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Blood Biochemistry BCH 577 Prof. Omar S. Al-Attas Professor of Biochemistry Biochemistry Department King Saud University.

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Presentation on theme: "Blood Biochemistry BCH 577 Prof. Omar S. Al-Attas Professor of Biochemistry Biochemistry Department King Saud University."— Presentation transcript:

1 Blood Biochemistry BCH 577 Prof. Omar S. Al-Attas Professor of Biochemistry Biochemistry Department King Saud University

2 Chapter 1

3 Introduction To Hematology Blood is the only tissue that flows throughout your body. This red liquid carries oxygen and nutrients to all parts of the body and waste products back to your lungs, kidneys and liver for disposal. It is also an essential part of your immune system, crucial to fluid and temperature balance, a hydraulic fluid for certain functions and a highway for hormonal messages. It represent about 8% of total body weight Average volume of 5 liters in women Average volume 5.6 liters in men 99% of blood cells are erythrocytes Plasma accounts for the remaining of the blood volume. Consist of three types of cellular elements (Erythrocytes, Leukocytes and Platelets) The cellular elements are suspended in plasma

4 Hematopoiesis The cells normally found in the circulatory blood are of three main types: The erythrocytes ( RBC) are largely concerned with oxygen transport. The leukocytes (White blood cell) play various roles in defense against infections and tissue injury. The thrombocytes (platelets) are immediately involved in maintaining the integrity of blood vessels and in the prevention of blood clots Hematopoiesis (poiesis= formation) Is the term used to describe the formation and development of blood cells. Cellular proliferation, differentiation and maturation take place in the hematopoietic tissue, which consists primarily of the bone marrow. Only mature cells are released to the peripheral blood. Hematopoietic begins in the nineteenth day after fertilization in the yolk sac of human embryo. Then the fetal liver becomes the chief site of blood cell production( at about this time the yolk sac discontinues its role in the hematopoiesis. Also at this time hematopoiesis also begins to lesser degree in the spleen, kidney, thymus and lymph nodes. At about 4-5 months of gestation hematopoiesis commences in the bone marrow where it is fully active by the seventh or eight month and at birth partially the whole bony skeleton contain active marrows. During childhood and adolescence there is a marked recession of marrow activity in the long bones so that in the adult activity is limited to the trunkcal skeleton and skull.


6 Site of Hematopoiesis


8 Cont… In the yolk sac, most of hematopoiesis activity at this site is confined to erythropoiesis (erythrocytes formation). Cell production at this site time is called Primitive erythropoiesis because this erythroblast and Hb are not typical of that seen in later developing erythroblasts Hematopoiesis in the bone marrow is called medullary hematopoiesis.

9 Hematopoietic Tissues 1. Spleen, is located in the upper left quadrant of the diaphragm (muscle near the stomach) It is enclosed by capsule of connective tissue which contains the largest collection of lymphocytes and mononuclear phagocytes in the body. Splenic function: Immune defense Culling(The removal of aging or abnormal red blood cells) Pitting (The ability of the spleen to clear inclusions while maintaining the integrity of the red cell)

10 Culling The discriminatory filtering and destruction of senescent or damaged red cells by the spleen ATP is important in cation pump of erythrocytes and because of entering the spleen through the slow transit become concentrated in the hypoglycemic due to low concentration of glucose and slow circulation in the splenic cords, the supply of glucose in the damaged or senescent erythrocytes is rapidly diminished. This decrease the availability of ATP and contributes to the demise of these red cells. Slow passage through a macrophage-rich route before allows the phagocytic cells to remove these old or damaged erythrocyte before or during their squeeze through 3µm pores to cords and sinuses. Normal RBC withstand this adverse environment and eventually reenter the circulation.

11 Cont… Pitting The spleens ability to “pluck out” particles from intact erythrocytes. The pinched off cell membrane can reseal itself, but the cell cannot synthesize lipids and proteins for new membrane because of its lack of cellular organelles. Therefore extensive pitting causes reduced surface- area to volume ratio resulting in the formation of spherocytes.

12 For Example: Howell Jolly Bodies: Small, round or oval structure pinkish or bluish in color, observed in erythrocytes in various anemias and leukemias and after splenectomy, they also represent unphysiological red cell nuclear remnant. Pappenheimer Bodies: Basophilic containing iron granules observed in various types of erythrocytes Heinz bodies: Resulting from oxidative injury to unprecipitation of Hb (abnormal Hb) and also erythrocytes with enzyme deficiency. Blood cells coated with Ab are also susceptible to pitting by macrophages. The macrophage removes the antigen-antibody complex and the attached membranes. The presence of spherocytes on a blood film is evidence that the red blood cells has undergone membrane assault in the spleen.

