Physiology Lab (1-2-3-4) PDF

Physiology lab (1) identification of different blood cells microscopically. 1 ‫اﻟﺤﺪاي‬ Red blood cells (R...

Physiology lab (1) identification of different blood cells microscopically. 1 ‫اﻟﺤﺪاي‬ Red blood cells (RBCs) / Erythrocytes M o r e t h a n 99% o f t h e f o r m e d e l e m e n t s in b l o o d are RBCs. They are a biconcave shape; contain the oxygen-carrying pigmient hemoglobin, which si respónsible for the red color of whole AS,bflex looible d. ,RCs anca bico ppevearshap ncawsveRdBisCc.s toThe as beicoallo htrough narrow capilaries. RBCs l a c k a ‫ﺿﻐﻂ‬ ezequs nucleus and other organelles. ‫اﻟﺸﻐﯿﺮات اﻟﺪﻣﻮﯾﺔ‬ 3 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 Blood composition. Blood is a niodified connective tissue composed of: I. Cellular component (45%): A. Red blood cells (RBCs) or erythrocytes. B. White blood cells (WBCs) or leukocytes..1 Granular leukocytes (granulocytes: Neutrophils - Basophils and Esinophils) 2. Agranular leukocytes (Lymphocytes, T cells and B cells, and Monocytes) C. Platelets or thrombocytes. II. Fluid component (plasma 55%). Composed of A. Water 90%. B. Dissolved substances 10% (SOLUTES):- 1. Plasma proteins 7% (most are synthesized by the liver include albumins, globulins, and fibrinogen). 2. Other organic substances as nutrients and waste products as urea, uric acid, ammon ‫ ﺻﻨﻲ‬-‫ واﻟﻠﺲ‬3. Inorganic substances e.g. Na, K, Ca, Cl ions etc..... 2 ‫أﺿﺮ‬ ‫ﻟﺘﺼﺮﯾﺮاﻟﻤﺴﺘﻨﺪات‬ RBC physiology As blood passes through the lungs, hemoglobin inside RBCs combines with oxygen to form oxyhemoglobin. Hb molecule consists of a protein called globin, * composed of 4polypeptide chains (2 called alpha and 2 called beta), plus 4 non-protein pigments called heme. * Each heme contains an IRON ion (Fe+2) that can combine reversibly with one oxygen molecule. The O2 is transpo rted in this s t a t e to tissues of b o d y I n t h e tissues, the iron-oxygen reaction reverses. Hb releases O2, which diffuses into the cells. 1 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  F u n c t i o n of RBCs ‫ﻗﻀﻤﯿﻦ‬ 1. Enclose hemoglobin. 2. Contain hemoglobin, which carry O2 and COz. 3. Contain carbonic anhydrase enzyme that catalyze the reaction between H20 and COz. 5 RBC number, shape, and size. Num ber: * Adult male 5-5.5 million/ mm'. * Adult female 4.5-4.8 million/ mm'. * More in newborn infants. Shape: *Circular, biconcave, non-nucleate discs. The biconcavity:.1 Increases the surface area in relation to volume. 2. Enable it to pass through narrow capillaries without rupt ure. Size: 90 p' in volume, 7.5 p in diameter, and 2 u ni thickness.  6/22/2024 heme g r o u p Quaternary Protein Sructue: Three-dimensional assembly of subunits N o r m a l Blood cells 10 200K E n : CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  N o r m a l b l o o d cells White blood cells (WBCs) (Leukocytes) Unlike RBCs, WBCs have a nucleus but do not contain Hb. Total count: 4000-11000/mm'. Leukocytes are divided into:.1 Granulocytes: contains granules in their cytoplasm, subdivided into: a. Neutrophils (60-70 %). b. Eosinophils (2-4 %). c. Basophils (1%). 2. Non-granulocytes, subdivided into: a. Lymphocytes (20-25 %). b. Monocytes (3-8%). CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 Life s p a n RBCs live only about (120 days) b e c a u s e of : -Dead RBCs are removed from blood and destroyed by fixed phagocytic Macrophages in spleen and liver and breakdown products (heme & proteins) are recycled. 01 Leukocytes white blood cells ~WBC agranular granular lymphocyles monocytes basophils neutrophils eosinophlls 20 - 25 % 3 - 8% 5. - 1% 60 - 70% 2 - 4% T-cell, B-cell, K N Cell 12. 