm (546) Temperature: 37°C, 30°C, 25°C Cuvette: 1cm light path Read against reagent blank. Mix and read the optical density (O.D) within 1 hour. Reference values: Adult females: 12-15 g/dL 120-150g/L Adult males: 13.9-16.3 g/dL 139-163 g/L New-born: 18-21.5 g/dL 180-215 g/L Infants (6 months): 12.8-16 g/dL 128-160 g/L Infants (1 year): 10-14 g/dL 100-140 g/L Children (8 years): 11.5-14.8 g/dL 115-148 g/L Children (14 years): 11.6-15 g/ dL 116-150 g/L  RBCs (Red blood cells) count The RBC count totals the number of red blood cells that are present in a person's sample of blood. RBCs typically make up about 40% of the blood volume. The typical life span of an RBC is 120 days; thus the bone marrow must continually produce new RBCs to replace those that age and degrade or are lost through bleeding. Changes in the RBC count usually mirror changes in the hematocrit and hemoglobin level. When the values of the RBC count, hematocrit, and hemoglobin decrease below the established reference interval, the person is said to be anemic. When the RBC and hemoglobin values increase above the normal range, the person is said to be polycythemic. Some common signs and symptoms associated with anemia that generally lead to a doctor ordering a CBC are: 1-Weakness or fatigue 2-Lack of energy 3-Paleness Some signs and symptoms that may appear with a high RBC count include: 1-Disturbed vision 2-Headache 3-Enlarged spleen Some causes of a low RBC count include: 1-Trauma 2-RBC destruction, for example hemolytic anemia caused by autoimmunity 3-Acute or chronic bleeding from the digestive tract 4-Nutritional deficiency (e.g., iron deficiency, vitamin B12 or folate deficiency) 5- Bone marrow damage (e.g., toxin, radiation or chemotherapy, infection, drugs) 6- Kidney failure—severe and chronic kidney diseases lead to decreased production of erythropoietin. Some causes of an increased RBC count (polycythemia) include: 1-Polycythemia 2-Smoking 3-Pulmonary disease 4-Congenital heart disease. 5-Dehydration. Normally high (RBC count)  People who live at high altitudes  Smokers  synthesis increases Specimen Whole blood, using EDTA as the anticoagulant. Principles to facilitate counting, whole blood is diluted with Gower's solution which hemolyze white blood cell and prevent red cell lysis. Procedure: 10 µL of blood + 2000 µL of reagent, mix and stand for 5 mins. The slide of counting is called ―hemocytometer‖  Calculations RBC blood cell count ( 5 squares) Find the sum of RBCs in 5 large squares, and divide it with 80 (5 x 16) small squares to find the average in one square, multiply it by 200 to allow for the dilution and then multiply by 4000 to obtain the number per cubic milliliter. ex: The sum of RBCs in 5 large squares = (84) + (71) + (63) + (93) + (83) = 394 cells The average of RBCs in one square= 394 / 80 = 4.9 cell RBC count= 4.9 x 200 x 4000 = 4 million/mm Normal range= 4.2-5 million/mm3 White blood cell (WBC) count: It is a count of the actual number of white blood cells per volume of blood. White blood cell differential looks at the types of white blood cells present. There are five different types of white blood cells, each with its own function in protecting us from infection. The differential classifies a person’s white blood cells into each type: neurophils, lymphocytes, monocytes, eosinophils, and basophils. White blood cells, also called leukocytes, are cells that exist in the blood. They are an important part of the body's defense system. They help protect against infections and also have a role in inflammation, allergic responses, and protecting against cancer When there is an infection or an inflammatory process somewhere in the body, the bone marrow produces more WBCs, releasing them into the blood, and through a complex process, they move to the site of infection or inflammation.  Total leukocytes count shows the number of WBC in a sample of blood.  A normal WBC count is between 4,500-11,000 cells per cubic millimeter (mm³).  The number of WBC is sometimes used to identify an infection or to monitor the body’s response totreatment. Low WBC count  Leukopenia A Condition in which the number of leukocytes is abnormally low and which is most commonly due tosever infections (such  as HIV) and radiation poisoning. High WBC count  Leukocytosis A condition characterized by an elevated the number of WBC occur as a result of an infection, or cancer(Leukemia). It can occur normally after eating fat-rich meals. Calculations WBC blood cell count ( 4 squares) Find the sum of WBCs in 4 large squares, and divide it with 64 (4 X 16) small squares to find the average in one square, multiply it by 20 to allow for the dilution and then multiply by 160 to obtain the number per cubic milliliter. ex: The sum of WBCs in 4 large squares = (16) + (21) + (17) + (15) = 69 cells. The average of WBCs in one square= 69/ 64 = 1.07 cells. WBC count= 1 x 20 x 160 = 3200 Cells/mm3. Normal range= 4500-11000 cells /mm3 Differential Count It determines the number of each type of WBC present in the blood.  