WINDSOR UNIVERSITY SCHOOL OF MEDICINE

WINDSOR UNIVERSITY SCHOOL OF MEDICINE
Blood Component and Function Dr.Vishal Surender.MD.

OBJECTIVES: BLOOD COMPOSITION
Describe the components of blood (cells, ions, proteins, platelets) giving their normal values. Describe the physico-chemical properties of the plasma. Define the term hematocrit, state its normal values. Explain the importance of maintaining normal hematocrit level. List the factors that affect the hematocrit value. Understand how dehydration, excessive water intake, RBC count and size affect Htc. Be able to calculate plasma volume for given Htc and total blood volume for given Htc and plasma volume. Describe the functions of plasma electrolytes. List the main fractions of plasma proteins and describe their properties and functions. Describe the reasons and consequences of hypoproteinemia. Understand why liver diseases with decreased synthesis of albumins cause edema formation. What is the meaning of the term ‘non-protein nitrogen’?

BODY FLUID Cells TOTAL BODY WATER = 42L ICF = 28L ECF = 14L
Interstitial Fluid (10L) Osmotic forces Red cell mass = 2L Plasma = 3L Starlings forces BLOOD BLOOD Transcellular fluid = 1L

THE COMPONENTS OF BLOOD
Blood is an opaque, red liquid consisting of several types of cells suspended in a complex, amber fluid known as plasma. When blood is allowed to clot or coagulate, the suspending medium is referred to as serum.

BLOOD FUNCTIONS Transportation
Dissolved or chemically bound matter (O2, CO2, nutrients, metabolites) Heat for heating and cooling (maintenance of the body temperature) Protection/Immunity Defense against foreign agents (specific and non-specific immunity) Hemostasis Prevention of hemorrhage - hemostasis Regulation/Hemostasis Transmission of signals (hormones) Maintenance of the homeostasis Concentration of dissolved substances Osmotic and oncotic pressure Acid-base balance - buffering the body fluids (maintenance of pH)

BLOOD VOLUME AND COMPOSITION
Males: 5000 – 6000 mL Females: 4000 – 5000 mL 6 - 8% of the total body mass 20% of the ECF. Composition Blood plasma - non-cellular portion of the blood - 55% 2. Formed elements (45 %): a. Red blood cells, RBC (erythrocytes) -99% of formd. Elmts. b. Platelets (thrombocytes) c. White blood cells, WBC (leukocytes) THE COMPONENTS OF BLOOD Blood is an opaque, red liquid consisting of several types of cells suspended in a complex, amber fluid known as plasma. When blood is allowed to clot or coagulate, the suspending medium is referred to as serum. Note that RBC constitute more than 99% of formed elements.

Blood Composition Blood Plasma HEMATOCRIT (Htc)
Note that hematocrit is also known as Packed Cell Volume (PCV) or Erythrocyte Volume Fraction (EVF)

Composition of Blood Plasma
Water Amino acids Albumin ions Globulin Proteins Fibrinogen Organic molecules Glucose such as is composed of PLASMA Lipids Trace elements and vitamins Nitrogenous wastes CO2 such as Gases O2 Figure 16-1 (1 of 2)

BLOOD PLASMA Physico-chemical properties Temperature: 38oC
pH: 7.35 (venous) – 7.45 (arterial) Relative whole blood viscosity - the internal friction of the blood (viscosity of water is 1.0): 3.5 – 5.4 Relative plasma viscosity: 1.9 – 2.6 Erythrocyte sedimentation rate- 2 to 8 mm/hr. Composition Water: 90-92% Functions Solvent & suspending medium for blood components Absorbs, transports and release heat Electrolytes (Na+, K+, Ca2+, Fe3+, Mg2+, Cl-, HCO3-, HPO42-, SO42-etc.) dissolved in water: 1% Create & maintain plasma osmotic pressure mOsm/L Essential role in cell functioning (i.e., electrical properties of the blood cells) Maintenance of the acid-base homeostasis*- is a steady state that provides an optimal internal environment for cell function Plasma Contains Many Important Solutes Plasma is composed mostly of water (93%) with various dissolved solutes, including proteins, lipids (fats), carbohydrates, amino acids, vitamins, minerals, hormones, wastes, cofactors, gases, and electrolytes. The solutes in plasma play crucial roles in homeostasis, such as maintaining normal plasma pH and osmolality

BLOOD PLASMA: COMPOSITION (cont.)
Organic substances (plasma proteins, nutrients, metabolites and waste products, regulatory substances, etc.) – 7-9% Plasma proteins – proteins confined to the blood Nutrients - products of digestion (AA, glucose, FA, glycerol, vitamins, minerals) Are transported by the blood for distribution in the body Waste products – breakdown products of protein metabolism (i.e., urea, uric acid, creatine, creatinine, bilirubin and ammonia) Are transported by the blood for excretion from the body Regulatory substances (i.e., hormones, enzymes, vitamins) Dissolved gases: O2, CO2, N2 Note that inorganic electrolytes and the proteins are functional elements of the plasma. Another group of plasma components (nutrients, vitamines, trace elements, hormones, enzymes, substances to be excreted, products of metabolism) are simply transported by the blood and under normal conditions they have little effect on physico-chemical properties of the plasma.

PLASMA PROTEINS 7-9% of the plasma, 65-90 g/L
Are synthesized by the liver (with the exception of γ-globulins) Fractions Albumins – plasma concentration – 45 g/L Globulins (α, β, γ) – 27 g/L Fibrinogen – 3 g/L Albumins have the smallest molecular mass whereas fibrinogen is the largest The most charged proteins Note that plasma protein deficiency is associated with edema – accumulation of fluid in the tissues. Note that binding of substances to plasma proteins prevents their rapid clearance from the blood. Note that the contribution of plasma proteins and blood cells to the total viscosity of the whole blood is almost equal. The least negatively-charged serum proteins

GENERAL FUNCTIONS OF PLASMA PROTEINS
Create colloid osmotic pressure (25 mmHg) → retaining water within the capillaries Binding and transport of hormones, enzymes, lipids, vitamins, metals, bilirubin, drugs, etc. Contribution to the blood viscosity Buffer properties – capability of accepting both H+ and base ions Protection of body against microorganisms and toxic substances Mediate blood coagulation Precursors of some hormones (angiotensinogen, erythropoietin) Protein reserve – source of AA for tissues in case of starvation

PROPERTIES & FUNCTIONS OF VARIOUS FRACTIONS OF PLASMA PROTEINS
Albumins 60% of the total plasma protein High concentration and small size → 80% of the total colloid osmotic P of the plasma 1 globulins (glycoproteins) Transport of glucose and lipids Include anti-protease 2 globulins Carriers for different substances (high affinity, low binding capacity) Ceruloplasmin – copper Thyroxin-binding protein Transcobalamin – Vit B12 Bilirubin binding globulin Transcortin – cortisol, etc.

PROPERTIES & FUNCTIONS OF VARIOUS FRACTIONS OF PLASMA PROTEINS (cont.)
-globulins Carriers for lipids (lipoproteins), polysaccharides and metals (i.e., transferrin – iron and cupper) -globulins Are immunoglobulins (antibodies) Quantity and composition fluctuate ↑ in almost all diseases (inflammation and infections) Fibrinogen A dissolved precursor of fibrin – blood clotting Serum – plasma without fibrinogen (and clotting factors)

HYPOPROTEINEMIA ↓ blood level of proteins Results from Malnutrition
Liver diseases (depression of protein synthesis) Intestinal disease (malabsorption) Kidney diseases (lost of albumins in urine) Results in ↓ plasma oncotic pressure (especially due to ↓ albumin concentration) and edema formation Depression of specific functions (i.e., ↓ in globulins – ↓ resistance to infections, ↓ in fibrinogen – defective blood clotting)

NON-PROTEIN NITROGEN OF THE PLASMA
Refers to nitrogen-containing substances other than proteins and AA (urea, uric acid, creatinine) ↑ in deranged kidney function

Plasma Proteins

Some components of Plasma
Class Substance Normal Concentration Range Cations Sodium (Na+) 136 – 145 mEq/L Potassium (K+) 3.5 – 5.0 mEq/L Calcium (Ca2+) 4.3 – 5.2 mEq/L Magnesium (Mg2+) 1.5 – 2.0 mEq/L Iron (Fe2+) 50 – 170 ug/dL Copper (Cu2+) 70 – 155ug/dL Hydrogen (H+) 35 – 45 mmoL/L Anions Chloride (CL-) 95 – 105 mEq/L Bicarbonate (HCO-3) 22 – 26 mEq/L Lactate 0.67 – 1.8 mEq/L Sulfate (SO42-) 0.9 – 1.1 mEq/L Phosphate 3.0 – 4.5 mg/dL

Class Substance Normal Concentration Range Protein Total 6 – 8 g/dL Albumin 3.5 – 5.5g/dL Globulin 2.3 – 3.5 g/dL Fats Cholesterol mg/dL Phospholipids 150 – 220 mg/dL Triglycerides 35 – 160 mg/dL Carbohydrates Glucose 70 – 110 mg/dL Vitamins Vitamin B12 200 – 800 ug/mL Vitamin A 0.15 – 0.6 ug/mL Vitamin C 0.4 – 1.5 mg/dL 2,3 DPG 3 – 4 mmo/L Transaminase (SGOT) 9 – 40 U/mL Alkaline phosphatase 20 – 70 U/mL Acid phosphatase 0.5 – 2 U/L

Class Substances Normal Concentration Range Other Substances Creatinine 0.6 – 1.2 mg/dL Uric Acid 0.18 – 0.49 mmo/L Blood Urea Nitrogen (BUN) 7 – 18 mg/dL Iodine 3.5 – 8.0 ug/dL CO2 23 – 30 mmol/L Bilirubin (total) 0.1 – 1.0 mg/dL Aldosterone 3 – 10nf/dL Cortisol 5 – 18 ug/dL Ketones 0.2 – 2.0 mg/dL

HEMATOCRIT (Htc)-Important Diagnostic Measurement
Is the fraction of the blood volume made up of the formed elements (mainly RBC) Is determined by the centrifuging heparinised/anticoagulated blood in a standard calibrated tube of a small diameter The contribution of the WBC to hematocrit is only 0.08%. WBCs are lighter than the RBCs, they form a thin whitish layer between the sedimented RBCs and the plasma. When blood is allowed to clot or coagulate, the suspending medium is referred to as serum

(Htc)-Important Diagnostic Measurement
Plasma (55% of whole blood) Buffy coat: Leucocytes and Platelets <1% of whole blood Formed Elements Erythrocytes (45% of whole blood)

HEMATOCRIT Values Males: 40 – 54 vol% (mean – 47%; 0.47)
Determination of hematocrit values is a simple and important screening diagnostic procedure in the evaluation of hematological disease Values Males: 40 – 54 vol% (mean – 47%; 0.47) Females: 38 – 46 vol% (mean – 42%, 0.42) ↑ in persons leaving at high altitudes, in dehydrated state, polycythemia, etc. ↓ in anemia, leukemia, bone marrow failure Importance Determines blood viscosity ↑ Htc → ↑ resistance to blood flow, load on the heart & BP Males have higher values of Htc due to testosterone which stimulates RBC production. Lower values of Htc in females of reproductive age may be also due to blood loss during menstruation. The contribution of the WBC to hematocrit is only 0.08%. WBCs are lighter than the RBCs, they form a thin whitish layer between the sedimented RBCs and the plasma.

