3Introduction 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 weightAverage volume of 5 liters in womenAverage volume 5.6 liters in men99% of blood cells are erythrocytesPlasma 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
4HematopoiesisThe 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 clotsHematopoiesis (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.
8Cont…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 erythroblastsHematopoiesis in the bone marrow is called medullary hematopoiesis.
9Hematopoietic Tissues 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 defenseCulling(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)
10CullingThe discriminatory filtering and destruction of senescent or damaged red cells by the spleenATP 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.
11Cont…PittingThe 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.
12For 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 erythrocytesHeinz 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.
14Cont…DefenseSpleen is rich supply of lymphocytes and phagocytic cells as well as its unique circulationReservoir for plateletsMassive 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 sequestrationRemoval 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 formationThe 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.
15HypersplenismUnder 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:Anemia, leukopenia or thrombocytopenia in the blood.2. Cellular or hyperplastic bone marrow ( increase in normal cells of the tissue)corresponding to the peripheral blood cytopenias.The occurrence of the splenomegaly4. The correction of cytopenia as following splenectomy.
16Hypersplenism Primary 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 splenomegalyPrimaryOccurs 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
17LymphnodesThe 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 lymphocytesThe cortex contains the T-lymphocytesThe 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
18Thymus3. ThymusThe 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.
19Bone 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 4:1For 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.
20The 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 activityCellular 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.
22Derivation 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.
24Cont…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.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.
25Cont…(Erythrocytic, myelocytic or megakaryocytic) It has been suggested that nodules appearing at 7th days were formed from more mature unipotent committed stem cellsIn 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
26ErythropoiesisThe 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.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.Any remaining nuclear material is removed by a process of pitting during passage through the splenic sinus walls.The successive cytoplasmic charge from blue to pink earning them name basophilic, polychromatic and orthochromaticAfter 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).
27Kinetics 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 hoursThis 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 erythropoiesisErythropoietin- 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.
28Cont…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, cobaltAmino acidsHormone: erythropoeitin, thyroxine and androgens
29ErythrocytesThe mature erythrocytes of the peripheral blood of a man are:Non-nucleated and contain organellesContain enzyme of both anearobic glycolytic pathway of Embden-Myerhoff and aerobic pentose P pathwayIt 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.
30Erythrocytes 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)
32Cont…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)SphengomyelinPhosphatidylserine
33Cont…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 membraneTwo 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 layerThese 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.
34Erythrocyte 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.
35Metabolic Pathways These pathways include: Embden-Meyerhof pathway Hexose-monophosphate (HMP) shuntMethemoglobin reductase pathwayRapoport-leubering pathwayThese pathways contribute:The first pathway for providing energy for maintaining high intracelllur K+ , low intracellular Na + and very low Ca2+ (cation pump)The second pathway provides reducing power to protect Hb in reducing state.The third pathway regulates oxygen affinity of HbMaintains Hb in reduced state
36Embden-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’sAre necessary to maintain red cell shape and flexibility.Membrane integrity through regulation of intracellular cation concentration:Na+ & Ca2+ are more concentrated in the plasma.K+ is more concentrated within the cell.Erythrocyte osmotic equilibrium is maintained by:The selective permeability of the membraneBy the cation pumps located in the cell membrane
37Cont… The Na+ & K+ pump ADP + Pi hydrolysisThe Na+ & K+ pump ADP + PiIn the expulsion of 3 Na+ and the uptake of 2K+ .Ca2+ is maintained in low concentration by the action of a similar but separate cation pump that utilized ATP for fuel.Excess leakage of Ca2+ 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 Ca2+ - 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 deathOne ATP
38Hexose 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 (H2O2).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.
39Methemoglobin 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 O2Methemoglobin 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.
40Rapoport-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 O2 to the tissues by causing a decrease in Hb affinity for oxygen. Thus, the red cell has built-in mechanism for regulation of O2 delivery to the tissues
44Lifespan and Faith of RBC Causes of reduction in the life span of RBC.Defects in RBC (curpuscular defects)e.g. Hereditary spherocytosisSickle cell anemia,Thalassemias2. Deficiency of redcell enzyme such as: G6PD,Pyruvate kinase def.,Autoimmune disorder,HypersplenismNormally, the average life span of red blood cells is 120 days. This cells live in blood circulation.In in typical adult produces 200 billion RBC per day.At the end of their lifespan, they become senescent, and are removed from circulation.In many chronic diseases, the lifespan of the erythrocytes is markedly reduced (e.g. patients requiring haemodialysis).The destruction of RBC is about 2-3 million per second on average.