13 Inclusion Bodies Pappenheimer bodies

14 Cont… Defense Spleen is rich supply of lymphocytes and phagocytic cells as well as its unique circulation Reservoir for platelets Massive splenomegaly may result in pooling of 80-90% of the platelets producing peripheral blood thrombocytopenia. Condition associated with an enlargement of the spleen are also frequently accompanied by leukopenia and anemia, the result of splenic pooling and sequestration Removal of the spleen results in transient thrombocytosis, with the platelet count returning to normal in about 10 days. Splenectomy does produce characteristic erythrocyte abnormalities that are easily noted on blood smears by experienced technologist. After splenectomy, the red cells contain granular inclusion such as Howell- jolly bodies, pappenheimer bodies. Target cells, cells with excess membrane to volume ratio, have several mechanism of formation The splenectomized patients, these cells are probably formed as a result of excess lipid on the membrane since the missing spleen would normally have groomed the excess lipid from reticulocytes in their maturation process.

15 Hypersplenism Under certain conditions the spleen may become enlarged; consequently exaggeration of its normal filtering and phagocytizing. So anemia leukopenia, thrombocytopenia, then plasma volume increases consequently cytopenias occurs. A diagnosis of hypersplenism is made when four conditions are met: 1. Anemia, leukopenia or thrombocytopenia in the blood Cellular or hyperplastic bone marrow ( increase in normal cells of the tissue)corresponding to the peripheral blood cytopenias. 3. The occurrence of the splenomegaly The correction of cytopenia as following splenectomy.

16 Hypersplenism Primary Occurs when no underlying cause identified. The spleen behaves normal but causes disease. The most common cause is congestive splenomegaly associated with liver cirrhosis and portal hypertension. Thrombosis of the splenic or portal veins may contribute Secondary Caused by a disorder. The causes: Inflammatory and infectious diseases increase the defense function of the spleen. Gaucher’s disease- macrophages accumulates large quantities of undigestable substance causes splenomegaly

17 Lymphnodes The lymphatic system is composed of lymph node (bean-shaped) 2. Lymph Vessels: Node are composed of lymphocytes, macrophages and reticular meshwork. The morphology: lymph nodes contain the following: an inner area called medulla, outer area called cortex. The medulla: surround the efferent lymphatics and contains the B lymphocytes The cortex contains the T-lymphocytes The lymph nodes act as filter removing foreign particles from the lymph by phagocytic cells. Antigens pass through the nodes, they contact and stimulate immune complex lymphocytes to proliferate and differentiate into effector cells

18 Thymus 3. Thymus The thymus is a well developed organ at birth and continues to increase in size until puberty, and eventually would begin to atrophy ( hardening). It is bilobular organ packed with small lymphocytes and a few macrophages. Thymus is to serve for maturation of T-lymphocytes. The thymic hormone, thymosin, is important in the maturation of virgin lymphocytes into immunocompetent T-cells.

19 Bone Marrow 4. Bone Marrow In adult the marrow normally consist of islands of cellular active marrow separated and supported by fat (Yellow fat). Red marrow contains both erythroid and myeloid precursors with ration of (M:E) of 1.5:1 to 4:1 For the first four years of life nearly all marrow cavities are composed of red hematopoietic marrow. After 4 years of age, the red marrow in shafts of long bones is gradually replaced by yellow fat tissues.

20 The Assessment of Marrow Activities In general if a patient has a normal peripheral blood count and a bone marrow aspirate contains what appears to be adequate number of cells, present in normal proportions it is reasonable to assume normal activity Cellular or even hypercellular marrow may be associated markedly defective output of cells to the peripheral circulation: e.g. megaloblast anemia is featured by excessive cellular destruction in the marrow itself. Other cases a defective peripheral cell count may be associated with hypercellular marrows, there being excessive destruction of cells in the circulation or sequestration in an enlarged spleen.

21 Chapter 2

22 Derivation of Blood Cells Replacement of effete peripheral hematopoietic cells is the function of more primitive elements in the bone marrow called stem cells. Stem cells are characterize by:  Ability to differentiate into distinct cells lines with specialize functions.  Ability to regenerate themselves in order to maintain the stem cell compartment.


