PH 184 CLINICAL MICROSCOPY

DEFINITION OF HEMATOLOGY
Hematology Scientific study of the structure and functions of blood in health and disease Analysis of concentration, components, and function of blood cells, plasma, serum A laboratory science in which the different components of blood are quantitatively and qualitatively observed in order to diagnose a great variety of diseases of humans and animals

Why study Blood? 1. 2. 3. 4. Easy to collect Easy to collect more if needed Still alive once collected & can still survive outside the body with minimum treatment Blood consists of a wide spectrum of different cells Each group with its own structure and functions and unique genetic markers on its membranes and inside its cells.

Importance of Blood in Homeostasis: Blood is an integral part of all other body tissues, any change in blood homeostasis will have an effect on the other tissues. Blood is in such close contact with all other body tissues, any change in these tissues will be reflected in the blood.

Scope of Hematology: 1. 2. 3. 4. 5. 6. 7. 8. Origin and development of the various components of blood. Structure of the various components of blood. Functions of the various components of blood. Regulations of levels of the various components of blood. Normal variations of blood components. Diseases associated with qualitative and quantitative variations of blood components. Understanding the hemostatic mechanism. Immunohematology and blood transfusions.

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BLOOD COMPOSITION
Blood highly specialized tissue, along circulatory system, adapted to meet needs of body tissues & organ systems Characteristics: Volume: 5 6 L ; total body weight pH: 7.35 7.45 slightly alkaline Viscosity: 3 5x thicker than water Components: Solid (2 L cells) Liquid (3 L plasma) Functions: Transportation Regulation Protection

Blood Components: A. LIQUID COMPONENT 1. Water 91 % of plasma Functions: a. solvent b. vehicle for transport of blood cells and other components c. temperature regulation Latent Heat of Vaporization heat absorbed when substance changes phase from liquid to gas keeps body temperature constant 2. Plasma proteins 7% of plasma Classification: a. Function Enzymes Immunoglobulins (antibodies) Enzyme inhibitors Coagulation factors Transport proteins Fibrinolytic factor Complement factors b. Structure Simple proteins made up of polypeptide material only Complex proteins w/ added materials to polypeptide core Glycoproteins Lipoproteins *Electrophoresis technique of separation of proteins in plasma *Densitometer quantitatively measures the plasma protein due to the stain Functions of Plasma Protein: a. Transport Carrier proteins where substances attach to, in order to reach target organs prevents degradation of substance before reaching goal of performed function as a stimulus b. Regulation of the movement of water between the intravascular and extravascular fluid compartments Mainly the function of albumins Albumins determine how much fluid stays w/in blood vessels & moves into interstitial spaces Osmotic pressure

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c. Coagulation Certain plasma proteins prevent excessive loss of blood upon vascular injury by plugging any breaks in the circulatory system Fibrinogen most important clotting factor Found in beta-globulin fraction of plasma Found in large quatities of blood: 200 400 mg/dL (=100mL) Fibrinolytic Factor prevent spontaneous clot in the blood vessels Ensures that coagulation process is limited to the area of the break in the circulation & does not spill over into other areas of the circulatory system Plasminogen most important fibrinolytic factor classified as an alpha2-globulin d. Immunoglobulins / Antibodies Produced by certain lymphocytes Play major role in bodys defense against invading pathogens Found mainly gamma-globulin fraction of the plasma e. Inflammation 3. Electrolytes function: for maintenance of acid-base balance of the body 4. Miscellaneous: sugars, fats, vitamins, hormones B. SOLID PHASE: CELLULAR ELEMENTS 1. Red Blood Cells / Erythrocytes Biconcave discs Thicker at edges than in middle Average: 7.5 m in diameter; 2 m thick at edges function: transport oxygen to the respiring tissues and carry CO 2 away from tissues Structure Function Relationship: biconcave shape gives greater surface area to facilitate gas exchange smooth round shape produces little friction through narrow capillary tubes large hemoglobin content Hemoglobin respiratory pigment that allows O2 & CO2 to be carried in RBC number present in bloodstream 1000 times more RBC than WBC in bloodstream 3 Women: 4.0 5.5 M/mm 3 Men: 4.5 6.0 M/mm 3 Newborn: 5.5 6.5 M/mm 2. White Blood Cells / Leukocytes form heterogeneous group of cells less numerous in blood than RBC 5,000 10,000 WBC/ L blood