3 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  Function of WBC Monocytes migrate out blood into tissues, enlarge and differentiate into (Macrophages). Some are fixed macrophages, which mean they reside in a particular tissue. Granulocytes and monocytes protect the body against invading organisms mainly by ingesting them by phagocytosis. Lymphocytes and plasma cells function mainly in connection with the immune system. Phagocytosis; attack and destroy the invading bacteria, parasites and foreign substances, slow and modulate allergic reactions and responsible for acquired immunity. 31 Platelets (Thrombocytes) * Count: 150,000-400,000/ mm'. * Shape: Non-nucleated rounded discs. * Size: 2-4 u in diameter. * Life span: 10 days. Function: Have an important role in homeostasis (Stoppage of bleeding from injured blood vessels) which is achieved by 3 mechanisms: 1) Vascular spasm: contraction of the smooth muscles of blood vessels 2) Platelets plug formation. 3) Blood coagulation (clotting). 51 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 WBC Lifespan Variable from few days to months and years. Lifespan of granulocytes in tissues is about 4-5 days. But, in serious tissue infections the lifespan is shortened to only few hours. Macrophages can live up to months or even years. Preparations of blood smear.1 Obtained of blood: - fresh blood is required from either finger stick or venous phlebotomy. a. Finger stick method: clean the finger with alcohol 70%, allow it to dry, Pierce fingertip with sterile lancet. Alow blood ot flow freely on a slid. b. Venous blood method: Obtain venous blood from volunteers, use a pipette to apply a drop of blood on a slide for blood smear..2 Touch a drop of the blood with edge of clean slide. 3. Bring the edge of the slide with the drop of the blood to the surface of the first slide, place it at the far end, and wait until the blood spread along the whole edge. 4. Put the slide at an angle of 45°, push it forward with a rapid gentle movement. 61 4 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  5. Let the smear to dry and Mark the slide with patient identification, date and time of collection. G. After the smear had dried, smear fixing is done in methanol 70% (deep in methanol for 5 seconds). 7. Place the slide flat in a staining rack. 8. Cover it with 1-2 ml of Giemsa solution, stand for 10 min. 9. Gently and carefully, rinse by floating excess stain by water. 10. Dry the smear and examine the smear under a microscope using oil immersion lens. ‫ﺑﯿﺔأﺑﯿﺪ‬ ‫إﯾﺮ ال‬.1 Microcytosis: RBCs size is less than normal (Microcytic) as in lead poisons and iron deficiency anemia. 2. Macrocytosis: RBCs size is more than normal (Macrocytic); folic acid and vitamin B12 deficiency anemia. 3.dP a htem alone heatni acco, nai lung diseases. 4. Poikilocytosis: variation in RBCs shape as in sever anemia. 5. Hypochromic: pale color cells, decrease Hb concentration. 6. Sickle shaped cells, heredity acquired disease. 91  6/22/2024 Blood smear preparation ‫ز‬ ‫اﻷﺳﺔ‬ ‫ﺑﻌﻮﯾﺮ‬ Sickle cell a n a e m i a ADAM. 5 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬ Normal and sickle shaped RBCS Sickla cells R e d biood cells 21 Pol ycy the mia CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 Spherocytosis rights F000 Micr ocyto sis CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  Hypo-chromic cells 251 Physiology lab. (2) Determination of haemoglobin content CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 Poikilocytosis Determination of haemoglobin content I t i s the determination o f the amount o f haemoglobin present i n 100 cc (ml) of blood, (gram/ 100 ml) o f t h e blood. Normalv a l u e : - Adult male 15-16 gm% (gm/dl). 