24 Cont… Myeloblasts and erythroblasts (pronormoblasts) because of the high mitotic index, were believed to responsible for maintaining normal numbers of mature blood cells. But these two types of cells have limited ability to proliferate into the billions of blood cells replaced daily. So two different theories on stem cells have been proposed to verify the existence of stem cell different from myeloblasts and erythroblasts. 1. Monophyletic theory: Pluripotential stem cell under unknown hormonal factors may give rise to each of the principle blood cell lines. These pluripotential cells have the capability to self-renewal, proliferation and differentiation into all hematopoietic cells lines. 2.Polyphylitic theory. Each blood cell type come from a separate stem cell. So each monopotential stem cell differentiates into only one type of blood cell.

25 Cont… (Erythrocytic, myelocytic or megakaryocytic) It has been suggested that nodules appearing at 7 th days were formed from more mature unipotent committed stem cells In the day 14 the nodules showed mixed cell populations. The cell that formed colony termed- Colony-forming unit spleen (CFU-S) Suggesting the nodules at this time were derived from more primitive multipotential stem cell. Stem cells number one per 1000 nucleated cells in the marrows

26 Erythropoiesis 0 The earliest recognizable erythroid cells in the bone marrow is the pronormoblast which is a long cell measuring µm in diameter. With dark blue cytoplasmin, a central rounded nucleus with nucleoli and slightly clumped chromatin. The deep color of the more immature cells due to the presence of large amount of RNS which is associated with active protein synthesis. 0 They also contain Hb (which stain pink) in the cytoplasm; the cytoplasm stain pale blue as it does its RNS and protein synthetic apparatus while the nuclear chromatin becomes more condensed. 0 Any remaining nuclear material is removed by a process of pitting during passage through the splenic sinus walls. 0 The successive cytoplasmic charge from blue to pink earning them name basophilic, polychromatic and orthochromatic 0 After extruding the nucleus from the normoblast the new stage of cells is called reticulocytes which still contain some ribosomal RNA and still able to synthesis Hb. This cell spends 1-2 days in the bone marrow and also circulates in the peripheral blood for 1-2 days before maturing mainly in the spleen when RNS is completely lost and completely pink-staining mature erythrocytes (red cell).

27 Kinetics of Erythropoiesis In man, time required for erythropoiesis to proceed from the undifferentiated stem cell to reticulocyte is about 7 days and the final maturation of these cells in the peripheral blood and spleen takes about 24 hours This means that some 210 thousand million erythrocytes must be produced by the marrow each day about 9 thousand million per hour. This requires the synthesis of 6.5g Hb and involves the turnover of about 22mg of iron. To maintain regular erythropoiesis 1. Erythropoietin- the principle site of erythropoietin production in the kidney. Erythropoiesis is regulated by hormone erythropoietin which: Acts primarily on more mature committed erythroid cells of erythroid colony- forming units. Increasing Hb synthesis in red cells precursors. Decreasing maturation time of red cell precursors. Releasing marrow reticulocytes into peripheral blood at an earlier stage than normal.

28 Cont… 2. For normal erythropoiesis to be established there must be adequate supply of stem cells in a satisfactory environment containing all the essential materials for their normal growth and differentiation. The materials include beside those require by all cells certain specific factors: Vit B12, folate, Vit C, Vit E, Vit B6, pyroxidine, thiamine, riboflavin. Metal: Iron, manganese, cobalt Amino acids Hormone: erythropoeitin, thyroxine and androgens

29 Erythrocytes The mature erythrocytes of the peripheral blood of a man are:  Non-nucleated and contain organelles  Contain enzyme of both anearobic glycolytic pathway of Embden-Myerhoff and aerobic pentose P pathway It stains pink to orange because of the large amount intracellular acidophilic protein called Hb. The 7 μm RBC must be flexible corpuscle to squeeze through the tiny 3 μm fenestrations of the capillaries of the spleen.

30 Erythrocytes Membrane The cells flexibility is a property of both the erythrocyte membrane and the fluidity of the cell’s content which is main hemoglobin. It is a biphospholipid protein composed of the following:  49 % protein (composed of contractile protein, enzyme, surface antigens)  43% lipid (95% of lipid is equal amounts of unspecified cholesterol and phospholipids. The remaining are glycolipids. The polar lipids on the external and internal surfaces and non polar at the center of the membrane.  8% CHO ( occurs on the external surface)


32 Cont… Cholesterol responsible for the passive cation permeability if the membrane. It appears that membrane cholesterol exist in free equilibrium with plasma cholesterol. Increase cholesterol in plasma (such as occur in lecithin-cholesterol acyltransferase LCAT deficiency) results in accumulation of cholesterol on membrane. These cholesterol laden erythrocytes appears distorted with formation of target cells and spicules (tiny spike-like structure) Increase in cholesterol: Phospholipids increases the microviscosity and the degree of order of the membrane. The phospholipids are: Phosphatidylethanolamine (Cephalin) Phosphatidylcholin (Lecithin) Sphengomyelin Phosphatidylserine

