PHYSIOLOGY LAB REVIEWER RBC Counting Erythrocytes most numerous of blood cells - Responsible for providing oxygen to tissues

s and partly for recovering CO2 produced as waste - 4-6 millions/cubic mm - Devoid of nucleus (man, mammals) w/ a shape of a biconcave lens - Nucleated (fishes, birds, reptilians and birds) - Shapes: Normal (discocyte), Berry (crenated), burr (echinocyte), target (codocyte), oat, sickled, helmet, pinched, pointed, indented, poikilocyte, etc. Hemoglobin protein able to bind in a reversible manner to oxygen Materials: 1. Thoma pipette with red bead (0.5 and 101 mark) 2. Hayems solution 3. Hemocytometer Procedure: a. Filling the pipette 1. Get fresh, unclotted blood from the animal subject 2. Using the thoma pipette with attached rubber tubing apply gentle suction on the mouthpiece to draw blood exactly to the 0.5 mark of the pipette 3. Wipe the tip of the pipette then thoroughly steady suction draw the diluting fluid up to 101 mark 4. Bring the pipette in a horizontal position and place the finger at the tip then remove the rubber tubing 5. Keep the pipette in horizontal position by holding it between the thumb and middle finger then carefully shake it using a figure of 8 for atleast 2-3 mins b. Counting of RBC 1. Clean the hemocytometer and the coverglass make sure these are free from grease before using 2. Place the coverglass laterally to and on the supporting tibs of the counting chamber 3. Discard at least one third of the contents of the pipette in a clean tissue paper and wipe off adhered excess fluid 4. Carefully touch tip of the pipette to the space between the chamber and the coverglass. Fill the space with the mixture up to three fourths of the chamber withdraw the pipette and let the fluid flow to completely fill the entire area 5. Stand the mixture for about 3 mins to allow the cells to settle care should be taken to avoid evaporation 6. Focus under LPO locate the central square of the nine primary large squares of the counting chamber 7. Shift to HPO observe all RBC in the area within the 5 squares of the 25 small squares in the center of the 9 primary squares 8. Note for even distribution of cells in the specified areas (4 corners and the center of the 25 secondary squares)

9. Each of the five small squares to be used for counting is lined by double or triple lines and further subdivided into 16 smaller squares (5X16=80 small squares) 10. To avoid any duplication in counting just count the cells following the sequence from the upper left corner to right then next row of 4 squares front right to left and so on Triple ruling count cells touching top and left center lines while cells touching bottom and right center lines are not Calculations: Sum of number of cells in 5 small squares X 10,000 = total RBC/ml of blood Questions 1. Define: a. Erythropoiesis - the production of red blood cells. - *hypoxia is the best stimulus for erythropoiesis b. Polycythemia - : An abnormally increased concentration of hemoglobin in the blood. c. Polycythemia vera - is a blood disorder in which the bone marrow makes too many red blood cells. Polycythemia vera may also result in the overproduction of white blood cells and platelets. d. Anemia - A condition marked by a deficiency of red blood cells or of hemoglobin in the blood, resulting in pallor and weariness Pernicious anemia deficiency of VitB12 Megaloblastic anemia deficiency of Folic Acid 2. Diagram of erythropoiesis Bone Marrow metarubricyte (nucleated) ; mammalian erythrocyte (non-nucleated) nucleus of metarubricyte is lost by extrusion or by absorption before it enters bloodstream with supravital stain the RNA is precipitated, giving a network of blue fibers known as reticulocyte during hemorrhage, parasitism, most animals respond w/ a release of young nucleated RBC and reticulocyte from bone marrow in circulating blood 3. What are the important factors needed for maturation of RBC Vit B12 & Folic Acid (DNA synthesis) 4. Give factors that may cause on invasion erythrocyte count a. Sex female have less RBC; they undergo estrus - Male have more RBC count; they are more active compared to females b. Age immature has increase RBC count c. Physical activities d. Altitude lower oxygen in air which causes erythropoiesis to occur e. Diet/nutrition obesity may have higher count of RBC because of increase degree of abdominal pressure f. Species poultry have higher level of RBC because of egg formation 5. Importance of erythropoietin