45Cont… The fate of the RBC After 120 days , the RBC becomes more fragile due to decrease NADPH activity.Younger RBC RBCs can easily pass through the capillaries which have the diameter smaller than the mature RBC’cWith a fragile membrane, the mature RBC are destroyed while trying to squeeze through capillaries.The destruction most occurs in the capillaries of the spleen. This is why spleen is called the “grave yard of the RBC”.The hemoglobin is released and taken up the macrophargesPathway of RBC destruction
47Bilirubin Is the yellow breakdown product of normal heme catabolism. Bilirubin is excreted in bile and urine, and elevated levels may indicate certain diseases. It is a toxic waste product in the bodyIt is extracted and biotransformed mainly in the liver and excreted in bile and liver.Elevation in serum and urine bilirubin is associated with juandice
504/13/2017Heme is degraded by reticuloendothelial cells (mononuclear phagocytes of the spleen, liver, and bone marrow).Bilirubin is insoluble in water and is responsible for the toxic effects. This unconjugated (indirect) bilirubin is transported in the serum bound to albumin.
51In BloodThe bilirubin synthesized in spleen, liver and bone marrow is unconjugated bilirubinIt is hydrophobic in nature so it is transported to the liver as complex with the plasma protein, albuminUnconjugated bilirubin(Free Bilirubin)Lipid soluble1gm albumin binds 8.5 mg of bilirubinFatty acids and drugs can displace bilirubinIndirect positive reaction in van den Berg test
52Unconjugated (indirect) Erythrocytes generated in the bone marrow are disposed of in the spleen when they get old or damaged.This releases hemoglobin, which is broken down to heme as the globin parts are turned into amino acids.The heme is then turned into unconjugated bilirubin in the reticuloendothelial cells of the spleen.This unconjugated bilirubin is not soluble in water, due to intramolecular hydrogen bonding. It is then bound to albumin and sent to the liver.Conjugated (Direct) bilirubin is released into the bile by the liver and stored in the gallbladder, or transferred directly to the small intestines.Bilirubin is further broken down by bacteria in the intestines, and those breakdown products contribute to the color of the feces.A small percentage of these breakdown compounds are taken in again by the body, and eventually appear in the urine
63Conjugated bilirubin - water soluble - direct reaction with dyes 4/13/2017Bilirubin Lab valuesBilirubin formNormal valueTotal (elderly, adult, child)(newborn)Critical value (a(newborn)0.1 to 1.0 mg/dL1.0 to 12.0 mg/dL>12 mg/dL>15 mg/dLPre-hepatic, unconjugated, indirect0.0 to 0.8 mg/dLPost-hepatic, conjugated, direct0.0 to 0.25 mg/dLFecal urobilinogen40 to 280 mg/dayUrine0.0 to 0.02 mg/dLConjugated bilirubin - water soluble - direct reaction with dyesUnconjugated bilirubin - water insoluble - alcohol is needed for dye (indirect) reactionObserve the color changes associated with heme degradation by watching the progress of a bruise (dark red to green to yellow).Elevated serum bilirubin levels - increased production, decreased conjugation, decreased secretion by the liver, or blockage of the bile ducts. In cases of increased production, or decreased conjugation, the unconjugated or indirect form of bilirubin will be elevated.Unconjugated hyperbilirubinemia - accelerated erythrocyte hemolysis in the newborn (erythroblastosis fetalis), absence of glucuronyl transferase, or hepatocellular disease.Conjugated hyperbilirubinemia - obstruction of the biliary ducts, as with gallstones or hepatocellular diseases such as cirrhosis or hepatitis.Elevated serum bilirubin test results may also be caused by the effects of many different drugs, including antibiotics, barbiturates, steroids, or oral contraceptives.Chronic acquired liver diseases, the serum bilirubin concentration is usually normal until a significant amount of liver damage has occurred and cirrhosis is present.Acute liver disease, the bilirubin is usually increased in relation to the severity of the acute process.
66Other causes: Inadequate production of mature red cells Deficiency of essential substances like iron, folic acid, vit B12, protein and other elements like copper, cobalt, etc.Deficiency of erythroblast.. i.e.Aplastic (anemia body's bone marrow does not make enough new blood cells.), Pure red cell aplasiaInfiltration of the bone marrow i.e. leukemia, lymphoma, carcinoma, myelofibrosisEndocrine abnormalities i.e. myxedema, Addison's disease, pituitary insufficiencyChronic renal diseaseChronic inflammatory diseaseCirrhosis of liver* Pure red cell aplasia (PRCA) is an uncommon disorder in which maturation arrest occurs in the formation of erythrocytes.
