Lab 7: Introduction to Blood (PDF)

Introduction to blood Biochemistry 1 Lab 7 Biochemistry department Blood  Blood is a constantly circulating fluid providing the body with nutrition, oxygen, and waste removal. 2  Blood is mostly liquid, with numerous cells and proteins suspended in...

Introduction to blood Biochemistry 1 Lab 7 Biochemistry department Blood  Blood is a constantly circulating fluid providing the body with nutrition, oxygen, and waste removal. 2  Blood is mostly liquid, with numerous cells and proteins suspended in it, making blood "thicker" than pure water.  The average person has about 5 liters of blood. Functions of blood 1. Transport: of gases, nutrients & metabolic waste products. 2. Communication: i.e transmission of signals e.g., hormones, enzymes. 3. Control and Regulation of the following: body temperature, water balance and 3 arterial blood pressure, acid-base balance (buffer system). 4. Hemostasis (blood clotting which prevents hemorrhage). 5. Defense (by antibodies and WBCs). Physical properties of blood: i. Color: Arterial blood is scarlet red due to the presence of oxy Hb. Venous blood is darker in color (bluish due to CO2). ii. Specific gravity: 1.05 at 37 °C. 4 iii. Viscosity: 5 times that of water. iv. pH: slightly alkaline 7.4 ± 0.05 (kept constant by the action of blood buffers). v. Osmotic pressure: as that of normal saline (0.9% NaCL). vi. Volume: 7.8 % of total body water (about 5 liters in normal healthy adult).  5 Ca+2 ions Difference between serum and plasma: Serum Plasma Method of preparation Blood is allowed to clot Blood is collected on an followed by centrifugation anti-coagulant followed by centrifugation Prothrombin Absent Present 6 Thrombin Present Absent Fibrinogen Absent Present Injection Never injected Can be injected Presence of excess Ca++ No clot Clot Anticoagulants: substances that suppress coagulation:  1-Heparin: It is the anticoagulant of choice in hematology since it does not alter the 7 shape of RBCs so they can be examined under the microscope. It is the naturally occurring cofactor for antithrombin III that inactivates thrombin & can be administered as a drug. 8  2-Dicumarol (Warfarin) (vit K analogue): It is a drug that interferes with the action of vit K (by preventing it from being reduced to its active reduced form )which is required for the synthesis of clotting factors by the liver. 9  3-EDTA: Chelates Ca++ needed for the conversion of prothrombin to thrombin  4- Oxalates: Form insoluble Ca oxalate so Ca++ is no longer available for the conversion of 10 prothrombin to thrombin.  5-Citrates: Form non-ionizable Ca citrate.  6-Fluorides: Used as anticoagulant together with potassium citrate in blood collected for sugar determination since it inhibits glycolysis ( conversion of glucose into lactate or pyruvate ) and so avoids false –ve 11 results during determination of blood glucose. Fluoride inhibits glycolysis since it inhibits enolase enzyme responsible for the conversion of 2- phosphoglycerate to phosphoenolpyruvate during glycolysis. N.B: Blood cells can be counted by taking a blood sample, diluting it by a known amount and then introducing a small volume into a special chamber on a microscope slide called a hemocytometer. This has a grid of fine squares etched on it, and the volume overlying each square is accurately known. The number of blood cells occupying 12 each of large number of squares is counted, and after correcting for the dilution factor, the number of cells in a given volume of blood is counted. Assessment of hematological parameters  A) Complete blood count [CBC]: It is one of the most common blood tests performed & it is ordered as a part of routine medical examination. In CBC, the various types of blood cells are analyzed using the following tests: 1. White blood cells count. 13 2. Differential white blood cells count. 3. RBCs count. 4. Hemoglobin content. 5. Hematocrit value. 6. Red cell parameters e.g. MCV, MCH. 7. Platelets count. 1- White blood cells count (leukocyte count):  WBCs are cells of the immune system, responsible for protecting the body from infection and malignancy.  They are larger and less numerous than erythrocytes. 14  WBC count: 4,500 to 10,000 cells/µL of blood.  Abnormal leukocyte count:  -Low count (leucopenia) indicates: AIDS, treatment of cancer (radiation or chemotherapy).  - High count (leukocytosis) indicates: infection, leukemia or sever emotional. 2- Differential leukocyte count:  To determine the number of different types of WBCs to aid in a variety of diagnosis.  There are 5 types of WBCs:  The granular leukocytes (granulocytes): 15 Neutrophils Eosinophils Basophils  The non-granular leukocytes (agranulocytes): Lymphocytes Monocytes Types of leukocytes 16 1.Neutrophils:  Function: Neutrophils play a central role in inflammatory processes. Receptors in their plasma membrane allow them to recognize foreign bodies, e.g. bacteria and tissue debris, which they begin to phagocytose and destroy. 17  Shape: They have a very characteristic nucleus. It is divided into 3-5 lobes which are connected by thin strands of chromatin.  High neutrophils count indicates: infection, certain type of cancer. 2.Eosinophils:  Function: The presence of antibody-antigen complexes stimulates the immune system. Eosinophils phagocytose these complexes and this may prevent the immune system from "overreacting". They may be also involved in the response of the body against parasitic infections. 