82 - A d u l t f e m a l e 14 om% 7 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬ - Each RBC contains about 280 million molecules of Hb. Each Hb molecule consists of four protein chains (globins). The chains are 2 alpha (141 amino acids) and 2 beta (146 amino acids) chains. Each chain attached to non-protein group called heme, which carries one molecule of 02. The whole Hb molecule can carry 4 oxygen molecules. A bout 20% of CO, in the blood binds to the globin group while 0, binds to heme group. Heme group is composed of iron protoporphyrine. 92 T h e r e a r e s e v e r a l types o f H b a c c o r d i n g to the a m i n o a c i d composition o f the peptide chains:- I. Physiological types: 1. Adult Hb (Hb-A): normal adult Hb contains 2a chains and 2ß chains (about 98% of normal adult Hb). 2. Hb-A2: A bout 2% of Hb is Hb-A2 in adult; contains 2a and 28 chains. 3. Fetal Hb (Hb-F): Fetal Hb present before birth, but soon after birth it is replaced by Eb-A; contains 2a and Zy chains. Hb-F has higher 02 binding capacity than adult Hb. Note: § and y chains are same in length but differ in amino acids sequences. 13 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 5 of Structure of haemoglobin d heme g r o u p bin ne. 29 Quaternary Protein Sructue: Three-dimensional assembly of subugits cid II. Pathological Hb: (Abnormal types).1 Haemoglobin-S (Hb-S): contains 2 ins normal a chains, but 2 abnormal B chains (number 6 amino acid glutamate s2 a replaced by valine), this will lead to precipitation of Hb and make RBCs and sickle in shape, results in sickle cell 1 anaemia. 2. Haemoglobin-H (Hb-H): contains 4 B ino chains (a-thalassemia) and B - thalass emia contain s 4 a chains. 23 8 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬ Old RBCs after 120 days are destroyed into: Hb which is further destroyed into globin & heme:- 1. Globin→ free amino acids, for protein synthesis. 2. Heme→ into: A. Ferrous (iron) - reused, storage or loss by injury, menstruation.. B. Bilirubin; un-conjugated in plasma, in liver it will conjugate with glucuronic acid to form conjugated bilirubin, in the small intestine bacteria modify bilirubin to stercobilinogen→ either reabsorbed (⅓) or pass with the stool and giving its normal colour (⅔). 33 ‫اﺑﯿﺚ‬ 2! CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 Determination of HB concentration by Sahli's method. Materials: 1. Sahli's hemoglobinometer. 2. A 20 mm* marked pipette. 3. 0.1 N HCl. 4. Distilled water. 5. Sterile lancet. 6. Alcohol 70%. 7. Cotto n. 43 Procedure: W. 1. Fill the dilution tube of Sahlis apparatus with 0.1 N HCl up to the mark 20. ‫اﻟﺘﺼﻮي‬ 2. Clean the finger with 70% alcohol, allow it to dry, prick with a sterile lancet or needle. 3. Suck up the blood with the pipette up to the mark 20 mm'; no air bubbles should enter. 4. Deliver the blood gently into the diluting tube, suck up and down 2 to 3 times to mix the blood with H C l and wait for 5 minutes for acid hem atin form atio n. 63 9 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬ 5. Add distilled water drop by drop until the colour is similar to that of the standard. 6. Note the level of the solution in the diluting tube; it gives Hb concentration in gm%. Note: Factors affecting Hb content as P.C.V. 73 PHYSILOGY LAB 3 Determination of hematocrite HCT% (PCV) 39 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  Debr weisung nometer sude agatons bat, encoinol mae, dent fabienion. ม Gl ont ens upa nee e dr e be D e t e r m i n a t i o no fhematocrite HCT% (PCV) Hematocrit 4 2- 5 0 %m a l e s 3 9- 4 8 %f e m a l e s CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  (Packed cell volume) P.C.V. Definition: It is the percentage ratio of RBCs volume to total blood volume. PC.V.. RBCS volume ×100 Total blood volume Normal value: - Adult male 45%. ‫ﻋﻠ‬ ‫ﺔ‬ ‫ﯿ‬ - Adult female 41%. ‫اﻟ‬ ‫ﻤﯿ ﺪ‬ - N e w b o r n 65%. Microhematocrite (Capillary tube method). Mat eria ls: 1. Alcohol 70%. 