67The etiologic possibilities are Iron deficiency Thalassemia Hypochromic Microcytic AnemiaNormochromic Normocytic AnemiaThe etiologic possibilities areIron deficiencyThalassemiaSideroblastic anemia (abnormal normoblasts with excessive accumulation of iron in the mitochondria)Anemias of chronic disease.Severe microcytic anemia (MCV <70 fL) iscaused mainly by iron deficiency or thalassemia.Chronic inflammatory disease— (1)infection (2)collagen vascular disease (3)inflammatory bowel diseaseRecent blood lossMalignancy/Marrow infiltrationChronic renal failureTransient erythroblastopenia of chidhood (A decrease in the number of erythroblasts in bone marrow, as seen in aplastic anemia)Marrow aplasia/hypoplasiaHIV infectionHemophagocytic syndrome
68Macrocytic 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, sulfaHemolysisMyelodysplastic 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 anemiaChronic liver diseaseHypothyroidismMacrocytic 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 lossThe 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
69Iron Deficiency Anemia 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.The incidence of anemia in the general population is about 1.5%.Iron deficiency related to inadequate replacement of lost iron is the most frequent cause of asymptomatic anemia and has a variety of causes.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).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.
70Iron Iron Store Function as electron transporter; Vital for life Must be in ferrous (Fe++) state of activity.In anaerobic condition, easy to maintain ferrous stateIron readily donates electrons to oxygen, superoxide radicals, H2O2 OH RadicalsFerric (Fe+++) ions cannot transport electrons or O2.Organisms able to limit exposure to iron had major survival advantageIron Store
71Cont… Iron absorption Duodenum Proximal jejunum (influenced by rate of erythropoiesis)Factor Affecting Iron AbsorptionForm of ironAcidsAmount of ironRate erythropoiesis
72Sickle-cell anemia is a molecular disease of Hb Comparison of normal and sickle-shaped erythrocytes
77Characterization of HbS HbS has between 2 & 4 more net + charges per molecule than net HbAASpI of Oxy Hb6.877.09= 0.22pI of deoxy Hb6.886.91=0.23Non-polar residue on the outside of HbS (due to Val) causing low solubilitySticky patch on the outside of its β chains & are present on both deoxy HbS & oxy HbS but not on HbA
79Signs and Symptoms of Sickle cell Episodes of painPain develops when sickle-shaped redblood cells block blood flow through tinyblood vessels to your chest, abdomen andjoints. Pain can also occur in your bones. Frequent infectionsSickle cells can damage your spleen, anorgan that fights infection. This may makeyou more vulnerable to infections. Vision problemsTiny blood vessels that supply your eyesmay become plugged with sickle cells. Thiscan damage the retina — the portion of theeye that processes visual images.
80Prevention of Sickle Cell Disease Screen for Hb S at birth. This method of finding allows institution of early treatment and controlPrenatal diagnosis. Prenatal testing is sensitive and rapid and must be accompanied with genetic testing and psychological counseling
81ThalassemiaDiverse group of disorders which manifest as anemia of varying degrees.Result of defective production of globin portion of hemoglobin molecule.Distribution is worldwide.May be either homozygous defect or heterozygous defect.Defect results from abnormal rate of synthesis in one of the globin chains. Globin chains structurally normal (is how differentiated from hemoglobinopathy), but have imbalance in production of two different types of chains.Results in overall decrease in amount of hemoglobin produced and may induce hemolysis.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.May contribute protection against malaria.
82Cont… Also called Alpha Thalassemia Minor. Caused by two missing alpha genes. May be homozygous (-a/-a) or heterozygous (--/aa).Exhibits mild microcytic, hypochromic anemia.MCV between fL.May be confused with iron deficiency anemia.Although some Bart's hemoglobin (γ4) present at birth, no Bart's hemoglobin present in adults.
84Alpha thalassemiaHas wide range clinical expressions.Is difficult to classify alpha thalassemias due to wide variety of possible genetic combinations.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 (γ4) or Hemoglobin H (β4), which are stable molecules but physiologically useless.Predominant cause of alpha thalassemias is large number of gene deletions in the alpha-globin gene.Are four clinical syndromes present in alpha thalassemia:Silent Carrier StateAlpha Thalassemia Trait (Alpha Thalassemia Minor)Hemoglobin H DiseaseBart's Hydrops Fetalis Syndrome
87ALPHA THALASSEMIA Silent Carrier state 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).