Circulating Blood Cell Levels
Blood Cell Type Approximate Normal Range Erythrocytes (cells/uL) Men 4.3 – 5.9 x 1006 Women 3.5 – 5.5 x 1006 Leukocytes 4,500 – 11,000 Neutrophils 4,000 – 7,000 Lymphocytes 2,500 – 5,000 Monocytes 100 – 1,000 Eosinnophils 0 – 500 Basophils 0 – 100 Platelets 150,000 – 400, 000

HEMATOCRIT Tube A Tube B Tube C Normal Anemia Polycythemia

Blood composition: summary

BLOOD 2 RED BLOOD CELLS JAUNDICE ANEMIA & POLYCYTHEMIA

CONTENT RED BLOOD CELLS (RBC) COUNT, FUNCTIONS, STRUCTURE
HEMOGLOBIN (Hb): CHEMISTRY, REACTIONS, FUNCTIONS, CONCENTRATION ERYTHROPOIESIS, CONTROL OF ERYTHROPOIESIS DESTRUCTION OF RBC, METABOLISM OF Hb AND IRON. HEMOSIDEROSIS JAUNDICE ERYTHROCYTE SEDIMENTATION RATE TYPES OF ANEMIA, SICKLE CELL DISEASE POLYCYTHEMIA

OBJECTIVES Describe the functional consequence of the lack of a nucleus, ribosomes, and mitochondria for a) protein synthesis and b) energy production within the red blood cell. Relate the three red blood cell concentration estimates, red blood cell count, hematocrit, and hemoglobin concentration. Know the importance of MCV and be able to calculate the mean corpuscular volume. Describe the structure of hemoglobin (Hb). Describe the differences between the major normal types of Hb (adult A and A2, glucosilated, fetal). Predict the changes in Hb types present in blood when synthesis of beta chains of globin is deficient. Describe the abnormal types of Hb (Hb S, thalassemias). Describe the normal and abnormal Hb reactions (oxyHb, MetHb, carboxyHb). Calculate the mean corpuscular Hb concentration and the mean corpuscular Hb. Identify the site of erythropoietin production, the adequate stimulus for erythropoietin release, and the target tissue for erythropoietin action. Describe the role of vitamin B12 & folic acid, and various hormones in regulation of RBC formation. Describe the dietary requirements for RBC production. Relate the rate of red blood cell production and the percentage of immature reticulocytes in the blood. Describe the metabolism of iron in the body. Describe the metabolism of Hb (pre-hepatic, hepatic, post-hepatic). Describe the three types of jaundice (pre-hepatic, hepatic and post-hepatic). Compare and contrast the laboratory findings and urine/stool color in the three types of jaundice. Describe physiological jaundice of the newborn. Discuss the normal balance of red blood cell synthesis and destruction, including how imbalances in each lead to anemia or polycythemia. Compare and contrast the main types of anemia (nutritional, hemolytic, aplastic, hemorrhagic). Be able to describe different types of anemia in terms of MCV and MCHC. Describe the main effects of anemia and polycythemia on body functions.

Blood Cells red blood cells (erythrocytes)
white blood cells (leucocytes) platelets (thrombocytes).

Blood Cells Lymphocytes Mnoocytes White Cellular blood cells elements
Neutrophils Eosinophils Basophils

RBC: Functions Transport of O2 from the lungs to the tissues and CO2 in the opposite direction Hemoglobin Carbonic anhydrase Catalyses the reaction H2O + CO2 ↔ H2CO3 Maintenance of pH homeostasis (globin, phosphate and bicarbonate buffers)-hemoglobin in the cells is an excellent acid-base buffer Contribution to the blood viscosity ↓ blood oncotic P (by keeping Hb-protein inside the cells)

RBC COUNT Normal values
Adult males: – /mm3 (5.4million/mL) Adult females: – /mm3 (4.8million/mL) Abnormally high count – polycythemia Abnormally low count – anemia

STRUCTURE OF THE MATURE RBC
Small size Excess of the plasma membrane & specific shape RBC - biconcave discs with central depression on each side High surface-to-volume ratio Deformation of the cells without stretching the plasma membrane Rapid diffusion of respiratory gases to and from the cell Easy passage through the small capillaries The RBC is a bag, which can be deformed into almost any shape.

Red Blood Cells Figure 16-5

STRUCTURE OF THE MATURE RBC (cont.)
Membrane contains special proteins and polysaccharides that differ from person to person – blood groups Lack of the nucleus and organelles Cannot undergo mitosis Generate ATP anaerobically → do not use oxygen they transport Can not synthesize new cellular components to replace damaged ones Life span days Contain a red pigment, hemoglobin (red color of the blood) Occupies 1/3 of cellular volume 280 million Hb molecules/RBC

MEAN CORPUSCULAR VOLUME
MCV: fL Mean volume of a RBC Values Normal range 82 – 99 femtolitre (fL) Low volume in microcytic anemia High volume in macrocytic anemia Calculation of the MCV Hematocrit x 10 RBC count (in millions/mL blood) fL= L Sample calculation: Htc = 40, RBC count = 5 (x 106/mL) MCV = (40 x 10)/5 = 80 fl

RBC Morphology In a normal individual RBCs show minimal anisocytosis(Excessive variation in the size of cells )and poikilocytosis(irregularly shaped erythrocytes). Larger than average RBCs are macrocytic (left), while those smaller than average are microcytic (right).

Pale cells (central pallor >1/3 dia) are referred to as hypochromic
(right), while cells without central pallor are called hyperchromic (left). Normal peripheral blood RBCs are normochromic normocytic.

RBC Morphology while cells without central pallor are called
Normal peripheral blood RBCs are normochromic normocytic. Pale cells (central pallor >1/3 dia) are referred to as hypochromic while cells without central pallor are called hyperchromic

RBC Morphology Abnormality Associated condition(s) Target Cells
Sickle-cell/Thalassemia Iron-deficient anemia Hyposplenism Liver disease Tear-drop poikilocytes Myelofibrosis Spherocytes Hereditary spherocytosis Autoimmune hemolytic anemia Basophilic stippling Lead poisoning Thalassemia Howell-Jolly bodies Heniz bodies G6PD deficiency Alpha-thalassemia Schistocytes (“helmet cells”) Intravascular hemolysis Mechanical heart valve Disseminated intravascular coagulation ‘Pencil’ poikilocytes Iron deficiency anemia Burr cells (echinocytes) Uremia Pyruvate kinase deficiency Pappenheimer bodies

Target cells Target cells (codocytes or leptocytes) have a "lump" of hemoglobinized cytoplasm within the area of normal central pallor, causing them to resemble a "bullseye" target

Tear-drop poikilocytes
An abnormally shaped red blood cell with a single point or elongation.

Spherocytes Spherocytes are red cells which have assumed the form of a sphere rather than the normal discoid shape. As a result, they appear on routine blood films as cells that are smaller and more dense than normal red cells of the species, and have a reduced area of central pallor

Basophilic stippling Basophilic stippling of erythrocytes (BSE) represents the spontaneous aggregation of ribosomal RNA in the cytoplasm of erythrocytes

Howell-Jolly bodies Howell-Jolly bodies in the blood of a (non-anemic) splenectomized dog. Howell-Jolly bodies (H-J) bodies are small fragments of non-functional nucleus which were not extruded as the cell left the marrow

Heniz bodies Heinz bodies are inclusions within red blood cells composed of denatured hemoglobin.

Schistocytes Schistocytes, or red cell fragments, are generally taken to reflect mechanical injury to erythrocytes. A wide variety of forms may be observed.

Burr cells (echinocytes)
Burr cells (echinocytes) are spiculated RBCs. The term crenation is also used to refer to cells of this type. The projections of the cell membrane may be sharp or blunt, are usually numerous, and tend to be evenly spaced around the circumference.

Pappenheimer bodies Pappenheimer bodies are basophilic erythrocytic inclusions that are usually located at the periphery of the cell.

HEMOGLOBIN: Chemistry
Protein – globin 4 polypeptide chains Normal adult Hb – HbA, Hbα2β2 A pair of α chains (141 AA) A pair of β chains (146 AA) Adult Hb – HbA2 (2.5% of Hb), Hbα2δ2 β chains are replaced by δ chains Fetal Hb – HbF, Hbα2γ2 β chains are replaced by γ chains (146 AA) Adult Hb glucosilated – HbA1c Has a glucose attached to each β chain (4% - 5.9%, 6.5%) Nonprotein pigment bound to each of the 4 chains – hem Each hem ring has 1 iron ion (Fe2+) that can combine reversibly to 1 O2 molecule Each Hb molecule can bind 4 O2 molecules Adult Hb – HbA, Hbα2β2 red, oxygen-carrying pigment in the red blood cells of vertebrates is hemoglobin, a protein with a molecular weight of 64,450. Hemoglobin is a globular molecule made up of four subunits (Figure 32–6). Each subunit contains a heme moiety conjugated to a polypeptide. Heme is an iron-containing porphyrin derivative (Figure 32–7). The polypeptides are referred to collectively as the globin portion of the hemoglobin molecule. There are two pairs of polypeptides in each hemoglobin molecule. In normal adult human hemoglobin (hemoglobin A), the two polypeptides are called chains, each of which contains 141 amino acid residues, and chains, each of which contains 146 amino acid residues. Thus, hemoglobin A is designated 22.,Formation of glucosilated Hb increases in untreated diabetes mellitus → abnormal dissociation of HbO2 and tissue hypoxia. Hb A 2α 2β HbA2 2δ HbF 2γ

SICKLE CELL DISEASE Inherited disease
High prevalence in the malaria belt Mutation causes formation of HbS instead of HbA HbS precipitates into long crystals when oxygen tension is low (hypoxia) → cell elongation (sickling) and damage to the cell membrane → hemolysis → hypoxia (vicious cycle) Rigid sickled RBCs occlude the microvasculature leading to vaso-occlusive crisis. HbS – HbαA2βS2 The production of each type of globin chain is controlled by an individual structural gene with five different loci. Mutations, which can occur anywhere in these five loci, have resulted in the production of over 550 types of abnormal hemoglobin molecules, most of which have no known clinical significance. Mutations can arise from a single substitution within the nucleic acid of the gene coding for the globin chain, a deletion of the codons, or gene rearrangement as a result of unequal crossing over between homologous chromosomes. Sickle-cell anemia, for example, results from the presence of sickle-cell hemoglobin (HbS), which differs from normal adult hemoglobin A because of the substitution of a single amino acid in each of the two chains. -Note. The gene responsible for sickle cell disease alters the permeability of the RBC membrane → ↑ leakage of K+ from the RBC →↑ resistance to malaria parasites. Negatively charged glutamate is substituted for nonpolar valine at position 6 in the β chain)

HEMOGLOBIN: Reactions
Oxyhemoglobin: Hb + 4 O2 (O2 attaches to Fe2+ in hem) Is produced in the lungs (oxygen loading) Reduced Hb (deoxyHb) Is produced in tissue capillaries after dissociation of O2 (oxygen unloading) Combines with H+ - acts as a buffer Combines with CO2 → Carbaminohemoglobin: Hb + CO2 (CO2 binds to globin, not to hem) OxyHb O2 carrying function CO2 carrying function Buffering function Oxyhemoglobin (HbO2), the oxygen-saturated form of hemoglobin, transports oxygen from the lungs to tissues, where the oxygen is released. When oxygen is released, HbO2 becomes reduced hemoglobin (Hb). While oxygensaturated hemoglobin is bright red, reduced hemoglobin is bluish-red, accounting for the difference in the color of blood in arteries and veins. The affinity of hemoglobin for O2 is affected by pH, temperature, and the concentration in the red cells of 2,3-bisphosphoglycerate (2,3-BPG). 2,3-BPG and H+ compete with O2 for binding to deoxygenated hemoglobin, decreasing the affinity of hemoglobin for O2 by shifting the positions of the four peptide chains (quaternary structure). COOH/COO- NH-COO- NH2/NH3+

HEMOGLOBIN (cont) Oxyhemoglobin (HbO2), the oxygen-saturated form of hemoglobin, transports oxygen from the lungs to tissues, where the oxygen is released. When oxygen is released, HbO2 becomes reduced hemoglobin (Hb). While oxygensaturated hemoglobin is bright red, reduced hemoglobin is bluish-red, accounting for the difference in the color of blood in arteries and veins. The affinity of hemoglobin for O2 is affected by pH, temperature, and the concentration in the red cells of 2,3-bisphosphoglycerate (2,3-BPG). 2,3-BPG and H+ compete with O2 for binding to deoxygenated hemoglobin, decreasing the affinity of hemoglobin for O2 by shifting the positions of the four peptide chains (quaternary structure).