33 Cont… Glycolipids is in the form of glycosphingolipids (cerebrosides and gangliosides) are responsible for some antigenic properties of the membrane in particular those coresponding to the A,B, H and lewis blood groups. Glycoprotien have similar antigenic properties. Proteins in the membrane Two types (both synthesized during cell development) Integral protein consist of two types: Glycophorin A and band 3.  Glycophorin A serves as receptors for certain viruses and lectins.  Band 3 (the name is from its migration with erythrocyte proteins on SDS polyacrylamide gel electrophoresis. It is responsible for an ion transport across the membrane. Peripheral proteins lack CHO moieties and are to the cytoplasmic side of the lipid bilipid layer These proteins include: Enzyme glycealdehyde-3-p dehydrogenase. Skeletal proteins e.g. spectrin actin viscoelastic properties and contribute to cell shape deformability and membrane stability. Calcium is a membrane component. 80% of intracellular calcium is found in membrane.  It is maintained at an extremely low intracellular concentration by the activity of an ATP pump.  The accumulation of calcium cation induces irreversible cross-linking and alteration of cytoskeletal proteins.

34 Erythrocyte Metabolism Although the binding, transport and release of oxygen and carbon dioxide is a passive process not requiring energy, a variety of energy- dependent metabolic processes occur that are essential to cell viability. The metabolism of the red cell is limited because of the absence of a nucleus, mitochondria and other subcellular organelles. The most important metabolic pathway in the mature erythrocytes require glucose as substrate.

35 Metabolic Pathways These pathways include: a. Embden-Meyerhof pathway b. Hexose-monophosphate (HMP) shunt c. Methemoglobin reductase pathway d. Rapoport-leubering pathway These pathways contribute:  The first pathway for providing energy for maintaining high intracelllur K +, low intracellular Na + and very low Ca 2+ (cation pump)  The second pathway provides reducing power to protect Hb in reducing state.  The third pathway regulates oxygen affinity of Hb  Maintains Hb in reduced state

36 Embden-Meyerhof Pathway 90-95% of the red cell’s glucose consumption is utilized by this pathway. Normal red cells have glycogen deposits. They depend entirely on environmental glucose for glycolysis. Glucose enters the cell by the facilitated diffusion an energy-free process. ATP’s Are necessary to maintain red cell shape and flexibility. Membrane integrity through regulation of intracellular cation concentration: Na + & Ca 2+ are more concentrated in the plasma. K + is more concentrated within the cell. Erythrocyte osmotic equilibrium is maintained by: The selective permeability of the membrane By the cation pumps located in the cell membrane

37 Cont… The Na + & K + pump ADP + Pi  In the expulsion of 3 Na + and the uptake of 2K +.  Ca 2+ is maintained in low concentration by the action of a similar but separate cation pump that utilized ATP for fuel. Excess leakage of Ca 2+ into the cell or failure of the pump causes rigid shrunken cells with protrusions (echinocytes) An increase in calcium is associated with excess K + leakage from cell. Magnesium is another major intracellular cation. It reacts with ATP to form the substrate complex, Mg-ATP for Ca 2+ - MgCT- ATPase (calcium cation pump). Upon the exhaustion of glucose, the fuel for the cation pumps is no longer available. Cells cannot maintain normal intracellular cation concentrations and this leads to cell death hydrolysis One ATP

38 Hexose Monophosphate Shunt (HMP shunt) 5% of cellular glucose enters the oxidative HMP shunt, an ancillary aerobic energy system. In the pathway glucose-6- P is converted to 6-phosphogluconate and so to ribulose-5- P. NADPH is generated and is linked with GSH which maintains sulfhydryl (-SH) groups intact in the cell including those in Hb and RBC membrane. Reduced glutathione (GSH) protects the cell from permanent oxidant injury (H 2 O 2 ). Oxidants, within the cell will oxidize Hb-SH groups, unless they are reduced by glutathione. This reduction oxidizes glutathione (GSSG), which in turn is reduced by adequate levels of NADPH. The red cell normally maintains a large ratio of NADPH to NADP +. Failure to maintain reducing power through levels of GSH or NADPH leads to oxidation of Hb - SH groups, followed by denaturation and precipitation of Hb in the form of Heinz bodies. Heinz bodies with a portion of the membrane are then plucked out by the macrophages of the spleen. Reduced GSH is also responsible for maintaining reduced-SH groups at the membrane level. Decrease of GSH lead to injury of membrane sulfhydryl groups resulting in leak of cell membrane.