91AnemiaPhysical Examination :General SignsOrganomegaly 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 SignsJaundice in hemolytic anemiasDiagnosis of Anemia:History- Information solicited by the physician which should include:Dietary habitsMedications takenPossible exposure to chemical or toxinsThe 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.
92Red Cell Indices MCH, MCV, MCHC , RDW- These indices are used for classifying anemias.MCH- Mean Cell Hemoglobin-is derived from the Hb devided by RBCHb (g/dl) x 10 ÷ RBC (10¹²/l)MCV-Mean Cell Volume-is calculated by deviding the PCV by RBCPCV (l/l) x 1000 ÷ RBC (10¹²/l)MCHC-Mean Cell Hemoglobin Concentration-is derived from the Hb, MCV, and RBCHb (g/dl) ÷ PCV (l/l)RDW- red Cell distribution Widthis derived from pulse height analysis can be expressed either as standarddeviation or as the coefficient variable (CV) (%)
98THE VASCULAR SYSTEM VEINS have thinner walls because blood in them is under less pressureCollapse more easilyDark bluish red (oxygen poor)ARTERIESHave thick walls to withstand the pressure of ventricular contraction, that creates a pulseNormal systemic arterial blood is bright red.Capillaryonly one cellCan easily be punctured to provide blood specimen
99M H 2 basic patterns of the veins VASCULAR ANATOMY (phlebotomy related)2 basic patterns of the veinsMH
100VASCULAR ANATOMY (phlebotomy related) H PATTERN
102OTHER VEINS: VASCULAR ANATOMY (phlebotomy related) 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
103SOURCE AND COMPOSITION OF BLOOD SPECIMENS ARTERIAL BLOODPrimarily reserved for blood gas evaluation and certain emergency situationsVENOUS BLOODaffected by metabolic activity of the tissue it drains and varies by collection sitechloride, glucose, pH, CO2, lactic acid and ammonia levels differ may from arterial bloodCAPILLARY BLOODContains arterial and venous blood plus tissue fluidCalcium, potassium and total protein are normally lower
104TYPES 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 suspendedWHOLE BLOOD
105VENIPUNCTURE EQUIPMENT Venipuncture can be performed by 3 basic methodsEvacuated 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 bloodNeedle and syringe – used on small, fragile and damaged veinsWinged infusion set (butterfly) – can be used with the ETS and syringeUsed to draw blood from infants and children, hand veins and other difficult to draw situations
106VENIPUNCTURE EQUIPMENT 1. TourniquetApplied to a patient’s arm during venipunctureDistends the veins, making them larger and easier to find, stretches the wall so they are thinner and easier to findMust not be left on longer than 1 minute because specimen quality may be affected2. Needles3. Evacuated Tube System (Vacutainer)
107The bevel is the slanted opening at the end of the needle. VENIPUNCTURE EQUIPMENTThe 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.
108Tube Additives Anticoagulants B. Antiglycolitic agents VENIPUNCTURE EQUIPMENTTube AdditivesAnticoagulantsPrevent blood from clotting and include EDTA, citrates, heparin and oxalatesB. Antiglycolitic agentsPrevent glycolysis which can decrease glucose concentration by upto 10 mg/dl per hourSodium fluoride : most common antiglycolitic agentPreserves glucose for upto 3 days, and inhibits bacterial growthC. Clot activatorsAre coagulation factors like thrombinGlass particles (silica)Inert clays ex. Diatomite (celite)Enhance clotting by providing more surface for platelet activation
109COMMON TESTS AFFECTED BY ADDITIVE CONTAMINATION ORDER OF DRAW AND ADDITIVE CARRY OVERCOMMON TESTS AFFECTED BY ADDITIVE CONTAMINATIONCitrate – ALP, Ca,PhosporusEDTA ALP, Ca, CK,PTT,K,PT,Serum Iron, NaHeparin – Activated CT, ACP, Ca, PT, PTT Na, LiOxalates- ACP, ALP, Amylase,Ca, LDH, PT, PTT, K, Red cellSilica (clot activator) – PTT, PTSodium fluoride – Na, BUN
110PROCEDURAL ERROS RISKS Hematoma formation- rapid swelling near the venipuncture site due to blood leaking into the tissuesSituations that can trigger hematoma formation?
111Capillary Specimen Collection Collection sitesFingers – adults and children over the age of 2Heels - infants
112Neonatal Bilirubin Collection – must be protected from light Neonatal Screeningscreens for phenylketonuria, a disorder which could be managed by dietary adjustment if diagnosed early.