HEMOGLOBIN: Reactions (cont.)
Methemoglobin (MetHb): Hb iron is oxidized from the ferrous (Fe2+) to the ferric state (Fe3+) Is incapable of carrying O2 and has a bluish color → cyanosis Limited amount of metHb can be converted back to Hb by methemoglobin reductase present in the RBCs In normal state, 1.5% of Hb is in MetHb state Methemoglobinemia: Met-Hb > 1.5% (results from oxidation by nitrates, drugs like phenacetin or sulfonamides and congenital deficiency of methemoglobin reductase). Carboxyhemoglobin: Hb + CO(carbon monoxide) → cherry-red color of the skin and mucous membranes CO has times the affinity to Hb as does O2 → HbCO is a very stable molecule CO ↓ the functional Hb concentration HbCO is unavailable for O2 transport → CO poisoning, acute onset anemia Certain chemicals readily block the oxygen-transporting function of hemoglobin. For example, carbon monoxide (CO) rapidly replaces oxygen in HbO2, resulting in the formation of the stable compound carboxyhemoglobin (HbCO). The formation of HbCO accounts for the asphyxiating properties of CO. Nitrates and certain other chemicals oxidize the iron in Hb from the ferrous to the ferric state, resulting in the formation of methemoglobin (metHb). MetHb contains oxygen bound tightly to ferric iron; as such, it is useless in respiration. Cyanosis, the darkblue coloration of skin associated with anoxia, becomes evident when the concentration of reduced hemoglobin exceeds 5 g/dL. Cyanosis may be rapidly reversed by oxygen if the condition is caused only by a diminished oxygen supply. However, cyanosis caused by the intestinal absorption of nitrates or other toxins, a condition known as enterogenous cyanosis, is due to the accumulation of stabilized methemoglobin and is not rapidly reversible by the administration of oxygen alone.

HEMOGLOBIN: Reactions (cont.)
Certain chemicals readily block the oxygen-transporting function of hemoglobin. For example, carbon monoxide (CO) rapidly replaces oxygen in HbO2, resulting in the formation of the stable compound carboxyhemoglobin (HbCO). The formation of HbCO accounts for the asphyxiating properties of CO. Nitrates and certain other chemicals oxidize the iron in Hb from the ferrous to the ferric state, resulting in the formation of methemoglobin (metHb). MetHb contains oxygen bound tightly to ferric iron; as such, it is useless in respiration. Cyanosis, the darkblue coloration of skin associated with anoxia, becomes evident when the concentration of reduced hemoglobin exceeds 5 g/dL. Cyanosis may be rapidly reversed by oxygen if the condition is caused only by a diminished oxygen supply.However, cyanosis caused by the intestinal absorption of nitrates or other toxins, a condition known as enterogenous cyanosis, is due to the accumulation of stabilized methemoglobin and is not rapidly reversible by the administration of oxygen alone.

HEMOGLOBIN: Concentrations
Concentration per unit volume of whole blood Mean corpuscular Hb concentration - concentration of Hb per unit packed cell volume MCHC = Hb amount / Volume of packed RBC Hb concentration = Hb amount (g)/Volume of whole blood (dL, L) Plasma Calculation: MCHC = Hb concentration x 100 Htc Sample calculation: [Hb] = 14.5 g/dL, Htc = 45 mL/dL MCHC = (14.5/45) x 100 = 32.2 g/dL packed cells Males – 16.0±2.0 g/dL Females – 14.0±2.0 g/dL RBC Normal range: 31-37 g/dL packed cells ↓ value – hypochromia (i.e., Hb deficiency) ↑ value – hyperchromia (i.e., spherocytosis)

Hb CONCENTRATION: Mean corpuscular Hb (MCH)
Is the total Hb content of a RBC Values Normal range – pg ↓ value – hypochromia (i.e., iron deficiency anemia) ↑ value – hyperchromia (i.e., vit B12 deficiency) Calculation MCH = Hb in grams/100 mL blood x 10 RBC count in million/L blood Sample calculation: [Hb] = 12 g/dL, RBC count = 4 x 106/mL MCH = 12/4 x 10 = 30 pg MCH

RBC CHARACTERISTICS: SUMMARY

Erythropoiesis Concept: The production of new red blood cells to replace the old and died ones In the adult, all the red cells are produced in bone marrow

Erythropoiesis- Pluripotent stem cells
in the bone marrow can produce any type of blood cells. is capable of both self-replication and differentiation to committed precursor cells that can produce only a specific cell line. CFU：colony-forming unit

Erythropoiesis-CPU-E
the committed red cell precursor undergoes several divisions. The daughter cells becomes progressively smaller, the cytoplasm changes color from blue to pink as hemoglobin is synthesized, the nucleus becomes small and dense and then extruded. Proerythroblast (Pronormoblast) Basophilic Normoblast Polychromatophilic Orthochromatophilic Reticulocyte Erythrocyte Early Intermediate Late

Erythropoiesis-CPU-E
The resulting non-nucleated cells is termed a reticulocyte since it still contains RNA. Within a few days of entering the circulation, the reticulocytes lose their RNA and becomes mature red cells Proerythroblast (Pronormoblast) Basophilic Normoblast Polychromatophilic Orthochromatophilic Reticulocyte Erythrocyte Early Intermediate Late

Regulation of Erythropoiesis
A. Erythropoietin, a glycoprotein released predominantly from the kidneys in response to tissue hypoxia. also produced by reticuloendothelial system of the liver and spleen. Effect: a, Stimulates the proliferation and differentiation of the committed red cell precursor b, Accelerates hemoglobin synthesis c, Shortens the period of red cell development in the bone marrow.

CONTROL OF ERYTHROPOIESIS: Hypoxia
Hypoxia stimulates production of EPO by the kidneys - the tubular epithelial cells and juxtaglomerular cells (90% of EPO) & the liver Biological effects of EPO: 1. ↑ production of proerythroblasts from hematopoietic stem cells 2. ↑ speed of erythropoietic stages ↑ ↑ ↓ Tissue oxygenation is the most powerful regulator of the RBC production (but not the RBC count in the blood) Note. The liver is an important source of EPO in the fetus and neonates but it is less sensitive to hypoxia than kidneys → smaller response to hypoxia. Erythropoietin is a very powerful stimulator of the RBC production. A significant increase in the erythropoietin secretion can rise the rate of RBC production 10 or more times.

ERYTHROPOIESIS Morpho-functional changes (proerythroblast → RBC)
Appearance of Hb Some Hb is present in the early erythroblasts Late erythroblasts are saturated with Hb Degeneration of the cell organelles Progressive ↓ in the cell size Degeneration of the nucleus Starts in the late erythroblast stage Disappeared by the reticulocyte stage Note that mature RBCs do not have nucleus and organelles. Reticulocytes contain remnants of RNA and cell organelles – reticulum or network of black spots in the cytoplasm. Reticulocytes enter the blood and within 1-2 days develop into mature RBC. Only mature RBC and reticulocytes are present in the blood

RETICULOCYTES & ERYTHROPOIESIS RATE
Normal reticulocytes count in the blood 1-4% of the circulating RBC in adults 2-6% in newborns ↑ reticulocytes count – indicator of rapid RBC production (i.e., hypoxia, hemorrhage, stress, effective therapy of anemia) ↓ reticulocytes count - ↓ erythropoiesis (↓ EPO production, ↓ ability of red bone marrow to respond to EPO, nutritional anemia, etc.)

CLINICAL FOCUS: BLOOD DOPING AND EPO
Beneficial effects of EPO ↑ RBC count and O2 carrying capacity of the blood → ↑ O2 delivery to tissues, ↑ muscular performance, ↓ muscular fatigue Recombinant EPO (rhEPO) is used for treatment of anemias associated with chronic renal failure, AIDS and cancer chemotherapy Dangers of excessive EPO Genetically engineered EPO (i.e., darbepoetin) has increased life time ↑ Htc → ↑ blood viscosity, ↑ peripheral resistance, ↑ blood pressure, ↓ heart rate (secondary to increased blood pressure), ↑ blood clotting Genetically engineered EPO often cause production of antibodies against natural EPO and destruction of the RBC

CONTROL OF ERYTHROPOIESIS: Vitamin B12 and folic acid
Are required for maturation of the RBC ↑ Synthesis of DNA (synthesis of thymidine triphosphate – DNA building block) → rapid proliferation of the erythroblastic cells Vitamin B12 (cyanocobolamin) Is required for action of folic acid on erythropoiesis Dietary B12 Parietal/oxyntic cells of gastric mucosa produce intrinsic factor (IF) B12+IF B12 binds with the IF – protection from digestion by GIT secretions Complex of Vit B12 +IF complex binds to the mucosal receptors in the ileum → transport across mucosa Dietary deficiency of the Vit B12 occurs partially in vegetarians because vegetables and fruits contain very little vitamin B12. Folic acid is easily destroyed during cooking. Note that macrocytes have normal oxygen carrying capacity. Release of B12 into the portal blood freed of IF Binding to the plasma globulins (transcobolamin I, II and III) → red bone marrow or storage in the liver (very large quantities – 3-4 years reserve)

CONTROL OF ERYTHROPOIESIS: Other factors
Testosterone Stimulates the release of EPO Adrenal cortical steroids and ACTH In physiological concentrations stimulate EPO production Large doses are inhibitory

DESTRUCTION OF THE RBC Sites of destruction Senescent RBC
Circulating blood (10% of senescent RBCs) Macrophage system (spleen and liver) Senescent RBC ↓ metabolic rate → ↑ fragility → rupture of the membrane when RBC pass through tight spots of the circulation (i.e., red pulp of the spleen)

METABOLISM OF Hb Prehepatic Takes places in the macrophages
Results in formation of bilirubin – a bile pigment Hepatic Takes place in the liver (hepatocytes) Conjugation of bilirubin with glucuronic acid – bilirubin mono- or bi-glucuronide and secretion of conjugated bilirubin into the bile Posthepatic Takes place in the GI and kidneys Formation of urobilinogen and stercobilinogen and excretion

PREHEPATIC METABOLISM OF Hb
RBC or remnant Macrophages Hemoglobin Cell remnant Hem Globin Pigment Fe++ CO Biliverdin Bilirubin Exhaled BLOOD Albumin Bilirubin-albumin Fe++ Liver 73