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Divisions (morphological point of view): a. Granulocytes Further divided accdg to staining characteristics of cytoplasmic granules: Neutrophils most numerous WBC 55 75% of total leukocyte population in normal individuals 90 95% of all granulocytes easily recognized in stained blood smear (using Wrights Stain) numerous small purple granules in the cytoplasm & nucleus divided into 3-5 interconnected lobes also called PMNs (Polymorphonuclear Neutrophils) function: ingest and destroy invading pathogenic organisms st 1 line of defense against microbes short life span makes them not very efficient in destroying infectious agents slows down infection and hold it locally Eosinophils many large red granules present in cytoplasm granules stain bright orange-red (because it contains many basic proteins w/ great affinity for red acidic dye eosin present in Wrights Stain) nucleus does not stain as deeply & lobulate as much as that of neutrophils nucleus usually has 2 lobes capable of phagocytosis (although ingestion not major function) function: attack parasitic worms, specifically larval forms of schistosomes or blood flukes number per microliter < neutrophils in every 100 WBCs counted in a blood smear, only 1 3 eosinophils classified

Basophils cytoplasmic granules stain a deep blue-black w/ methylene blue (granules contain acidic molecules) nucleus appears deeply indented rather than lobulated least common WBC in bloodstream <1% of total leukocyte population not considered phagocytic also called Mediator Cell closely related to Mast cell found in connective tissue have granules containing vasoactive amines (histamine & serotonin) function: play najor role in infection & certain types of allergies by producing states of acute inflammation

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b. Lymphocytes nd 2 most common group of WBC in blood 20 35% of total leukocyte population relatively small cells, only slightly larger than RBC has a large densely stained nucleus surrounded only by a tiny amount of cytoplasm large round or oval nucleus almost completely fills the cell function concerned with immune system

Division (functional point of view): Natural Killer Cells LGLs (Large Granular Lymphocytes) o lymphocytes with more cytoplasm surrounding the nucleus & cytoplasm containing reddish-purple granules o usually confused with monocytes < 10% of blood lymphocytes lack specific B- and T-cell receptors attack most virus-infected & neoplastic cells without needing to be primed for attack by specific antigens derived from bone marrow B Lymphocytes / B Cells 10 15% of blood lymphocytes Function: produce antibodies for humoral immunity st 1 described as originating from bursa of Fabricius in birds (bursal equivalent in mammals is the bone marrow)

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T Lymphocytes / T Cells most lymphocytes in peripheral bloodstream 75 80% of all lymphocytes in the blood originally derived from Thymus Gland a. TC Cells (Cytotoxic T Cells) mainly responsible for cell-mediated immunity (CMI) attack and destroy cells that have been invaded by viruses and other pathogenic organisms also attack neoplastic / cancerous cells b. TH Cells (Helper T Cells) activate B cells and TC Cells c. Monocytes largest of the circulatory WBCs diameter: 17 - 24m nucleus is twisted into a C or S shape few in peripheral bloodstream 2 6% of all WBC Few in blood but many in tissues (macrophages) Functions: 1. long-lived cells efficient in phagocytosis and killing of microbial organisms 2. antigen-presenting cells (APCs) - activate immune system by engulfing & presenting to appropriate B cells and T cells & start producing antibodies & lymphokines 3. clean up and remove dead pathogens and destroyed cells after an infection is over 3. Platelets / Thrombocytes not cells but small packages of cytoplasm nipped off from large mother cells in the bone marrow (Megakaryocytes) smaller than red blood cells (2m in diameter) more fragile than average blood cell average person: 250,000 (or 150,000 400,000) platelets/L blood usually round or oval with very invaginated surface area, allowing adherence of clotting factors Functions: a. Instrumental in maintaining the integrity of the endothelial lining of the blood vessels b. Repair of any breaks in the vascular system, especially at the microcirculation level