39 Methemoglobin Reductase Pathway The methemoglobin reductase pathway, an offshoot of the Embden-Meyerhof pathway, is essential to maintain heme iron in the reduced state, Fe++ Hemoglobin with iron in the ferric state Fe ++ is known as methoglobin. This form of Hb cannot combine with O 2 Methemoglobin reductase together with NADPH produced by the Embden-Meyerhof pathway protect the heme iron from oxidation. The absence of this system, the 2% of heme methemoglobin formed daily will eventually raise to 20-40% surely limiting the oxygen-carrying capacity of the blood.

40 Rapoport-Luebering Pathway The Rapoport-Luebering Pathway is a shunt of Embden- Meyerhof pathway. DPG is present in the erythrocytes in a conclusion of 1,ol DPG/ 1 mol Hb, and it binds exclusively to deoxyHb. As more DPG binds to deoxy Hb, glycolysis is stimulated to produce more DPG and ATP. Increase in DPG concentration facilitate the release of O 2 to the tissues by causing a decrease in Hb affinity for oxygen. Thus, the red cell has built-in mechanism for regulation of O 2 delivery to the tissues


42 Hexose monophophate shunt pathway

43 Chapter 3

44 Lifespan and Faith of RBC 0 Normally, the average life span of red blood cells is 120 days. This cells live in blood circulation. 0 In in typical adult produces 200 billion RBC per day. 0 At the end of their lifespan, they become senescent, and are removed from circulation. 0 In many chronic diseases, the lifespan of the erythrocytes is markedly reduced (e.g. patients requiring haemodialysis). 0 The destruction of RBC is about 2-3 million per second on average. Causes of reduction in the life span of RBC. 1. Defects in RBC (curpuscular defects) e.g. Hereditary spherocytosis Sickle cell anemia, Thalassemias 2.Deficiency of red cell enzyme such as: G6PD, Pyruvate kinase def., Autoimmune disorder, Hypersplenism

45 Cont… The fate of the RBC 0 After 120 days, the RBC becomes more fragile due to decrease NADPH activity. 0 Younger RBC RBCs can easily pass through the capillaries which have the diameter smaller than the mature RBC’c 0 With a fragile membrane, the mature RBC are destroyed while trying to squeeze through capillaries. 0 The destruction most occurs in the capillaries of the spleen. This is why spleen is called the “grave yard of the RBC”. 0 The hemoglobin is released and taken up the macropharges Pathway of RBC destruction

46 Cont…

47 Bilirubin 0 Is the yellow breakdown product of normal heme catabolism. 0 Bilirubin is excreted in bile and urine, and elevated levels may indicate certain diseases. 0 It is a toxic waste product in the body 0 It is extracted and biotransformed mainly in the liver and excreted in bile and liver. 0 Elevation in serum and urine bilirubin is associated with juandice




51 In Blood 0 The bilirubin synthesized in spleen, liver and bone marrow is unconjugated bilirubin 0 It is hydrophobic in nature so it is transported to the liver as complex with the plasma protein, albumin Unconjugated bilirubin(Free Bilirubin) 0 Lipid soluble 0 1gm albumin binds 8.5 mg of bilirubin 0 Fatty acids and drugs can displace bilirubin 0 Indirect positive reaction in van den Berg test

52 0 Conjugated (Direct) bilirubin is released into the bile by the liver and stored in the gallbladder, or transferred directly to the small intestines. 0 Bilirubin is further broken down by bacteria in the intestines, and those breakdown products contribute to the color of the feces. 0 A small percentage of these breakdown compounds are taken in again by the body, and eventually appear in the urine 0 Unconjugated (indirect) Erythrocytes generated in the bone marrow are disposed of in the spleen when they get old or damaged. 0 This releases hemoglobin, which is broken down to heme as the globin parts are turned into amino acids. 0 The heme is then turned into unconjugated bilirubin in the reticuloendothelial cells of the spleen. 0 This unconjugated bilirubin is not soluble in water, due to intramolecular hydrogen bonding. It is then bound to albumin and sent to the liver.

53 Jaundice








61 Treatment for Neonatal Jaundice


63 Bilirubin Lab values Bilirubin formNormal value Total (elderly, adult, child) (newborn) Critical value (a(newborn) 0.1 to 1.0 mg/dL 1.0 to 12.0 mg/dL >12 mg/dL >15 mg/dL Pre-hepatic, unconjugated, indirect0.0 to 0.8 mg/dL Post-hepatic, conjugated, direct0.0 to 0.25 mg/dL Fecal urobilinogen40 to 280 mg/day Urine0.0 to 0.02 mg/dL Conjugated bilirubin - water soluble - direct reaction with dyes Unconjugated bilirubin - water insoluble - alcohol is needed for dye (indirect) reaction Observe the color changes associated with heme degradation by watching the progress of a bruise (dark red to green to yellow).