HEPATIC & POSTHEPATIC METABOLISM OF BILIRUBIN
In the liver Replacement of albumin with glucuronic acid – bilirubin mono- or bi-glucuronide (water soluble) Excretion of conjugated bilirubin into the small intestine via the bile In the small intestine Conversion of bilirubin to urobilinogen by the intestinal bacteria Conversion to stercobilinogen → oxidation and excretion in the feces as stercobilin Absorption from the small intestine & either re-excretion by the liver or oxidation & excretion by the kidneys as urobilin. Transport of bilirubin from plasma into the hepatocytes Liver Glucuronic acid Albumin Bilirubin-glucuronide Urobilinogen (in the small intestine) Reabsorption Stercobilinogen Re-excretion in bile Excretion as urobilin in urine Excretion as stercobilin in feces

BILIRUBIN: Concentration in plasma
Concentration in plasma, mg/dL Free bilirubin = unconjugated bilirubin 0.1 – 1 Conjugated bilirubin 0 – 0.3 Total bilirubin 0.3 – 1.2

JAUNDICE Refers to the yellow color of the skin, conjunctivae and mucous membranes caused by the presence of excessive bilirubin in the plasma and body fluids (jaune (French) = yellow) Blood bilirubin level must exceed three times the normal values, for the coloration to be easy visible Types of jaundice: Pre-hepatic – the pathology occurs prior to the liver Hepatic – the pathology is located in the liver Post-hepatic – the pathology occurs after the conjugation of bilirubin in the liver

PRE-HEPATIC JAUNDICE N Urobilinogen (small intestine) Reabsorption
Cause: Excessive hemolysis of the RBCs – hemolytic jaundice Pigment Biliverdin Bilirubin Inc. bilirubin production ↑ unconjugated (indirect) bilirubin Blood Albumin Capacity of the liver to conjugate bilirubin is exceeded (saturation of enzyme glucuronyl transferase) Bilurubin-albumin Liver Normal conjugated (direct) bilirubin Bilurubin-glucuronide N Note that unconjugated bilirubin can enter the brain  toxic effect in neonates. The yellowish discoloration is less marked than in hepatic and posthepatic jaundice. In infants urobilinogen formation is not increased because gut flora is not developed. Urobilinogen (small intestine)  urobilinogen formation   urobilinogen → dark urine Reabsorption Stercobilinogen ↑ stercobilinogen → dark feces Excretion as stercobilin in feces Excretion as urobilin in urine Re-excretion in bile

HEPATIC JAUNDICE Results from infective or toxic damage to the liver cells (hepatocellular damage) Uptake, conjugation and/or excretion of bilirubin is affected ↑ unconjugated bilirubin Normal/decreased conjugated bilirubin ↑ urobilinogen in blood (↓ enterohepatic circulation and hepatic extraction of blood urobilinogen by damaged hepatocytes) In obstructive jaundice, serum level of cholesterol is also elevated. Patients often complain of severe itching or "pruritus". In posthepatic and hepatic jaundice fecal fat increases and liver function tests are impaired. ↑ urobilinogen filtration and excretion in urine Pale/N stool Dark urine

POSTHEPATIC JAUNDICE N ↓ or absent urobilin in urine
Results from obstruction of the bile ducts by stones, tumors, etc. Functioning of the hepatic cells is normal N Normal unconjugated bilirubin  plasma level of conjugated bilirubin due to the bile entry into the blood from ruptured congested canaliculi and ↑ total bilirubin  urobilinogen formation Conjugated bilirubin in urine (kidney can excrete small quantities of highly soluble conjugated bilirubin) → dark urine ↓ or absent urobilin in urine ↓ stercobilin content in feces → pale feces

PHYSIOLOGICAL JAUNDICE OF THE NEWBORN
Hemolysis of the excess RBC when the infant is suddenly exposed to a high oxygen environment and hence does not need so many RBC as in the uterus Immaturity of the liver (inability to conjugate significant quantities of bilirubin with glucuronic acid for excretion into the bile) to handle the excess bilirubin (especially in premature babies) ↑ plasma total bilirubin concentration (less than 1 mg/dL → 5 mg/dL) Mild jaundice (yellowness) of the infant’s skin and the sclerae for 1-2 weeks Is observed in 60% of normal babies during the first 1-2 weeks.

IRON METABOLISM Dissociation of Fe from the hem → plasma → binding to transferrin, transport in the blood → Detachment from transferrin & storage in the liver, muscle cells & macrophages attached to ferritin or hemosiderin → Release from the storage sites, transport in the blood by transferrin Transport into the RBC precursor cells by receptor mediated endocytosis → Hem synthesis 2 1 3 4 ↓ quantities of transferrin → ↓ Hb content in the RBC – hypochromic anemia Note that transferrin strongly binds to receptors on the erythroblasts plasma membrane and is transported inside the cell by endocytosis. In the cell the transferrin delivers iron to the site of hem synthesis – mitochondria. Note that intestinal absorption of non-hem iron is facilitated by ascorbic acid. Synthesis of transferrin increases with iron deficiency but decreases with any type of chronic disease.

FORMS OF IRON IN THE BODY
Recommended daily intake - 15 – 18 mg ( μmol) Minimal absorption to balance iron loss Adult males - 35 μmol Adult females μmol Distribution of body iron in an average man Hb, 2100 mg Ferritin - water soluble protein-iron complex , 700 mg (in the liver, spleen, marrow and plasma) Hemosiderin - water insoluble complex (macrophages of the liver and bone marrow), 300 mg Myoglobin - local oxygen reserve, 200 mg Tissue (heme and nonheme) enzymes, 150 mg Transport-iron compartment in plasma (transferrin), 3 mg. Note that ferrous iron (Fe2+) is absorbed much readily than ferric iron (Fe3+); reducing substances such as ascorbic acid facilitate iron absorption. Ferritin is also present in the plasma and its plasma concentration is an excellent indicator of the iron stored in the body, because of a dynamic balance, which exists between intra- and extracellular ferritin iron and iron used. Insoluble hemosiderin tends to form large clusters in the cells that can be observed microscopically.

HEMOCHROMATOSIS Reasons
Primary - one of the most common autosomal recessive genetic disorders characterized by excessive absorption of dietary iron resulting in a pathological increase in total body iron stores Failure to reduce iron reabsorption in response to increased iron level in the body Secondary – is not genetic (results from anemia, alcoholism, transfusion iron overload –hemosiderosis, etc.) Consequences Deposition of iron in the body tissues (liver, heart, pancreas, pituitary, joints, and skin) initially as ferritin and than as hemosiderin Toxic action on organs and damage of cells due to action as a pro-oxidant (↑ formation of free radical formation, i.e., the hydroxyl radical and the superoxide radical) → DNA cleavage, impaired protein synthesis, and impairment of cell integrity and cell proliferation, leading to cell injury and fibrosis. Cirrhosis, hyperpigmentation of skin, diabetes mellitus, impotence, joint diseases, etc. Normally only 10% of iron from diet is reabsorbed in the GI. In hemochromatosis, up to 30% of iron is reabsorbed. Over time, the patients absorb and retain between five to 20 times more iron than the body needs. Because the body has no natural way to rid itself of the excess iron, it is stored in body tissues, specifically the liver, heart, and pancreas.

ERYTHROCYTE SEDIMENTATION RATE (ESR)
Specific weight of the RBC is higher than that of the plasma  in a stabilized blood, RBC slowly sink towards the bottom of the test tube -sedimentation Factors increasing ESR ↓ Htc, ↓ blood viscosity ↑ concentration of fibrinogen (i.e., pregnancy, vascular diseases, heart diseases), haptoglobulin, lipoproteins, immunoglobulins Macrocytic RBC Extreme elevation of WBC count (leukemia) Factors decreasing ESR ↑ Htc Change in the RBC shape (i.e., sickle-cell anemia, poikilocytosis – nonuniformity of shape) ↑ albumin concentration Males – 3-6 mm/h Females – 8-10 mm/h ESR Clumps of RBCs Gathering of the RBCs into clumps increases ESR. Negative charge of the RBC membrane prevents clamps formation.

ANEMIA Deficiency of blood Hb due to
↓ RBC count (too rapid loss or/and too slow production) ↓ Hb quantity in the RBC WHO's Hemoglobin thresholds used to define anemia (1 g/dL = mmol/L) Age or gender group Hb threshold (g/dl) Hb threshold (mmol/l) Children ( yrs) 11,0 6,8 Children (5-12 yrs) 11,5 7,1 Children (12-15 yrs) 12,0 7,4 Women, non-pregnant (>15yrs) Women, pregnant Men (>15yrs) 13,0 8,1 Note that under normal conditions about 1% of the RBCs are destroyed daily and equal numbers are produced.

ANEMIA: CONSEQUENCES ↓ oxygen-carrying capacity of the blood → hypoxia → vasodilation ↑ in pulse and respiratory rates (effort to supply sufficient oxygen to tissues) ↓ exercise & cold tolerance Pale skin (↓ red colored oxyHb) ↑ fatigue and lassitude ↓ blood viscosity → ↓ peripheral vascular resistance → ↑ blood flow, venous return, cardiac output and work load on the heart Eyes - yellowing

ANEMIAS: Classifications
Classification according to etiological ground Nutritional Aplastic Hemorrhagic Hemolytic Anemia: classification according to MCV Macrocytic anemia (MCV>100) Normocytic anemia (80<MCV<100) Microcytic anemia (MCV<80) Deficiency of vit B12, folic acid, or IF. Hypothyroidism. Alcoholism. Liver diseases. Drugs that inhibit DNA replication (i.e., methotrexate, zidovudine) Hem synthesis defect (i.e., iron deficiency, chronic diseases) Globin synthesis defect (i.e., thalassemia) Sideroblastic defect Acute blood loss, chronic diseases, bone marrow failure, hemolysis Sideroblastic defect. The body has iron available, but cannot incorporate it into hemoglobin. This results in production of sideroblasts, which are nucleated erythrocytes with granules of iron in their cytoplasm.

ANEMIA: Nutritional Iron deficiency Is the most common type Reasons
Premenopousal women: Blood loss during menses (20% of all women of childbearing age have iron deficiency anemia, compared with only 2% of adult men) Males and postmenopausal females: Excessive iron loss due to chronic occult bleeding (peptic ulcer, tumor, etc.) Increased iron demands (i.e., pregnancy and lactation) Inadequate iron intake or absorption (i.e., vit. C deficiency) Parasitic infestation (hookworm, amebiasis, schistosomiasis) Chronic intravascular hemolysis (if the amount of iron released during hemolysis exceeds the plasma iron-binding capacity) Iron deficiency anemia develops slowly after the normal stores of iron have been depleted in the body and in the bone marrow. Women, in general, have smaller stores of iron than men and have increased loss through menstruation, placing them at higher risk for anaemia than men. In men and postmenopausal women, anaemia is usually due to gastrointestinal blood loss associated with ulcers or the use of aspirin or nonsteroidal anti-inflammatory medications (NSAIDs). Dietary sources of iron are red meat, liver, and egg yolks. Flour, bread, and some cereals are fortified with iron. If the diet is deficient in iron, iron should be taken orally. During periods of increased requirements such as pregnancy and lactation, increase dietary intake or take iron supplements. Oral iron supplements are in the form of iron salts (ferrous sulphate, gluconate, etc.) or saccharated iron . The best absorption of iron is on an empty stomach, but many people are unable to tolerate this and may need to take it with food. Milk and antacids may interfere with absorption of iron and should not be taken at the same time as iron supplements. Vitamin C can increase absorption and is essential in the production of haemoglobin. Parenteral iron causes the same therapeutic response as oral iron. It is reserved for patients who do not tolerate or who will not take oral iron or for patients who steadily lose large amounts of blood because of capillary or vascular disorders.