2. Sterile lancet. 3. Heparinized capillary tube. 4. Wa x. 5. Microhematocrite centrifuge. 6. Cotton. 7. Graph-paper. 34 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 Factors affecting P.C.V:-. Physiological factors: A. Higher in fetus and newborn than adult. B 1. Higher in male t h a n female. 2. Higher in venous blood than arterial blood. B. Pathological factors:- 1. Decreased in: a. Anemia because of low number of RBCS. b. Over-hydration due to increase plasma volume. 2. Increased in: 2a d la a. Polycythemia due to increased RBCs. b. Dehydration due to decrease plasma volume). ،‫اﻟﺘﺼﻮﯾﺎ‬ 24 Heparinized capillary tube 44 11 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  Micro hema tocrit e centr ifuge Micro hemat ocrite centri fuge CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 Procedure:.1 Clean the finger tip with 70% alcohol and allow it to dry. 2. Make a single puncture at the tip of the finger and wait till a drop of blood is collected. 3. Place the heparinized capillary tube on the drop of the blood, the blood will ascend in the tube by the capillary action. Let the tube to be filled up to ¾ its length. 12 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  +. Seal the end of the tube with wax; place the tube in the centrifug e for 10 minutes with aspeed of 3000 round /min, this wil separate the coloured packed red blood cells fro m t h e stra w col our ed pla sm a. 5. Remove t h e tube and measure RBCS column (A) a n d total blood column (B) using the graph-paper 6. P.C.V. = RBCs column (A) × 100 Total blood column (B) 49 H E M AT O C R I T \PLASMA BLOOD 1003% RBCs: 3 9 - 47% CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 Plasma Whole Blood White Blood Cells (V1) (WBC) Red Blood Cells (RBC, V2) Centrifugation Het = (V2/V1) x 100 How to measure PCV% 1000 a d 8 0 7 4 5 0 4 0 13 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  PHYSIOLOGY LAB 4 DETERMINATION OF BLOOD INDICES 53.I Normocytic normochromic anaemia: RBCs size (MCV) is normal →Normocytic. Hb in each RBC (MCH) is normal (normochromic). - It is due to:.1 Acute blood loss (sever haemorrhage) →haemorrhagic anaemia. RBCs haemolysis (excessive RBCs destruction) → haemolytic anaemia; as in lead & arsenic poisons, snake venoms, bacterial toxins, drugs as sulphonamides, excessive exposure to X-ray, some viral infections, deficiency of glucose-6- phosphate dehydrogenase enzyme and incompatible blood transfusion. 3. Sickle cel anaemia, thalassemia and spherocytosis. C a m S c a n n e r ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 Blood indices (Haematological indices) Anaemia: It is decreased in RBCs count, decrease in PCV o r decrease in Hb content or all below the normal range. Types: 1. Normocytic normochromic anaemia. 2. Microcytic hypochromic anaemia. 3. Macrocytic hyperchromic anaemia. 45 II. Microcytic hypochromic anaemia: RBCs size (MCV) is less than normal →Microcytic. Amount of Hb in each RBC (MCH) is less than normal → hypochromic. It is due to iron-deficiency anaemia, which due to:- 1. Trequirement (pregnancy, lactation). 2. s t o r a g e (liver disease). 3. I intake (malnutrition & starvation). 4. 1 HCl in stomach & vitamin C in food. 5. Bile deficiency (apotransferrin necessary for iron absorption). 6. Excess phytic acid (in tea) - form insoluble complex. 7. Chronic blood loss (as in peptic ulcer bleeding, hematuria, some parasitic infestation and excessive menstruation. 