64 Chapter 4

65 Classification of anemia Morphologic 0 Normocytic: MCV= fL 0 Macrocytic: MCV > 100 fL 0 Microcytic : MCV < 80 fL Pathogenic (underlying mechanism) 0 Blood loss (bleeding) 0 Decreased RBC production 0 Increased RBC destruction/pooling

66 Other causes: Inadequate production of mature red cells 1. Deficiency of essential substances like iron, folic acid, vit B12, protein and other elements like copper, cobalt, etc. 2. Deficiency of erythroblast.. i.e.Aplastic (anemia body's bone marrow does not make enough new blood cells.), Pure red cell aplasia 3. Infiltration of the bone marrow i.e. leukemia, lymphoma, carcinoma, myelofibrosis 4. Endocrine abnormalities i.e. myxedema, Addison's disease, pituitary insufficiency 5. Chronic renal disease 6. Chronic inflammatory disease 7. Cirrhosis of liver Pure red cell aplasia (PRCA) is an uncommon disorder in which maturation arrest occurs in the formation of erythrocytes. * Pure red cell aplasia (PRCA) is an uncommon disorder in which maturation arrest occurs in the formation of erythrocytes.

67 The etiologic possibilities are Iron deficiency Thalassemia Sideroblastic anemia (abnormal normoblasts with excessive accumulation of iron in the mitochondria) Anemias of chronic disease. Severe microcytic anemia (MCV <70 fL) is caused mainly by iron deficiency or thalassemia. Chronic inflammatory disease— (1)infection (2)collagen vascular disease (3)inflammatory bowel disease Recent blood loss Malignancy/Marrow infiltration Chronic renal failure Transient erythroblastopenia of chidhood (A decrease in the number of erythroblasts in bone marrow, as seen in aplastic anemia) Marrow aplasia/hypoplasia HIV infection Hemophagocytic syndrome Hypochromic Microcytic Anemia Normochromic Normocytic Anemia

68 Macrocytic Anemia Normocytic Anemias Megaloblastic anemias Vit.B12 def. - (1) pernicious anemia (2) malabsorption Folate def. - (1) malnutrition (2) malabsorption (3) chronic hemolysis (4)drugs - phenytoin, sulfa Hemolysis Myelodysplastic syndrome ( A group of conditions that occur when the blood-forming cells in the bone marrow are damaged. This damage leads to low numbers of one or more types of blood cells.) Marrow failure - Aplastic anemia Chronic liver disease Hypothyroidism Macrocytic anemia may be the result of megaloblastic (folate or vitamin B12 deficiency) or nonmegaloblastic causes. Folate deficiency can in turn be due to either reduced intake or diminished absorption. Severe macrocytic anemia (MCV >125 fL) is almost always megaloblastic. These may be classified as follows: underproduction of erythrocytes due to (1) the anemia of chronic disease (2) marrow failure (3) renal failure (decreased erythropoietin) loss or destruction of erythrocytes due to (1) hemolysis (2) acute blood loss The causes of normocytic anemias include aplastic anemia, bone-marrow replacement, pure red-cell aplasia, anemias of chronic disease, hemolytic anemia, and recent blood loss. A number of anemias have a genetic etiology. Examples of such inherited disorders include hereditary spherocytosis, sickle-cell (SC) anemia, and thalassemia

69 Iron Deficiency Anemia 0 It is a condition when supply of iron in the body to bone marrow falls short of that required for the production of red blood cells. It is the commonest cause of anemia throughout the world. 0 The incidence of anemia in the general population is about 1.5%. 0 Iron deficiency related to inadequate replacement of lost iron is the most frequent cause of asymptomatic anemia and has a variety of causes. 0 Iron deficiency is common among women of childbearing age; 10% to 20% of menstruating women have abnormally low concentrations of hemoglobin (usually <12 g per 100 mL). 0 Between 20% and 60% of pregnant women have hemoglobin levels <11 g per 100 mL. Anemia was found in 6% of white women and 17% of black women during the first trimester and in 25% of white women and 46% of black women during the third trimester.