IRON DEFICIENCY ANEMIA: CONSEQUENCES
Low serum ferritin (serum iron) level Plasma ferritin concentration is an excellent indicator of the iron stored in the body, because of a dynamic balance between intra- and extracellular ferritin iron ↓ bone marrow iron stores (ferritin and hemosiderin) ↓ saturation of transferrin ↓ RBC count & Htc RBC are small and look pale - microcytic hypochromic anemia Abnormal fissuring of the angular (corner) sections of the lips (angular stomatitis). Abnormal craving to eat substances (eg, ice, dirt, paint).

DEFICIENCY OF IRON UTILIZATION: SIDEROBLASTIC ANEMIA
Inadequate marrow utilization of iron for Hb synthesis despite the presence of adequate or increased amounts of iron Reasons: Hereditary or acquired, including lead and ethanol toxicity, pyridoxine deficiency Deficient reticulocyte production, intramedullary death of RBCs, and bone marrow erythroid hyperplasia (and dysplasia) Presence of polychromatophilic, stippled RBCs (siderocytes) Hipochromic, microcytic RBCs, variations in RBC size Ring sideroblasts are erythroid precursors whose mitochondria (located around the nucleus) are loaded with nonheme iron.

ANEMIA: Nutritional (cont.)
Deficiency of vitamin B12 and/or folic acid Reasons Inadequate intake (a strict vegetarian diet excluding all meat, fish, dairy products, and eggs; chronic alcoholism) Inadequate GI absorption Lack of IF - pernicious anemia Autoimmune destruction of parietal cells (atrophic gastric mucosa) or AB against IF Removal of the functional portion of the stomach, such as during gastric bypass surgery Crohn's disease intestinal malabsorption disorders Resection (or inflammation) of the ileum (site of B12 reabsorption) Consequences Maturation failure Failure of DNA synthesis with preserved RNA synthesis, which result in restricted cell division of the progenitor cells. Production of large precursor cells – megaloblasts and larger irregular oval erythrocytes – macrocytes fully saturated with Hb – macrocytic (megaloblastic) anemia ↑ fragility of the plasma membrane → ↓ life span → anemia Vitamin B12 deficiency only results in peripheral neuropathy and spinal cord degeneration Dietary deficiency of the Vit B12 occurs partially in vegetarians because vegetables and fruits contain very little vitamin B12. Folic acid is easily destroyed during cooking. Note that macrocytes have normal oxygen carrying capacity.

ANEMIA: Hemorrhagic Results from abnormal blood loss (mild or severe; acute or chronic) Replacement of lost fluid within 1 – 3 days (much faster than the replacement of lost RBC) → dilution of the RBC Is normocytic Prolonged but mild loss of the blood causes microcytic hypochromic anemia (iron deficiency)

ANEMIA: Aplastic Results from suppression or destruction of the bone marrow (i.e., overexposure to ionizing radiation, adverse drug reaction, toxic chemicals, severe infections) Is usually normocytic Panhypoplasia of the marrow is associated with leukopenia and thrombocytopenia

ANEMIA: Hemolytic Spherocytosis
Is caused by an abnormally high rate of the RBCs destruction (hemolysis) due to: Structural abnormalities of the RBC (more fragile cells) Hereditary spherocytosis – cells are spherical and can not be compressed Sickle cell anemia – cells have sickle shape → hemolysis Bacterial toxins, parasitic infections (i.e., malaria) Adverse drug reactions Autoimmune reactions The bone marrow is unable to compensate for premature destruction of RBC by increasing their production. Thalassemias (α, β) Hereditary hemolytic anemia Abnormal or nonfunctional genes → globin chains are normal in structure but are produced in reduced amounts Cells are microcytic and hypochromic Spherocytosis

ANEMIA OF CHRONIC DISEASE
Occurs as part of a chronic disorder (i.e., infection, inflammatory disease, or cancer) Pathophysiologic mechanisms Shortened RBC survival ↓ EPO production and marrow responsiveness to EPO Impaired intracellular iron metabolism Is microcytic or marginal normocytic

POLYCYTHEMIA ↑ RBC count, Htc and Hb concentration Reasons
Hypoxic erythropoietic drive (i.e., high altitudes, chronic pulmonary or cardiac disease) Hemoconcentration - dehydration (i.e., heavy sweating, vomiting or diarrhea) Polycythemia vera or erythremia – uncontrolled RBC production (i.e., neoplastic disease condition of hemocytoblastic cells) Results in ↑ blood viscosity ↑ peripheral resistance → ↓ venous return to the heart ↑ blood volume tends to ↑ venous return ↑ arterial BP Ruddy skin and mucosa membranes with cyanotic tint (sluggish blood flow → ↑ blood deoxygenation in the skin circulation)

CLINICAL CASE A 14-year-old girl complained of fatigue and loss of stamina. Her appetite was marginal, as she was very conscious of maintaining her body weight at 96 pounds. Her monthly menstrual flow was always heavy and long from its onset at twelve years of age. Relevant laboratory findings included the following: Hematocrit (Hct) - 28% Hemoglobin (Hgb) - 9 g/dL Iron 16 µg/dL Bone marrow iron - absent Erythrocytes - small and pale Suggested treatment included ferrous sulfate or ferrous gluconate for six months orally between meals, since food may reduce absorption. A well-balanced diet was also suggested, as well as a gynecological examination. Questions. 1. What is the primary disorder of this individual? 2. What does the ferrous sulfate or ferrous gluconate provide? Why is it necessary? 3. What dietary inclusions would you suggest? 4. Why is the gynecological examination important? 5. Why is bone marrow iron an important clinical indicator in this individual?

PAST EXAMS QUESTION A 51-year old male complains of generalized weakness and weight loss over the past 6 months. His blood pressure and pulse rate are elevated. Laboratory values revealed a hematocrit of 35% and hemoglobin level of 10.9 g/dL. A blood smear shows hypochromic and microcytic cells. A stool test for occult blood is positive. Which of the following would be the most likely cause of the findings? a. Acute blood loss b. Iron deficiency c. Spherocytosis d. Folic acid deficiency B e. Autoimmune reactions

HEMOSTASIS Normal hemostasis is a consequence of tightly regulated process that maintain blood in a fluid state in normal vessels, yet also permit the rapid formation of a hemostasis clot at the site of a vascular injury.

Endothelium and Hemostasis
Endothelia cells are key players in regulation of hemostasis. Before injury, they exhibit: Antiplatelets – Production of Prostacyclin (PGI2) and Nitric Oxide Anticoagulant and Fibrinolytic properties They however acquire numerous pro coagulant activities as a result of injury.

Endothelium Antiplatelets effects
Endothelium produce Prostacyclin (PGI2) and Nitric oxide which impede platelets adhension It also elaborate adenosine diphosphatase which degrades adenosine diphosphate (ADP) and further inhibits platelets aggregation

Endothelium Anticoagulant effects
Endothelium inhibit coagulation via production of: Thombomodulin which binds to thrombin and converts it from procoagulant to an anticoagulant via its ability to activated Protein C which inhibits clotting by inactivating factors Va and VIIIa Protein S a cofactor for protein C, and tissue factor pathway inhibit (TFPI) a cell surface protein that directly inhibits tissue factor VIIa and factor Xa activities

Endothelium Fibrinolytic effects
Endothelia cells synthesize tissue type plasminogen factor (t-PA), a protease that cleaves plaminogen to form plasmin, plasmin in turn cleaves fibrin to degrade thrombi

Endothelium Prothrombotic Properties
As a result of trauma, inflammation and other factor, endothelia cells can induce a prothrombotic state via: Secretion of von Willebrand factor (vWF) Secretion of tissue factor (the major activator of the extrinsic clotting cascade) Section of inhibitor or plasminogen activator (PAIs) which limit fibrinolysis and tend to favor thrombosis

HEMOSTASIS The process of hemostasis can be divided into two distinct stages namely: Primary hemostasis – platelet plug formation Secondary hemostasis – coagulation cascade The goal of both primary and secondary hemostasis is to arrest bleeding from damaged blood vessels (hemo = blood, stasis = standing) Is counter-balanced by reactions, which prevent blood coagulation in uninjured vessels and maintain the blood in a fluid state Balance between procoagulants and anticoagulants 4 overlapping processes or stages Local vasoconstriction Formation of a platelet plug Formation of a web of fibrin proteins that penetrate and surround the platelet plug – blood coagulation or clotting Clot retraction. HEMOSTASIS Circulating in a high-pressure, closed system that communicates with all tissues and cells in the body, blood exchanges oxygen, nutrients, and wastes and provides necessary components for host defense. This communication takes place largely in the complex and dynamic networks of capillary beds that provide oxygen to almost every cell in the body (only the cornea and intervertebral disks are avascular; these tissues receive oxygen by diffusion). Disruption of the integrity of the fragile capillaries may result from minor tissue injury associated with normal physical activity or from massive tissue trauma as a result of serious injury or infection, and may quickly lead to death. Any opening in the vascular network may lead to massive bruising or blood loss if left unrepaired. To minimize bleeding and prevent blood loss after tissue injury, components of the hemostatic system are activated. The components of this dynamic, integrated system include blood platelets, endothelial cells, and plasma coagulation factors. They may be activated on exposure to foreign surfaces during bleeding, or by torn tissue at the site of injury, or by products released from the interior of damaged cells. Hemostasis can be viewed as four separate but interrelated events: • Compression and vasoconstriction, which act immediately to stop the flow of blood • Formation of a platelet plug • Blood coagulation • Clot retraction

LOCAL VASOCONSTRICTION
Results from Release of vasoconstrictor substances (paracrine & autocrine agents) from Platelets (i.e., serotonin & thromboxane A2) Traumatized tissue Local myogenic spasm initiated by direct tissue damage Reflex vasoconstriction initiated by activation of nociceptors and other sensory endings Effects ↓ blood flow and Pressure in the damaged area Last for many minutes or even hours, during this time the ensuing processes of platelet plugging and blood coagulation can take place Physical Factors Immediately Act to Constrain Bleeding Immediately after tissue injury, blood flow through the disrupted vessel is slowed by the interplay of several important physical factors, including compression or back-pressure exerted by the tissue around the injured area, and vasoconstriction. The degree of compression varies in different tissues; for example, bleeding below the eye is not readily deterred because the skin in this area is easily distensible. Back-pressure increases as blood which leaks out of the disrupted capillaries accumulates. In some tissues, notably the uterus after childbirth, contraction of underlying muscles compresses blood vessels supplying the tissue and minimizes blood loss. Damaged cells at the site of tissue injury release potent substances that directly cause blood vessels to constrict, including serotonin, thromboxane A2, epinephrine,

FORMATION OF A PLATELET PLUG (temporary hemostatic plug, white plug)
Factors that prevent/limit formation of a plug Intact blood vessel wall Damaged blood vessel wall Collagen fibers are exposed to the blood and coated with WF* 1. Prostacyclin (prostaglandin I2). Inhibits platelet aggregation; vasodilator - Adhesion of the platelets + - Platelet release reaction & activation Secretion of prostacyclin & nitric oxide 2. Nitric oxide* (NO). Inhibits platelet adhesion, activation and aggregation and stimulates local vasodilation + - Platelet aggregation & plug retraction Local vasoconstriction + stimulation - inhibition Temporary hemostatic (platelet) plug * Von Willebrand factor, a protein synthesized by endothelial cells and megakaryocytes, enhances platelet adherence by forming a bridge between cell surface receptors and collagen in the subendothelial matrix.