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HEMATOPOIESIS
Hematopoiesis Process of development of blood cells (Greek) haima Blood + poiesis making or creating Ontogeny hematopoiesis in embryonic point of view Phylogeny evolution of blood cells in animal kingdom Erythropoiesis proliferation and differentiation of red blood cells Leukopoiesis development of WBCs Thrombopoiesis development of platelets Pluripotent Stem Cell (PPSC) common ancestral cell in bone marrow where blood cells are derived form; can develop either a: a. Lymphoid Stem Cell from which all lymphocytes are ultimately derived b. Myeloid Stem Cell precursor of all other blood cells Development of early lymphocytes take place either in: a. Bone Marrow NK cells & B Cells b. Thymus Gland T Cells ONTOGENY OF BLOOD CELLS / HEMATOPOIESIS Mesenchymal cells present in the yolk sac; where all human blood cells are ultimately derived from 3 week of Gestation: mesodermal cells aggregate to cluster of cells (blood islands) Liver 1 organ stem cells migrate to becomes the main source of hematopoiesis after the yolk sac disappears rd th major organ of blood cell formation from 3 Lunar month of pregnancy to 6 month 6
th st rd

month of Gestation: bone marrow becomes major organ of hematopoiesis

rd th

Spleen becomes organ of hematopoiesis on the 3 month of gestation to 7 month At birth: bone marrow becomes sole source of blood cell formation Lymph nodes and Thymus Gland th invaded by stem cells on the 4 month of gestation remain as secondary source of lymphocyte production throughout life

Types of Ontogeny of Hematopoiesis: Medullary Hematopoiesis blood cell production in the bone marrow Flat bones:sternum, ribs, skull, vertebrae, & innominates Extramedullary Hematopoiesis blood cell production outside the bone marrow

lymphoid organs

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PHYLOGENY OF BLOOD CELLS / HEMATOPOIESIS Techniques for Bone Marrow Study: 1. Aspiration Most common Needle inserted into to the soft center of the bone and small quantity of bone marrow is aspirated Sample is smeared on a slide, fixed, stained and examined Adult: iliac rest Children / infants: tibia Disadvantages: disturbs the bone marrow architecture 2. Biopsy A piece of bone marrow is removed intact w/o disturbing the architecture of the bone Done w/ fairly thick, hollow biopsy needle & a core sample of bone marrow is taken Sample is fixed, embedded in paraffin, sectioned, stained, & examined Disadvantages: more dangerous & traumatic due to much wider needle used Advantage: gives better picture of real structure of bone marrow Paul Erlich German scientist Postulated that there were two cell lines: 1. Myeloid Line from which all cells produced in the bone marrow were derived 2. Lymphoid Line from which the lymphocytes originated Theories of Hematopoiesis 1. Polyphylogeny / Dualistic Theory there are different stem cell for each of the major blood cell lines 2. Monophylogeny / Unitarian Theory all blood cells develop from a single ancestral cell Cytokinetics study of methods by which cell populations proliferate concerned with: mass or size, maturation time, life span, and turnover rate 3 Categories of Proliferation: 1. Constantly Dividing Cells skin epidermis, sperm cells, hematopoietic cells of bone marrow & lymphocytes 2. Nonproliferating Cells nerve cells & muscle cells 3. Cells that proliferate only on demand & divide only when called upon to do so by an external stimulus 2 Patterns of Bone Marrow Cell Proliferation: 1. Steady-State Pattern proliferation at constant rate maintained by growth factors or cytokines produced by stromal cells of bone marrow 2. Non-Steady-State Pattern proliferation behavior is changed and results in either increase or decreased cell production, depending on the physiological and pathological condition of the individual

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RED BLOOD CELL STUDIES

Erythropoiesis process of development and maturation of the red blood cells

Erythropoietin (EPO) humoral agent which regulates the feedback lop between the oxidizing tissues and the bone marrow when the amount of oxygen decreases, the rate of erythropoiesis is stepped up hormone discovered in blood and urine of anemic rats by Erslev function: a. stimulate committed stem cells to differentiate into erythroblasts or normoblasts b. stimulate synthesis of messenger RNA

Maintain Normal Red Blood Cell Levels: 1. Erythropoietin 2. IL-1 3. IL-3 4. IL-4 5. IGF (Insulinlike Growth Factor) 6. PDGF (Platelet-derived growth factor) 2 Types of Erythropoiesis: 1. Effective 2. Ineffective extent that the process of erythropoiesis fails to deliver cells to the circulating blood