70 Iron Function as electron transporter; Vital for life Must be in ferrous (Fe ++ ) state of activity. 0 In anaerobic condition, easy to maintain ferrous state 0 Iron readily donates electrons to oxygen, superoxide radicals, H2O2 OH Radicals 0 Ferric (Fe +++ ) ions cannot transport electrons or O2. 0 Organisms able to limit exposure to iron had major survival advantage Iron Store

71 Cont… Iron absorption 0 Duodenum 0 Proximal jejunum (influenced by rate of erythropoiesis) Factor Affecting Iron Absorption 0 Form of iron 0 Acids 0 Amount of iron 0 Rate erythropoiesis

72 Sickle-cell anemia is a molecular disease of Hb Comparison of normal and sickle-shaped erythrocytes


74 Mutation of Sickle Cell Gene



77 Characterization of HbS  HbS has between 2 & 4 more net + charges per molecule than net HbA AS pI of Oxy Hb = 0.22 pI of deoxy Hb =0.23  Non-polar residue on the outside of HbS (due to Val) causing low solubility  Sticky patch on the outside of its β chains & are present on both deoxy HbS & oxy HbS but not on HbA


79 Signs and Symptoms of Sickle cell Episodes of pain Pain develops when sickle-shaped red blood cells block blood flow through tiny blood vessels to your chest, abdomen and joints. Pain can also occur in your bones. Frequent infections Sickle cells can damage your spleen, an organ that fights infection. This may make you more vulnerable to infections. Vision problems Tiny blood vessels that supply your eyes may become plugged with sickle cells. This can damage the retina — the portion of the eye that processes visual images.

80 Prevention of Sickle Cell Disease 0 Screen for Hb S at birth. This method of finding allows institution of early treatment and control 0 Prenatal diagnosis. Prenatal testing is sensitive and rapid and must be accompanied with genetic testing and psychological counseling

81 Thalassemia 0 Diverse group of disorders which manifest as anemia of varying degrees. 0 Result of defective production of globin portion of hemoglobin molecule. 0 Distribution is worldwide. 0 May be either homozygous defect or heterozygous defect. 0 Defect results from abnormal rate of synthesis in one of the globin chains. 0 Globin chains structurally normal (is how differentiated from hemoglobinopathy), but have imbalance in production of two different types of chains. 0 Results in overall decrease in amount of hemoglobin produced and may induce hemolysis. 0 Two major types of thalassemia:  Alpha (α) - Caused by defect in rate of synthesis of alpha chains.  Beta (β) - Caused by defect in rate of synthesis in beta chains. 0 May contribute protection against malaria.

82 Cont… 0 Also called Alpha Thalassemia Minor. 0 Caused by two missing alpha genes. May be homozygous (- a/-a) or heterozygous (--/aa). 0 Exhibits mild microcytic, hypochromic anemia. 0 MCV between fL. 0 May be confused with iron deficiency anemia. 0 Although some Bart's hemoglobin (γ 4 ) present at birth, no Bart's hemoglobin present in adults.


84 Alpha thalassemia 0 Has wide range clinical expressions. 0 Is difficult to classify alpha thalassemias due to wide variety of possible genetic combinations. 0 Absence of alpha chains will result in increase of gamma chains during fetal life and excess beta chains later in life; Causes molecules like Bart's Hemoglobin (γ 4 ) or Hemoglobin H (β 4 ), which are stable molecules but physiologically useless. 0 Predominant cause of alpha thalassemias is large number of gene deletions in the alpha-globin gene. 0 Are four clinical syndromes present in alpha thalassemia: 0 Silent Carrier State 0 Alpha Thalassemia Trait (Alpha Thalassemia Minor) 0 Hemoglobin H Disease 0 Bart's Hydrops Fetalis Syndrome

85 Alpha thalassemia

86 Cont…

87 ALPHA THALASSEMIA  Deletion of one alpha gene, leaving three functional alpha genes.  Alpha/Beta chain ratio nearly normal.  No hematologic abnormalities present.  No reliable way to diagnose silent carriers by hematologic methods; Must be done by genetic mapping.  May see borderline low MCV (78- 80fL). Silent Carrier state

88 Beta thalassemia



91 Anemia Diagnosis of Anemia: History- Information solicited by the physician which should include: Dietary habits Medications taken Possible exposure to chemical or toxins The most complaint is tiredness. Muscle weakness and fatigue when there is not enough oxygen available to burn fuel for production of energy. When no oxygen to the brain, headache vertigo and syncope may occur. Physical Examination : General Signs Organomegaly of the spleen and liver may develop since they are important in hematopoietic system production and destruction. Heart abnormalities may occur as a result of increased cardiac workload associated with the physiologic adaptations to anemia. Specific Signs Jaundice in hemolytic anemias