FORMATION OF A PLATELET PLUG (cont.)
Stage 1. Platelets adhesion A. vWF - von Willebrand factor (soluble plasma protein) binds to collagen of subendothelial matrix Failure of this step may be due to: - Absence of von Willebrand factor - Malfunction of collagen - Scurvy B. vWF exposes multiple intrinsic binding sites for the platelet specific membrane glycoprotein Ib (GPIb) vWF binds to glucoprotein Ib receptors of platelets and to collagen

Stage 2-3 Platelets release reaction and activation. Binding of the platelets to the collagen → Release of agents from secretory granules (degranulation) – serotonin, adrenaline, several clotting factors, thromboxane A2, tissue factor and ADP Serotonin, adrenaline and ADP act locally → changes in the metabolism, shape, and surface proteins of the platelets. Serotonin and thromboxane A2 stimulate local vasoconstriction

Primary Hemostatsis Deficiency: Bernard-Soulier syndrome
Step 1: Transient vasoconstriction (endothelin) Step 2: Platelet adhesion - von willebrand factor bind to the disrupted blood vessel via GpIb Step 3: Platelet release ADP and thromboxane A2 which stimulate adhesion of the next layers of platelets (recruitment) through a positive feedback mechanism and formation of a platelet plug inside the vessel ADP induces platelet to express GpIIB-IIIa which is needed to platelets aggregation via fibrinogen Step 4: Platelet aggregation (Platelt plug) Deficiency: Glanzmann thombasthenia GpIb Platelet GpIIb-IIIa complex Fibrinogen GpIb Endothelium Von willebrand factor Subendothelium

Stage 4: Recruitment and loose platelets aggregation Platelet Fibrinogen ADP and thromboxane A2 stimulate adhesion of the next layers of platelets (recruitment) through a positive feedback mechanism and formation of a platelet plug inside the vessel Deficiency : Glanzmann thombasthenia GpIIb-IIIa complex Platelets begin to swell, assume irregular form with numerous pseudopods. Contractions of the contractile proteins facilitate release of the secretory granules. Failure of this step: - Insufficient number of platelets - Dysfunctional platelets (prior activation occurs during cardiopulmonary bypass, storage, exposure to aspirin, uraemia and acute and chronic alcohol exposure) 111

Stage 5- irreversible platelet aggregation Destruction of the platelets membrane (stimulated by thrombin) → release of BAS from thrombocytes → secondary vasoconstriction Release of factor 3 (platelet thromboplastin) facilitates activation of blood coagulation Stage 6. Plug retraction Contraction of actin and myosin in the aggregated platelets → compression and strengthening of the platelet plug

Normal Blood Vessel

Exposed collagen binds and activates platelets
Injured blood vessel Attracts more platelets Aggregate into platelet plug Release of Platelet factors Exposed collagen binds and activates platelets

Secondary Hemostasis This involve the conversion of the fibrinogen (solube) in the platelet plug to fibrin (insoluble). Fibrin is then cross-linked to yield a stable platelet-fibrin thrombus. Secondary hemostasis involve the activation of coagulation cascade factors in both intrinsic and extrinsic pathways

BLOOD COAGULATION (CLOTTING)
Is the transformation of the blood into a solid gel (a clot or thrombus) Occurs locally around the platelet plug; supports and reinforces the plug Requires 12 plasma clotting factors and platelets Involves a cascade of biochemical reactions in which each factor that is activated in turn activates the next factor The fundamental reaction is conversion a soluble protein, fibrinogen to an insoluble protein, fibrin In coagulation a series of plasma proteins called blood-clotting factors play major roles. Most of these are inactive forms of proteolytic enzymes. When converted to the active forms, their enzymatic actions cause the successive, cascading reactions of the clotting process.

The two pathways Two separate coagulation cascades result in blood clotting in different circumstances. The two systems are: 1 The Intrinsic coagulation pathway (Contact activation pathway) The Extrinsic coagulation pathway (tissue factor pathway) However, the final steps in fibrin formation are common to both pathways. Phospholipids are required for activation of both coagulation pathways – provide a surface for the efficient interaction of several factors.

Clotting pathways In the intrinsic pathway, all the factors required for coagulation are present in the circulation. For the initiation of extrinsic pathway, a factor extrinsic to blood but released from injured tissue, called tissue thromboplastin or tissue factor (factor III), is required. The common pathway is initiated by the conversion of inactive clotting factor X to its active form factor Xa and results in the conversion of prothrombin to thrombin – calalysing the generation of fibrin

PLASMA CLOTTING FACTORS
Scientific Name Common Name Main Function Factor I Fibrinogen Converted to fibrin Factor II Prothrombin Enzyme Factor III Tissue thromboplasm Cofactor Factor IV Calcium Factor V Proaccelerin Factor VII Proconvertin Factor VIII Antihemophilic factor Factor IX Christmas factor Factor X Stuart factor Factor XI Plasma thromboplatin antecedent Factor XII Hageman factor Factor XIII Fibrin stabilizing factor

3 PHASES OF BLOOD COAGULATION
Formation of a complex of activated substances - prothrombinase (prothrombin activator) Formation of active thrombin from prothrombin Is catalyzed by prothrombin activator Formation of insoluble fibrin from soluble fibrinogen Is catalyzed by thrombin

PHASE 1 – FORMATION OF PROTHROMBINASE
INTRINSIC PATHWAY EXTRINSIC PATHWAY XII XIIa XIa III Ca2+ VIIa VII XI IX IXa PF-3 Ca2+ VIII Ca2+ Xa PF-3 Ca2+ V X X XIII II Thrombin XIIIa Fibrinogen Fibrin Stable fibrin polymer

PHASE 3 – FORMATION OF FIBRIN
Thrombin catalyses release of 2 pairs of polypeptides from each fibrinogen molecule and formation of fibrin monomers Ca++ and platelet factors are also required Monomers join together to form insoluble fibrin polymers – a loose mesh of stands Stabilization of fibrin – formation of covalent cross-bridges, which is catalyzed by factor XIII (+ Ca++) In the early stages of polymerization, monomers molecules are held together by weak non-covalent hydrogen bonds and fibers are not cross-linked with one another → weak reticulum of fibers can be easily broken.

FINAL EVENTS OF HEMOSTASIS
Fibrin forms a meshwork, which supports the platelet plug Clot occludes the damaged blood vessel and ↓ or stops bleeding Retraction of the clot due to contraction of fibrin fibers and contractile proteins of the platelets ↑ clot density Occlusion of the damaged vessel Bringing the edges of wound together → facilitation of wound heeling Fate of the blood clot Invasion by fibroblasts → formation of connective tissue through the clot Fibrinolysis and destruction of the clot Note that a clot consists of fibrin (the most essential component of the clot), platelets, RBC and WBC. Note that further heeling of the wound involves proliferation of fibroblasts, formation of connective tissue, scar formation and regeneration of the endothelium.

Overview of Hemostasis and Tissue Repair
Damage to wall of blood vessel Collagen exposed Tissue factor exposed Platelets adhere and release platelet factors Coagulation cascade Vasoconstriction Platelets aggregate into loose platelet plug Thrombin formation Converts fibrinogen to fibrin Temporary hemostasis Reinforced platelet plug (clot) Fibrin slowly dissolved by plasmin Cell growth and tissue repair Clot dissolves Intact blood vessel wall

ROLE OF VITAMIN K IN CLOTTING
Vitamin K acts as a cofactor of the enzyme γ-glutamyl carboxylase Is required for γ carboxylation in the liver of Prothrombin and factors VII, IX and X Proteins S and C (natural anticoagilants) Vit K is activated by epoxide reductase in the liver γ carboxylation (introduction of a carboxylic acid group) of certain glutamate residues in target clotting factors → binding sites for Ca++ and PF3 most of clotting factors are synthesized by the liver. Therefore, liver diseases (i.e., hepatitis, cirrhoses, atrophy) depress the clotting system. Decreased dietary intake of vit K has limited consequences on blood clotting because Vit K is continuously synthesized by the intestinal flora. Note that Vit K is fat soluble and requires fats for absorption. Lack of the bile decreases fat digestion and absorption. Note that most of clotting factors are synthesized by the liver. Therefore, liver diseases (i.e., hepatitis, cirrhoses, atrophy) depress the clotting system. Decreased dietary intake of vit K has limited consequences on blood clotting because Vit K is continuously synthesized by the intestinal flora. Note that Vit K is fat soluble and requires fats for absorption. Lack of the bile decreases fat digestion and absorption.

ROLE OF Ca++ IN COAGULATION
Ca++ is required for all steps of coagulation (except first 2 steps of the intrinsic pathway) ↓ in the plasma [Ca++] below the threshold level for clotting → ↓ blood clotting by both pathways

ROLE OF THE PLATELETS IN COAGULATION
Activated platelets Display specific plasma membrane receptors that bind several of the clotting factors → several cascade reactions take place on the surface of activated platelets Display phospholipids (platelet factors), which act as cofactors of the bound clotting factors

ROLE OF THE LIVER IN BLOOD COAGULATION
Synthesis of the plasma clotting factors Synthesis of the bile salts, which are required for intestinal absorption of lipid soluble vitamin K Note that most of clotting factors are synthesized by the liver. Therefore, liver diseases (i.e., hepatitis, cirrhoses, atrophy) depress the clotting system.

FIBRINOLYTIC SYSTEM Fibrinolysis - dissolution or disposal of blood clots Fibrin is digested by an enzyme, plasmin (fibrinolysin) into fibrin degradation products Plasmin also degrades factors Va, VIIIa and GPIb In the blood, plasmin is present as an inactive precursor, plasminogen Plasminogen is activated by plasminogen activators Adrenaline, urokinase, thrombomodulin-thrombin complex, kallikrein, tissue plasminogen activators (t-PAs) t-PAs are secreted by the endothelial cells urokinase is produced by kidney. Plasminogen activation Plasminogen Plasmin Fibrin Soluble fibrin fragment Note that urokinase is secreted by the epithelial cells in the kidneys. Note that plasmin also digests fibrinogen, factors V, VIII & XII and prothrombin. The fibrin formed within blood vessels is gradually dissolved to restore the fluidity of the blood. The process of liquefaction or lysis of the fibrin is called fibrinolysis There are two types of plasminogen activator Vessel activator. Some clotting factors in the intrinsic pathway, such as XIIa, XIa, prekallikrein , HMW kininogen, IIa (thrombin) et al. Tissue activator. Released by the injured tissue and endothelium tissue-type plasminogen activator (t-PA) urokinase synthesized by the kidney Plasmin is a most powerful proteolytic enzyme. digest the fibrin, fibrinogen, Factor V, VIII, prothrombin and Factor XII

Fibrinolysis Clinical application-
Human t-PA is produced by recombinant DNA technology and available for clinical use. lyses clots in the coronary arteries if given to patients soon after the onset of myocardial infarction. Streptokinase (from bacteria-streptococcci) and urokinase are also fibrinolytic enzymes used in the treatment of early myocardial infarction

ANTICLOTTING MECHANISMS
Removal of activated clotting factors from the blood by the liver Factors that reduce the adhesiveness of platelets The smooth lining of the intact vessel walls Mucopolysaccharides on the surface of endothelial cells (glycocalyx) – repulsion of clotting factors and platelets Circulation of the blood Antiplatelet-aggregation effect of the prostacyclin by the intact endothelial cells Note that blood does not normally coagulate in undamaged blood vessels. This is due to the action of intravascular anticlotting mechanisms.