18  Shape: Their nucleus has only two lobes. The cytoplasm appears filled with the specific granules which stain red or pink when eosin is used in the staining process.  High Eosinophils count indicates: Allergies, parasitic infections. 3.Basophils:  Function: They synthesize and store histamine (important in allergic reactions) and heparin (prevents blood clotting).  Shape: They have a 2 or 3 lobed nucleus. Their 19 granules are stained deeply bluish or reddish-violet. Their color corresponds closely to the colour of the nucleus which sometimes is difficult to see amongst or behind the granules. 4. Monocytes:  Function: Once monocytes enter the connective tissue they differentiate into macrophages. At sites of infection macrophages phagocytose microorganisms & tissue debris.  Shape: They have a C-shaped nucleus. 20  High Monocytes count indicates: Bacterial infection. 5. Lymphocytes:  Function:  -B-lymphocytes: upon exposure to antigens, they differentiate into antibody producing plasma cells. 21  -T-lymphocytes: represent the "cellular arm" of the immune response and may attack foreign cells, cancer cells and cells infected by virus.  Shape: The nucleus is large and fills the entire cell.  Low lymphocyte count may be a symptom of AIDS. Assessment of hematological parameters continu. A. Complete Blood Count 1. White blood cells count 2. Differential white blood cells count 3. RBCs count 22 4. Hemoglobin content 5. Hematocrit value 6. Red cell parameters e.g MCV, MCH. 7. Platelets count B. Erythrocyte Sedimentation Rate C. Blood Groups  3. Red blood cells count (erythrocyte count)  RBCs, the most abundant type of blood cells ( 90% of blood cells), are responsible for transporting oxygen thought out the body. 23  The range of normal RBCs count is: Male: 4.7 to 6.1 million cells/µL Female: 4.2 to 5.4 million cells/µL  Erythrocytes do not contain a nucleus.  They are biconcave disks although their shape is influenced by osmotic forces.  However, under light microscope they appear as circular homogenous discs of nearly uniform size ranging from 6-8 µm, the center of each is somewhat paler than the periphery. 24 Abnormal erythrocyte count :  Increased count indicates: Polycythemia, Low oxygen tension in the blood : e.g. in Congenital heart disease , pulmonary fibrosis. 25  Decreased count indicates: Anemia, hemolysis (RBC destruction), Hemorrhage (blood loss), erythropoietin deficiency (secondary to renal disease).  4. Hemoglobin assay:  Hb is a conjugated protein present only in RBCs & gives blood its red color.  It is the only O2 binding protein inside the RBCs & constitutes about 95% of its dry weight.  It is composed of a colorless protein globin and a red pigment 26 Heme.  The globin of adult human (HbA) consist of 2 pairs of unlike polypeptide chains (2α & 2β chains) which differ in amino acid composition and sequence. 27 28  Heme is a complex of iron and porphyrin.  The Hb consists of 4 heme groups one heme attached to each of the 4 polypeptide chains.  Normal value: Adult female: 12- 16 gm% 29 Adult male: 14- 18 gm% Children: 12-16 gm%  5. Hematocrit Value( HCV) = Packed cell volume (PCV):  It is the percentage of blood volume occupied by the erythrocytes.  The HCV test, like the RBC count, is performed to check whether the red cell count is abnormally high or low. 30  HCV = Red blood cell volume X 100 Total blood volume Determination of HCV:  Blood sample is obtained from the patient by finger prick.  Blood is placed in a special tube called Wintrobe tube that contains an anticoagulant (heparin or EDTA) & is then centrifuged.  The heavier cellular elements slowly settle to the bottom and the lighter 31 plasma rises to the top.  The RBCs are packed at the bottom of the tube while WBCs and platelets which are less dense than RBCs are packed in a thin, cream-colored layer (buffy coat) on the top of the packed red cell column. 32  Normal value: Adult male 40-47 % Adult female 36-42 % Newly born infants 60% , why? 33  Because partial pressure of placental O2 is low so there is relative hypoxia in the intra-uterine life thus there is polycythemia as a compensatory mechanism. In the first few weeks after birth this excess amount of RBCs is hemolyzed releasing their iron content which is also needed by the infant as mother’s milk is deprived of iron.  Interpretation: o It increases in: a) Dehydration: Burns, Diarrhea. b) Polycythemia. c) Low oxygen tension (smoking, congenital heart disease, living at high altitudes). 34 o It decreases in: a) Anemia. b) Blood loss (hemorrhage). c) Hemolysis (RBCs destruction) related to transfusion reaction.  6. Red cell parameters: a) Mean corpuscular volume (MCV):  It is the average volume of a red cell Volume of RBCs in 100 ml blood (hematocrit)  MCV = 35 Number of RBCs in 100 ml blood  MCV ↑ in: megaloblastic anemia (large cell)  MCV ↓ in: iron deficiency anemia (small cell) b) Mean corpuscular hemoglobin (MCH):  It is the average amount of Hb in a red cell Hb in gm / 100 ml blood 36  MCH = Number of RBCs / 100 ml blood  MCH ↑ in macrocytic cell with large amount of Hb  MCH ↓ in iron deficiency anemia  7. Platelets count;  They are cell fragments essential for the process of blood clotting.  Normal platelet count is 150,000 to 450,000 / µl of blood.  Low platelets count is termed thrombocytopenia, 37  while increased platelets count is termed thrombocytosis.  38 THANK YOU