92 Red Cell Indices MCH, MCV, MCHC, RDW- These indices are used for classifying anemias. MCH- Mean Cell Hemoglobin -is derived from the Hb devided by RBC Hb (g/dl) x 10 ÷ RBC (10¹²/l) MCV-Mean Cell Volume -is calculated by deviding the PCV by RBC PCV (l/l) x 1000 ÷ RBC (10¹²/l) MCHC-Mean Cell Hemoglobin Concentration -is derived from the Hb, MCV, and RBC Hb (g/dl) ÷ PCV (l/l) RDW- red Cell distribution Width is derived from pulse height analysis can be expressed either as standard deviation or as the coefficient variable (CV) (%)


94 Chapter 5

95 Collecting and Handling of Blood

96 Phlebotomy Technician


98 THE VASCULAR SYSTEM ARTERIES Have thick walls to withstand the pressure of ventricular contraction, that creates a pulse Normal systemic arterial blood is bright red. VEINS have thinner walls because blood in them is under less pressure Collapse more easily Dark bluish red (oxygen poor) Capillary only one cell Can easily be punctured to provide blood specimen

99 VASCULAR ANATOMY (phlebotomy related) 2 basic patterns of the veins

100 VASCULAR ANATOMY (phlebotomy related)


102 OTHER VEINS: Veins on the back of the hand or at the ankle may be used, although these are less desirable and should be avoided in diabetics and other individuals with poor circulation. Leg, ankle and foot veins are sometimes used but not without permission of the patient’s physician due to potential medical complications

103 SOURCE AND COMPOSITION OF BLOOD SPECIMENS ARTERIAL BLOOD Primarily reserved for blood gas evaluation and certain emergency situations VENOUS BLOOD affected by metabolic activity of the tissue it drains and varies by collection site chloride, glucose, pH, CO2, lactic acid and ammonia levels differ may from arterial blood CAPILLARY BLOOD Contains arterial and venous blood plus tissue fluid Calcium, potassium and total protein are normally lower

104 TYPES OF BLOOD SPECIMENS SERUM- Serum is that part of blood which is similar in composition with plasma but exclude clotting factors of blood. PLASMA- Plasma is considered as the medium of blood in which RBCs (Red Blood Cells), WBC (White Blood Cells) and other components of blood are suspended WHOLE BLOOD

105 VENIPUNCTURE EQUIPMENT Venipuncture can be performed by 3 basic methods Evacuated tube system (ETS) – most preferred because blood is collected directly from the vein in the tube, minimizing the risk of specimen contamination and exposure to the blood Needle and syringe – used on small, fragile and damaged veins Winged infusion set (butterfly ) – can be used with the ETS and syringe Used to draw blood from infants and children, hand veins and other difficult to draw situations

106 VENIPUNCTURE EQUIPMENT 1. Tourniquet Applied to a patient’s arm during venipuncture Distends the veins, making them larger and easier to find, stretches the wall so they are thinner and easier to find Must not be left on longer than 1 minute because specimen quality may be affected 2. Needles 3. Evacuated Tube System (Vacutainer)

107 The bevel is the slanted opening at the end of the needle. bevel of the needle must face upward when the needle is inserted into the vein. VENIPUNCTURE EQUIPMENT

108 Tube Additives A.Anticoagulants Prevent blood from clotting and include EDTA, citrates, heparin and oxalates B. Antiglycolitic agents Prevent glycolysis which can decrease glucose concentration by upto 10 mg/dl per hour Sodium fluoride : most common antiglycolitic agent Preserves glucose for upto 3 days, and inhibits bacterial growth C. Clot activators Are coagulation factors like thrombin Glass particles (silica) Inert clays ex. Diatomite (celite) Enhance clotting by providing more surface for platelet activation

109 ORDER OF DRAW AND ADDITIVE CARRY OVER COMMON TESTS AFFECTED BY ADDITIVE CONTAMINATION Citrate – ALP, Ca,Phosporus EDTA - ALP, Ca, CK,PTT,K,PT,Serum Iron, Na Heparin – Activated CT, ACP, Ca, PT, PTT Na, Li Oxalates- ACP, ALP, Amylase,Ca, LDH, PT, PTT, K, Red cell Silica (clot activator) – PTT, PT Sodium fluoride – Na, BUN

110 PROCEDURAL ERROS RISKS 1.Hematoma formation - rapid swelling near the venipuncture site due to blood leaking into the tissues Situations that can trigger hematoma formation?

111 Capillary Specimen Collection Collection sites 1.Fingers – adults and children over the age of 2 2.Heels - infants

112 Neonatal Bilirubin Collection – must be protected from light Neonatal Screening screens for phenylketonuria, a disorder which could be managed by dietary adjustment if diagnosed early.


114 End

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