ANTICLOTTING MECHANISMS: Natural anticoagulants
Antithrombin III (antithrombin-heparin cofactors) Is a plasma α globulin its binding to heparin increases its activity. Inactivates thrombin and some other clotting factors (IX, X, XI, XII) Heparin Is produced by the mast cells and blood basophils By itself, it has little or no anticoagulant property, but when it combines with antithrombin III, it increases a hundred-fold the effectiveness of antithrombin III Activated protein C Inactivates factors Va and VIIIa and activates plasminogen

NATURAL ANTICOAGULANTS (cont.)
Thrombin/thrombo-modulin/protein C pathway Endothelial cell Thrombomodulin is a thrombin-binding endothelial cell receptor Thrombomodulin Thrombin Binds thrombin and inactivates it Protein C Complex of thrombin+thrombo-modulin binds protein C and activates it Activated Prot C Protein S Protein C in collaboration with protein S inactivates factors Va and VIIIa and activates plasminogen and fibrinolysis Inactivation of inhibitors of plasminogen activator Va V ViIIa VIII Plasmin Plasminogen Note: Mutated factor V cannot be inactivated (switched off) by activated protein C, and this will lead to hypercoagulable state Thrombin Fibrinolysis

ANTICLOTTING MECHANISMS: SUMMARY
Tissue factor pathway inhibitor AT III-Heparin Proteins C & S

DRUGS THAT INHIBIT BLOOD CLOTTING (ANTICOAGULANTS)
Heparin: Heparin binds to the enzyme inhibitor antithrombin III (AT), causing a conformational change that results in its activation. The activated AT then inactivates thrombin and other proteases involved in blood clotting such as XIIa, XIa, Xa and IXa Coumarin derivatives (i.e., warfarin) Block stimulatory effects of vitamin K on synthesis of clotting factors II, VII, IX, and X by the liver (inhibit epoxide reductase which activates vit K in the liver: K → K1) Aspirin Low doses inhibit prostaglandins and thromboxanes synthesis by the platelets → inhibition of platelet release reaction and platelet aggregation Is effective in preventing of heart attack and reduction of the incidence of sudden death Note that some rat poisons contain Warfarin.

IN VITRO INHIBITION OF BLOOD CLOTTING
Keeping of blood in seliconized containers – prevention of contact activation of platelets and factor XII Substances that bind ionized calcium to produce un-ionized calcium compound or to form insoluble salts with calcium Sodium citrate or oxalate Ammonium or potassium citrate EDTA (ethylenediaminetetraacetic acid) Is ability to "sequester" di- and tricationic ions (Ca2+ & Fe3+) Is widely used as an anticoagulant for blood samples for complete blood count/full blood examination Heparin Note that citrate anticoagulants have an important advantage over the oxalate anticoagulants – they are less toxic. Therefore, moderate amounts of citrate can be injected intravenously without causing toxic effects. Citrate is rapidly removed from the blood by the liver and metabolized for energy or for glucose synthesis. In case of the liver damage, the citrate ions may not be removed from the circulation quickly enough and they can depress calcium level in the blood → tetany and convulsive death.

PROTHROMBIN TIME (protime, PT test)
Measures the clotting time of plasma from the activation of factor VII, through the formation of fibrin clot Assesses the integrity of the extrinsic/tissue factor pathway and common pathways of coagulation (factors VII, X, V, II, I) The PT test is widely used to monitor patients taking anticoagulants as well as to help diagnose clotting disorders The PT test is widely used to monitor patients taking anticoagulants as well as to help diagnose clotting disorders.

PROTHROMBIN TIME (cont.)
Depends on [prothrombin] in the blood Normal range 12 – 14 sec Increased ↓ prothrombin (less than 10% of normal) Deficiency of fibrinogen or factors V, VII, or X Therapeutic anticoagulants (i.e., heparin, warfarin, aspirin), some drugs (i.e., antibiotics, anabolic steroids, estrogens, etc.) Liver diseases Vit K deficiency Disseminated intravascular coagulation Decreased Vit K supplementation Thrombophlebitis Newborns normally have prolonged PTs in comparison with adults. However, newborns and infants do not normally experience bleeding, because a balance between procoagulants and natural anticoagulants is maintained. A PT time that exceeds approximately two and a half times the control value (usually 30 seconds or longer) is grounds for concern, as abnormal bleeding may occur.

ACTIVATED PARTIAL THROMBOPLASTIN TIME (aPTT)
Assesses the integrity of the intrinsic and common pathways of coagulation Measures the clotting time of plasma, from the activation of factor XII by a reagent through the formation of fibrin clot Normal range 25 – 38 sec Prolonged time Use of heparin Antiphospholipids antibodies Coagulation factors deficiency (intrinsic and common pathways; i.e., hemophilias) Apart from detecting abnormalities in blood clotting, it is also used to monitor the treatment effects with heparin. Heparin or warfarin causes both the PT and aPTT to become prolonged because both drugs inhibit factors in the final common pathway.

2 TYPES OF ABNORMALITIES OF HEMOSTASIS
Excessive bleeding (hemorrhagic disease) caused by deficiency of a clotting factor/s or platelets Excessive clotting: thrombosis, embolism, disseminated intravascular coagulation

CONDITIONS THAT CAUSE EXCESSIVE BLEEDING
Vitamin K deficiency Deficiency of clotting factors (i.e., hemophilia) Deficiency of thrombocytes – thrombocytopenia Deficiency of von Willebrand factor

VITAMIN K DEFICIENCY Results from
↓ intestinal absorption of fats due to ↓ bile secretion (i.e., liver disease or obstruction of the bile ducts) ↓ dietary intake of vit K (limited importance) Results in ↓ hepatic gamma carboxylation of Prothrombin (II) Factors VII, IX and X Protein C and S Bleeding tendency Prolonged prothrombin time and partial thromboplastin time Normal platelets count and serum fibrinogen split products Decreased dietary intake of vit K has limited consequences on blood clotting because Vit K is continuously synthesized by the intestinal flora. Note that Vit K is fat soluble and requires fats for absorption. Lack of the bile decreases fat digestion and absorption. Note that Vit K is often given to patients with liver diseases before performing a surgical procedure. Note that without gamma carboxylation clotting factors are unfunctional.

HEMOPHILIA Is a hemorrhagic disease that results from deficiency of
Factor VIII (the smaller component) - hemophilia A or classical Factor IX – hemophilia B, Christmas disease Factor XI – hemophilia C Is a genetic disease Hemophilia A and B are sex linked (X chromosome) Occur in males Females are hemophilia carriers Results in ↑ aPTT (PT, thrombocytes count, fibrin split products are normal)

HEMOPHILIA Deficiency Factor Clinical Syndrome Cause Factor I
Afibrinogenemia Depletion during pregnancy with premature separation of placenta: Congenital Factor II Hypoprothrombinemia (Hemorrhagic tendency in liver diseases Decreased hepatic synthesis (secondary to vitamin K deficiency) Factor V Parahemophila Congenital Factor VII Hypoconvertinemia Factor VIII Hemophilia A (classical hemophilia) Congenital recessive sex-linked defect due to abnormalities of the gene that codes for factor VIII (X chromosome) Factor IX Hemophilia B (Christmas disease) Congenital recessive trait carried on X chromosome Factor X Stuart-Prower factor deficiency Hemophilia C (PTA deficiency Hageman trait

THROMBOCYTOPENIA Petechiae – small punctate hemorrhages(1-3 mm)
Low thrombocytes count (below /m l) → poor plug formation, deficient clot retraction, deficient platelet phospholipids, poor constriction of ruptured vessels → bleeding tendency from many small venules and capillaries Multiple hemorrhages in the skin and mucous membranes – thrombocytopenic purpura Petechiae – small punctate hemorrhages(1-3 mm) Echymoses - large hemorrhages (bruises) Other causes of purpura ↓ plasma level of 1 or more clotting factors ↑ fragility of capillary walls (congenital, Vit C deficiency, adrenal failure, toxins, drugs, allergic reactions) Thrombocytopenia can be treated with fresh whole blood transfusion and splenectomy.

von Willebrand’s disease
Is the most common genetic bleeding disorder Results from defect in vWF ( quantitative or functional) Results in combination of Platelet function abnormality (vWF) - impaired adhesion Clotting factor deficiency (factor VIII) - ↑ aPTT (PT is normal) Remember: vWF is normally produced by endothelial cells and megakaryocytes 146

THROMBO-EMBOLIC CONDITIONS
Thrombosis - blood clotting within the CVS which obstruct the blood flow through the CVS (Should be distinguished from extravascular clotting, clotting in wounds and clotting that occurs in the CVS after death). Thrombosis is rather a pathological condition. Common causes Roughened endothelial surface (i.e., atherosclerosis, infections, traumas) Slow blood flow Hypercoagulobility Acquired refers to transient or acquired conditions that increase the tendency to clot. This might include antiphospholipid antibodies or a temporary hypercoagulable state such as pregnancy. Also, advanced carcinomas of the pancreas or lung may produce a hypercoagulable state. Congenital refers to hereditary conditions that increase the tendency to clot. These include Factor V Leiden, prothrombin ,protein C, protein S and antithrombin deficiencies

Consequences Formation of emboli (thromboembolism) – braking down of the thrombus and spreading of its particles particles throughout the CVS Thrombosis in the left side of the heart and large arteries → emboli in the brain, kidneys, etc Thrombosis in the venous system and in the right side of the heart → emboli in the pulmonary circulation

DESSIMINATED INTRAVASCULAR COAGULATION
Reasons Large areas of necrotic tissue (release of tissue factors into the blood) Septicemia (activation of clotting by circulation bacteria and bacterial toxins) Consequences Consumption coagulopathy ↓ fibrinogen, thrombocytopenia ↑ fibrin split products ↑ PT and PTT

CHALENGE YOURSELF 1/5 A baby is born prematurely at 28 weeks gestational age with a birth weight of 1200 g. A few weeks after birth his mother noticed a bleeding tendency in the infant. Blood test revealed a low prothrombin level. Which vitamin can be given to the baby to reduce or to prevent the bleeding tendency? a. Vitamin B12 b. Vitamin B6 c. Vitamin K d. Folic acid e. Vitamin A Answer is C

CHALENGE YOURSELF 2/5 A 72-year-old African-American man undergoes hip surgery. On his third hospital day he experiences chest pain, tachycardia, dyspnea, and a low-grade fever. The man goes into cardiac arrest, and efforts to resuscitate him are unsuccessful. On autopsy a massive pulmonary embolus is discovered. Which of the following, if present, would most likely predispose the patient to this event? (A) Factor VIII defi ciency (B) Low serum homocysteine levels (C) Mutation in the Factor V gene (D) Overproduction of protein C (E) von Willebrand factor defi ciency Answer is C

CHALENGE YOURSELF 3/5 Taking aspirin every day can reduce the risk of heart disease because a. it is a powerful vasodilator b it stimulates fibrinolysis c. it prevents atherosclerosis d. it loosens atherosclerotic plaque on arterial walls it prevents platelet aggregation Answer is E

CHALENGE YOURSELF 4/5 A one month old Caucasian with a history of persistent jaundice experiences muscle rigidity, lethargy and seizure. Which of the following causes of hyperbilirubenimia is most likely to produce this patient’s neurological abnormalities. A. Absent liver onjugation enzymes B. Deficient bilirubin excretion into bile canaliculi C. Impaired canalicular bile transit D. Increased gut deconjugation of bilirubin Impaired gut reabsorption of bilurubin The answer is A