Stages of Erythrocyte Development: 1. Pronormoblast / Proerythroblast / Rubriblast Stage name given to committed erythroid stem cell fairly large, varying in diameter from 12 14 m nucleus prominent ; contains coarse chromatin & one or more nucleoli nucleus large relative to cytoplasm & round with smooth nuclear membrane stained with Wrights stain, cytoplasm is deep blue and lack inclusions

2. Basophilic Normoblast / Basophilic Erythroblast / Prorubricyte Stage may be similar in size or smaller than pronormoblast chromatin of nucleus is coarser cytoplasm also stains deep blue with Wrights stain called such due to high affinity to basic dye methylene blue distinguishable feature: absence of nucleoli in the nucleus

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3. Polychromatophilic Normoblast / Polychromic Erythroblast / Rubricyte Stage smaller than 2 previous cell types diameter about 10 m distinguishable in 2 Major Changes: a. nucleus more condensed & stains much darker; chromatin extremely coarse & shows a checkboard appearance b. cytoplasm no longer deep blue but appears much paler; produces lavender color when stained with Wrights stain stage where hemoglobin appears the first time in the cytoplasm hemoglobin causes pinkish shine between lighter blue of the cytoplasm called such due to mixture of hues in cytoplasm 4. Orthochromic Normoblast / Pyknotic Erythroblast / Metarubricyte cytoplasm almost completely salmon-pink, traces of blue have disappeared distinguishing feature: shape & size of nucleus nucleus appears as blue-clack sphere (indicating dense chromatin mass) Pyknotic Nucleus dense nucleus still called blast cell & can undergo blast formation and mitosis

5. Reticulocyte last immature erythrocyte in the maturation series called such for when stained with supravital dyes (methylene blue or brilliant cresyl blue), reticular network of strands can be observed in the cell strands visible are remnants of RNA strands irregular tangle of threadlike material disappears in a day or two, cell becomes fully matured slightly larger and less regular than the erythrocyte 6. Erythrocyte maturation of reticulocyte to adult erythrocyte: 24 48 hours ribosomes & mitochondria disappears cell loses capacity for hemoglobin synthesis & oxidative metabolism ribosomal RNA gradually degraded to extracellular ribonucleosides maturing RBC enter circulation as reticulocyte normal erythrocyte life span: 120 days

Structure of Erythrocyte Membrane Fluid Mosaic Model chemical composition of cell membrane: 50% of membrane is protein 40% is fat 10% is carbohydrate

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Lipid Matrix made up of different group of fats 60% phospholipids, 30% neutral lipids (mainly cholesterol), 10% glycolipids composed of double layer of phospholipids Phospholipids made up of polar phosphate head & nonpolar hydrophobic tail; made up of 2 fatty acids polar heads always found outside & inside of membrane nonpolar point toward the center of the membrane must have certain degree of rigidity to permit lateral movement cholesterol stabilizes the two layer structure Membrane Proteins Classified accrdg to: 1. Position a. Peripheral Proteins loosely connected with membrane may easily be separated by ultracentrifugation & other methods consist of Structural Proteins which gives erythrocytes its shape Spectrin Actin b. Integral Proteins penetrate phospholipid bilayer & cannot be separated from it w/o destroying membrane includes: Receptor Proteins Transmembranal Transport Proteins 2. Structure a. Glycoproteins - proteins that have 1 or more carbohydrates moieties attached to them 3 major types: Simple glycoproteins Large glycoproteins Protruding glycoproteins b. Lipoproteins c. Glycoplipoproteins d. Simple Polypeptides 3. Function a. Structural Proteins maintain shape of RBC (i.e. Spectrin; 20% of all membrane proteins) b. Transport Proteins multiple polypeptide structures; regulate entrance of materials into cells c. Receptor Proteins bind to hormones and other messenger molecules, forming complex that initiate changes in metabolic processes of cell Carbohydrate Moieties Present as part of other membrane molecules, i.e.: Proteins forming glycoproteins Lipids forming glycolipids Mixtures of both forming glycolipoproteins Act as antigenic determinants Important in recognition of RBC belonging to self

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