14 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  ROLE O F I R O N I r o n i s an e s s e n t i a l component of Hb. The total quantity of iron in the body is 4 to 5 grams, about 65% in the form of Hb and 15 to 30 % is s t o r e d in t h e liver. Daily intake: 10-20 mg. Daily need: 1mg in males and 2mg in fem ales. III. Macrocytic hyperchromic anaemia: RBCs size (MCV) is more than normal → Macrocytosis. Amount of Hb in each RBC (MCH) is more than normal → hyperchromic. - It is due to vitamin B12 or folic acid-deficiency anaemia. - Daily requirement 1-2 ug vit B12. - Liver stores 3000ug (3mg) vit B12, 5 years body need. 95 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 Transport and storage of iron When iron is absorbed from the small intestine, it immediately combines with a plasma beta globulin (apotransferrin)to form (transferrin), which is then transported to plasma. The iron can be released to any tissue cells at any point in the body. Excess iron in blood is deposited in all cells of the body especially in the liver hepatocytes. In the cell cytoplasm, it combines mainly with (apoferritin) to form(ferritin), which called storage iron. People with low transferrin develop ( hypochromic anemia) that is decreased number of RBCs that contain less Hb than normal. 58 Causes of vitamin B12 deficiency: 1. I intake (rare). ( intrinsic factor) - pernicious anaemia..2 I absorption L 3. I storage (liver disease). 4. 1 requirement (rare). Causes of folic acid deficiency: 1. Lintake. 2. Labsorption. 3. I st o ra g e. 4. 1 requirement. 60 15 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬ Also a n e m i a c a n b e classifi ed into 4 types 1. Blood loss anem ia (hemo rrhagi c anemi a). 2. Aplastic anemia (bone marrow aplasia) lack of function of b o n e marrow d u e t o t h e explosion to abnormal conditions (excessive X-ray), gamma ray radiation and certain industrial chemicals. 3. Megaplastic anemia (slow erythroblasts repro ductio n in bone marro w d u e vitam in B12 or folic acid or pernicious anemia). 16 An Anemia effects: 1. 102 supply →hypoxia (anaemic hypoxia). 2. I blood viscosity→ Iperipheral resistance →Theart rate, cardiac output this will lead to heart failure. 3. Jaundice in haemolytic anaemia. 4. Pernicious anaemia →lead to spinal cord degeneration. 5. Pallor, palpitation, fatigue, fainting, headache and blurring of vision 63 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 4. Hemolytic anemia: easily rupture of RBCs, even through their number is normal (life span become s o short causes: 5. Polycythemia: disorder characterized by Hematocrite and RBCs count above the normal level (2 types):.i Secondary Polycythemia: production of large quantities of RBCs (6-7 millions/ ml) due to little 02 in atmosphere as high altitude or failure delivery of 02 to the tissue, as cardiac failure. ii. Polycythemia Vera (Erythremia): The blast cell no longer stops producing RBCs. 62 e calculate: RBCs count, **Diagnosis of anemia: w Hb content, HCT%. **Diagnosis hte type of anemia: w e calculate blood indices. 46 16 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  1. Mean Corpuscular Volume : ( MCV) It si hte average volume of a single RBC. MCV= Hematocrite X 10 RBCs in million/mm' Normal= 45 x 10 =90 * (Femtoliter). 5 Normal range = 90 $ 7 u3. 3. Mean Corpuscular Hemoglobin concentration percent :( MCHC) It is amount of Hb in 100 ml packed RBCs. MCHC= Hemoglobin content x 100 Hematoc rite Normally= 15 × 100 = 33 % 45 Normal range= 33 % $ 3 1114.1 1. 11 41171 76 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬  6/22/2024 2. Mean Corpuscular hemoglobin: (MCH) It si the average amount of hemoglobin in a single RBC. MCH= Hemoglobin content X 10 RBCs in million/mm' Normally= 15 x10 = 30 P. g. (1012 gram). Normal range = 30 ¢ 3 P.g. ‫ﻣﻜﻠﯿﮫ‬-‫ﺑﺮاﺑﻠﺲ‬ ‫زع‬ 56 Significance of blood indices determination→to diagnose type of anemia:.1 Increased in macrocytic hyperchromic anemia. 2. Decreased in Microcytic hypochromic anemia. 3. Normal in Normocytic normochromic anemia. 63 17 CamScanner ‫اﻟﻤﻤﺴﻮﺣﺔ‬ ‫ب‬