Erythropoietin stimulates red bone marrow to produce more RBC. Thus, when O2 levels in the blood decrease the production of erythropoietin increases red blood cells production the increased number of RBC increases the ability of the blood transport oxygen. This mechanism returns blood O2 levels to normal and maintains homeostasis by increasing the delivery of tissues. Platelet Counting Platelets (thrombocytes) not true cells - Geminate from big leukocytes called megakaryocytes - Appear purple color and are more intense than red cells Materials 1. Hemocytometer 2. Thoma pipette w/ red bead (0.5 101 mark) 3. Rees & Ecker solution Procedure: 1. Make a venipuncture and draw blood into an RBC diluting pipette to the 0.5 mark and then draw Rees and Ecker solution up to the 101 mark 2. Mix vigorously using the figure 8 direction for 5 minutes 3. Discard the 4 first drops and then fill both chambers of the hemocytometer similara to erythrocyte count 4. Let it stand for 15 minutes to allow cells to settle 5. Focus the 9 primary squares under LPO then locate the center square (w/ 25 squares) 6. Count the thrombocytes in the entire central rules area on both chambers Results: total number of platelets X 10,000 = thrombocytes/microliter of blood Questions 1. Differentiate thrombocytopenia and thrombocytosis Thrombocytopenia is any disorder in which there is an abnormally low amount of platelets. Platelets are parts of the blood that help blood to clot Thrombocytosis increase in the number of platelets in the blood which tends to cause clots to form; associated with many neoplasms and chronic infections and other diseases. 2. Discuss importance of platelets Platelets play a great role in the prevention of blood loss. This prevention is accomplished in 2 ways: a. Formation of platelet plus b. Formation of clots 3. What will happen to the individual w. thrombocytopenia An individual w/ thrombocytopenia will undergo excessive bleeding time because of the loss of platelets

Leukocyte Count Leukocyte density 5000-7000/cu. mm - Two categories: granulocytes, agranulocytes - Responsible for the defense of the organism B lymphocyte is the originator of 6 types of wbc (plasma cells, monocyte, lymphocyte, basophil, neutrophil, eosinophil) Materials: 1. Thoma pipette w/ white bead (0.5-11 mark) 2. WBC diluting fluid 3. hemocytometer Procedure: a. filling the technique 1. follow the technique described under RBC except for the diluting pipette that has 11 mark above the bulb. 2. Draw blood exactly to the 0.5 mark and wipe the tip of the tube with a clean tissue paper 3. With the attached rubber tubing suck the wbc diluting fluid up to the 11 mark above the bulb 4. Shake well for 3 minutes b. Counting of leukocytes 1. Discard 2-3 drops from the pipette before filling the hemocytometer 2. Stand for about a minute to allow lysis of erythrocyte and for the wbc to settle 3. Under LPO count the number of cells in each of the 4 large corners of the 9 primary squares c. Calculation Cells counted in 4 large squares multiply by 10 then X 20 then divided by 4 which equals to leukocyte per microliter Questions 1. Morphology Function Distribution Neutrophil Nucleus w/ 2-4 lobes Phagocytizes 54-62% connected by thin microorganisms and other filaments; substances; cytoplasmic granules stain purple, a light pink or reddish purple; Monocyte largest leukocyte; diapedesis; become the 2-10% nucleus is round, macrophage and is the kidney or horse shoe weakest WBC shaped Eosinophil telephone shaped Releases chemicals that 1-6% nucleus; often reduce inflammation; bilobed attacks certain worm or parasites Lymphocyte Immune response Produces antibodies and 28-33% other chemicals responsive for destroying

Basophil

Nucleus w/ 2 lobes cytoplasmic stain blue-purple

microorganisms;contributes to allergic reactions, graft rejection, tumor control and regulation of immune system Vasodilators. Release Less than 1% heparin, histamine and bronchodilators; promotes inflammation

2. Leukemia - A malignant progressive disease in which the bone marrow and other bloodforming organs produce increased numbers of immature or abnormal leukocytes, suppressing the production of normal blood cells. 3. Leukocytosis vs. Leukemia Leukocytosis - An increase in the number of white cells in the blood, esp. during an infection Leukemia - A malignant progressive disease in which the bone marrow and other bloodforming organs produce increased numbers of immature or abnormal leukocytes, suppressing the production of normal blood cells. 4. Leukocytosis vs. leucopenia Leukopenia - A reduction in the number of white cells in the blood, typical of various diseases. Leukocytosis - - An increase in the number of white cells in the blood, esp. during an infection Erythrocytes Leukocytes Non-mobile Mobile Presence of hemoglobin Absent Smaller than wbc Larger than rbc Non-nucleated (mammals); nucleated (birds) Polymorphonucleated Red in color Color depends on the staining granules Segmented vs. Non-segmented neutrophil Nucleus of each mature cell is generally divided into lobes or segmented connected on filaments. The cells that are immature appear as curved or called bands, reduce or even deeply indented but w/ segmentation are known as band cells