CHALENGE YOURSELF 5/5 A 65-year-old man presented with history of acute chest pain that radiates to his left arm. Coronary angiography demonstrates more than 75% occlusion of his coronary artery. He was administered a thrombolytic agent for reestablishment of blood flow to the dying myocardium. The thrombolytic agent activates: Heparin Throbin Plasminogen Kininogen Prothrombin The Answer is C

Fill in the gap please Hemophilia A N No Hemophilia B vWF disease
PT PTT Platelet Count Bleeding Time Reticulocyte Megakyryocyte D-Dimer Schicocyte Hemophilia A N No Hemophilia B vWF disease Vit K Def Liver Disease

Section 3 Blood Group and Transfusion
Agglutination, hemolysis and the transfusion reaction

Blood Groups Based on the types of special antigens on the surface of the red blood cells Two particular groups of antigens are more likely than the others to cause blood transfusion reactions. ABO system Rh system, Antigenicity Causes Immune Reactions of Blood When blood transfusions from one person to another were first attempted, immediate or delayed agglutination and hemolysis of the red blood cells often occurred, resulting in typical transfusion reactions that frequently led to death. Soon it was discovered that the bloods of different people have different antigenic and immune properties, so that antibodies in the plasma of one blood will react with antigens on the surfaces of the red cells of another blood type. If proper precautions are taken, one can determine ahead of time whether the antibodies and antigens present in the donor and recipient bloods will cause a transfusion reaction. Multiplicity of Antigens in the Blood Cells. At least 30 commonly occurring antigens and hundreds of other rare antigens, each of which can at times cause antigenantibody reactions, have been found in human blood cells, especially on the surfaces of the cell membranes. Most of the antigens are weak and therefore are of importance principally for studying the inheritance of genes to establish parentage. Two particular types of antigens are much more likely than the others to cause blood transfusion reactions. They are the O-A-B system of antigens and the Rh system.

ABO Blood classifications depend on the presence or absence of the agglutinogens (antigens) on the surface of the red cell membrane.

Agglutinogens Two antigens- type A and type B occur on the surface of the red blood cells. These antigens are called agglutinogens because they often cause blood cell agglutination, causing blood transfusion reactions Two antigens—type A and type B—occur on the surfaces of the red blood cells in a large proportion of human beings. It is these antigens (also called agglutinogens because they often cause blood cell agglutination) that cause most blood transfusion reactions. Because of the way these agglutinogens are inherited, people may have neither of them on their cells, they may have one, or they may have both simultaneously. Major O-A-B Blood Types. In transfusing blood from one person to another, the bloods of donors and recipients are normally classified into four major O-A-B blood types, as shown in Table 35–1, depending on the presence or absence of the two agglutinogens, the A and B agglutinogens.When neither A nor B agglutinogen is present, the blood is type O. When only type A agglutinogen is present, the blood is type A.When only type B agglutinogen is present, the blood is type B.When both A and B agglutinogens are present, the blood is type AB. Group AB Group A Group B Group O

Agglutinins The agglutinins (antibodies) are gamma globulin in the plasma. When type A agglutinogen is not present in a person’s red blood cells, antibodies known as anti-A agglutinins develop in the plasma. when type B agglutinogen is not present in the blood cells, antibodies known as anti-B agglutinins develop in the plasma. type O blood, although containing no agglutinogens,does contain both anti-A and anti-B agglutinins. Finally, type AB blood contains both A and B agglutinogens but no agglutinins. Origin of Agglutinins in the Plasma. The agglutinins are gamma globulins, as are almost all antibodies, and they are produced by the same bone marrow and lymph gland cells that produce antibodies to any other antigens. Most of them are IgM and IgG immunoglobulin molecules. But why are these agglutinins produced in people who do not have the respective agglutinogens in their red blood cells? The answer to this is that small amounts of type A and B antigens enter the body in food, in bacteria, and in other ways, and these substances initiate the development of the anti-A and anti-B agglutinins. For instance, infusion of group A antigen into a recipient having a non-A blood type causes a typical immune response with formation of greater quantities of anti-A agglutinins than ever.Also, the neonate has few, if any, agglutinins, showing that agglutinin formation occurs almost entirely after birth.

Major types of ABO blood group
The bloods are normally classified into four major types, depending on the presence of absence of the two agglutinogens, A and B

ABO blood group But why are these agglutinins produced in people who do not have the respective agglutinogens in their red blood cells? Small amounts of type A and B antigens enter the body in food, in bacteria, and in other ways, and these substances initiate the development of the anti-A and anti-B agglutinins.

Blood Type Agglutinogen Agglutinin O -- Anti-A, Anti-B A Anti - B B Anti-A AB A and B

Agglutination and hemolysis in transfusion reaction
When bloods are mismatched so anti-A or anti-B plasma agglutinins are mixed with red blood cells that contain A or B agglutinogens respectively, agglutinins will make the red cells adhere to each other – agglutination. The clumps of red cells will plug small blood vessels throughout the circulatory. Because the agglutinins have two binding sites (IgG type) or 10 binding sites (IgM type), a single agglutinin can attach to two or more red blood cells at the same time, thereby causing the cells to be bound together by the agglutinin. This causes the cells to clump, which is the process of “agglutination.

Group B Group B anti-B anti-B anti-B Group B Group B anti-B

Agglutination and hemolysis in transfusion reaction
During the ensuring few hours to a few days, either physical distortion of the cells or attack by phagocytic white cells destroys the agglutinated cells, releasing hemoglobin into the plasma, which is called “hemolysis” of the red blood cells. Type II hypersensitivity reaction

agglutination During severe hemolytic reaction, fever, chills and shock may occur. One of the most lethal effects of transfusion reactions is kidney shutdown, which can begin within few minutes to a few hours. If the shutdown is complete and fails to open up, the patient die of renal failure.

Blood typing Before giving a blood transfusion to a person, it is necessary to determine the blood type of the recipient’s blood and the blood type of the donor blood so that the bloods can be appropriately matched. This is called blood typing and blood matching. Performed in the following way: The red blood cells are first separated from the plasma and diluted with saline. One portion is then mixed with anti-A agglutinin and another portion with anti-B agglutinin. After several minutes, the mixtures are observed under a microscope. If the red blood cells have become clumped— that is, “agglutinated”—one knows that an antibody-antigen reaction has resulted.

Rh Blood Groups Blood Typing, Showing Agglutination of Cells of the Different Blood Types with Anti-A or Anti-B Agglutininsin the Sera Red Blood Cell Types Sera Anti-A Anti-B O A B AB O red blood cells have no agglutinogens and therefore do not react with either the anti-A or the anti-B agglutinins. Type A blood has A agglutinogens and therefore agglutinates with anti-A agglutinins. Type B blood has B agglutinogens and agglutinates with anti-B agglutinins. Type AB blood has both A and B agglutinogens and agglutinates with both types of agglutinins.

Blood typing and blood matching
AB O A Serum from Contains Agglutinin B B Serum from Contains Agglutinin A O Serum from Contains Agglutinin A &B AB Serum from Contains No Agglutinin N = No Agglutination A = Agglutination

Blood typing and blood matching
AB O N A A N A Serum from Contains Agglutinin B A N A N B Serum from Contains Agglutinin A A A A N Serum from O Contains Agglutinin A &B AB N N N N Serum from Contains No Agglutinin N = No Agglutination A = Agglutination

Rh BLOOD GROUP SYSTEM Rh (rhesus factor)
Is named after the rhesus monkey in which it was found Rh is an antigen present on the RBC membrane – transmembrane proteins, which may act as ionic channels 6 types of Rh AG C, D, E, c, d*, and e (+ 43 other Rh antigens, which are less common) Antigen D is the most common and potent Presence of D antigen - Rh+ (RhD+) Lack of D antigen - Rh- (RhD-) Population data on RhD+ 85% of Caucasians 90% of African Americans 99% of Asians 100% of Africans Note that antigen d is hypothetical. Lowercase “d” indicates the absence of the D antigen (the gene is either deleted or nonfunctional).

Rh BLOOD GROUP SYSTEM: Antibodies
Blood of normal individual does not contain anti-Rh antibodies (with exception of anti-E) Production of anti-Rh antibodies can be evoked by Transfusion of Rh- individual with Rh+ blood (only 0.5 ml may suffice) The presence of Rh+ fetus in a Rh- mother Note that contrary to the OAB system, in the Rh system agglutinins do not develop spontaneously. A person should be exposed to an Rh AG to produce AB.

Rh BLOOD GROUP SYSTEM: Typing
Mixing of the blood sample with anti-Rh serum (D AB) Agglutination – RhD+ No agglutination – RhD-

Rh IMMUNE RESPONSE Transfusion of RhD+ blood to un-immunized RhD- recipient No immediate reaction Sensitization of recipient’s blood to RhD AG - slow formation of anti-RhD AB Delayed transfusion reaction – hemolysis of the donor’s RBC that still circulate in the recipient’s blood Transfusion of RhD+ blood to immunized RhD-recipient Enhanced transfusion reaction – acute hemolysis

ERYTHROBLASTOSIS FETALIS
Note that the Rh-antibodies in the maternal plasma do not reach high enough level with the first pregnancy (gravida 1 or para 1) to cause significant complications. In the second or later Rh positive pregnancies (gravida 2 or para 2) concentration of Rh-antibodies increases, which causes an increase in hemolysis of the fetal RBCs. Note. Gravida - the total number of times a woman has been pregnant, regardless of whether these pregnancies were carried to term (includes aborted pregnancies and current pregnancy). Para – the number of births. ↑ risk starting from gravida 2 or para 2.

ERYTHROBLASTOSIS FETALIS (cont.)
Rh+ fetus/RH- mother Some fetal RBC enter the maternal circulation Formation of anti-Rh AB in the maternal blood Hemolysis of fetal Rh+ RBC Some anti-Rh antibodies enter the fetal circulation Gross edema (hydrops fetalis) Hemolytic jaundice, anemia, high reticulocyte count, erythroblasts Bile pigments pass into the brain and are deposited in the basal ganglia → impairment of motor functions - Kernicterus Gross edema

ERYTHROBLASTOSIS FETALIS (cont.)
Treatment Replacement of the neonate’s blood with Rh- blood – removal of Rh AB and damaged RBC Prevention Administration of anti-D AB to the Rh- pregnant woman who expects RhD+ baby Destruction of Rh+ RBC, which cross the placenta and enter the maternal circulation Inhibition of AG-induced AB production by B lymphocytes in the pregnant woman

Past exams question Fred's blood type is A, Rh- and Linda is B, Rh+. Fred and Linda have a 4-year-old son who is AB, Rh+. Which one of the following conclusions is correct? a. During the second pregnancy Ginger needs to take anti-D antibodies b. There is no risk to a second child to develop erythroblastosis fetalis c. If the son needs a blood transfusion Fred could provide it safely, but not Ginger d. Fred is not the boy’s father B

WINDSOR UNIVERSITY SCHOOL OF MEDICINE

Similar presentations

Presentation on theme: "WINDSOR UNIVERSITY SCHOOL OF MEDICINE"— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

WINDSOR UNIVERSITY SCHOOL OF MEDICINE

Similar presentations

Presentation on theme: "WINDSOR UNIVERSITY SCHOOL OF MEDICINE"— Presentation transcript:

Similar presentations

About project

Feedback