Leukocyte Classes 1- Granulocytes: - Neutrophil (also called a polymorphonuclear cell) - diameter = 10-14 m - Most commonly seen white blood cells in the circulating blood. - They have small cytoplasmic granules and a complex, multilobed nucleus. - The granules take a neutral (purple or pink) color with various stains such as Wright’s stain. - lobed nuclei (3-6 lobes) ↑ in bacterial infections - Granules contain lysosomal enzymes - Neutrophils are quick acting phagocytes (1st responders) - They are quick but weak! Eosinophil - bilobed nucleus - Less common in the bloodstream than neutrophils. - They are characterized by a dumbbell-shaped Nucleus (bi-lobed) and large, prominent, red (eosinophilic) granules. - increased ↑ in parasitic infections - Granules contain digestive enzymes - Are antiparasitic & Anti-allergy Basophil - diameter = 8-10 m - large granules - The rarest of all white blood cells found in the blood. - It is a large cell filled with prominent blue (basophilic) granules. - These large granules contain Heparin, an anticoagulant, and Histamine, which increases the permeability of capillary walls. The nucleus is somewhat hidden behind these large granules. - U or S shaped nucleus - Granules contain: - histamine which induces inflammation and vasodilation - heparin an anticoagulant 2- Agranuloctes : lack visible cytoplasmic granules Lymphocyte: - diameter 5-8 m (small), 10-12 m (medium) or 14-17 m (large) - Small, spherical cells with large, round nucleus in each of them. - The cytoplasm of these cells does not contain any granules. - The Nucleus occupies most of the volume of the cell, leaving only a thin crescent of Cytoplasm around it. - large, deep blue - T lymphocyte (T cells) fight antigens directly - B lymphocytes (B cells) divide to produce plasma cells that secrete antibodies Monocytes - diameter = 18 m - nucleus is U shaped or kidney shaped - Easily the largest of all white blood cells in size.  - The cytoplasm of these cells does not contain any granule - They possess a large, horseshoe-shaped (kidney-shaped) Nucleus. - abundant pale blue cytoplasm - Are phagocytes – slow but strong - Develop into when they migrate into connective tissue The normal values of each different type of white blood cells NEUTROPHILS 50 – 70% EOSINOPHILS 1 – 3% BASOPHILS 0.4 – 1% MONOCYTES 4 – 6% LYMPHOCYTE 25 – 35% Conditions the percentages of the various types of white blood cells are increased: NEUTROPHILS è will increase in acute bacterial or fungal infections. EOSINOPHILS è will increases in parasitic infections and allergies. BASOPHILS è will increase in allergies and malignancies. MONOCYTES è will increase in chronic infections. LYMPHOCYTE è will increase in acute viral infections and malignancies. Stains are used in the preparation of blood films:  Leishman’s stain  Wright’s stain Platelets count The platelet count is the number of platelets in a given volume of blood. Both increases and decreases can point to abnormal conditions of excess bleeding or clotting. Platelets, also called thrombocytes, are formed from the bone marrow and released into the blood to circulate, are essential for normal blood clotting. The platelet count is a test that determines the number of platelets in a person's sample of blood. If there are insufficient platelets, or if platelets are not functioning normally, a stable clot may not form and a person may be at an increased risk of excessive bleeding. Calculation P x 1000  Preparation of Blood Smear The blood film or peripheral blood smear is a thin layer of blood smeared on microscopic slides then stained in such a way to allow the various blood cells to be examined microscopically. Blood films are usually examined to investigate haematological problems (disorders of the blood) and occasionally, to look for parasites within the blood such as malaria and filaria. How to prepare a blood smear : Capillary blood obtained by fingerstick :  Wear gloves.  Microscopic slide is cleaned using 70 to 90% alcohol and is left till dry.  Do not touch the surface of the slide where the blood smear will be made.  Select the finger to puncture, usually the middle or ring finger. In infants, puncture the heel.  Clean the area to be punctured with 70% alcohol; allow to dry.  Puncture the ball of the finger and put a drop of blood in the edge of the slide.  Put the spreader slide behind the blood drop at a 30° - 40°angle and Draw in carefully back to the drop of blood.  Increasing the angle results in a thicker smear, whereas a smaller angle gives a thin smear.  draw the spreader slide rapidly and smoothly over the entire length of the smear slide pulling a thin even film behind it. The smear should cover 1/2-2/3 of the slide and finish with a "feathered" edge.  Prepare a second slide.  Allow slides to dry on a flat surface.  Label the slides with patient's name (or other identifier), date and time of collection  Place slides in a plastic slide container. Stain the slide with Lishman stain for one minute.  Leishman Stain is a neutral stain for blood smears which was devised by the British surgeon W. B. Leishman (1865–1926). It consists of a mixture of eosin (an acidic stain), and Methylene blue (a basic stain) in Methyl alcohol and is usually diluted and buffered during the staining procedure  Wash gently with water and allow the slide to dry.  Examine under microscope on 10X - 40X at the thin part (feather edge).  When we use a peripheral blood, we must use EDTA anticoagulant tube for blood collection. Smear must be made immediately. All specimen should be free of clots. Specimen should be checked with two applicator sticks to be sure no clots are present. Common Causes of a Poor Blood Smear As soon as the drop of blood is placed on the glass slide, there should be no delay in the making of the smear. Any delay, whatsoever, results in abnormal distribution of the white cells with many of the white cells accumulating at the thin edge of the smear. Rouleaux of the red cells and clumping of the platelets may also occur. Drop of blood too large or too small. Spreader slide pushed across the horizontal slide in a jerky manner. Failure to keep the entire edge of the spreader slide against the horizontal side while making the smear. Failure in using appropriate angle for the spreader slide. As a rule of thumb, if your patient has a low hemoglobin, increase the angle of your spreader slide; if your patient has a high hemoglobin, then decrease the angle.