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Peripheral blood & b.m. lecture 6  2011
 

Peripheral blood & b.m. lecture 6 2011

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    Peripheral blood & b.m. lecture 6  2011 Peripheral blood & b.m. lecture 6 2011 Presentation Transcript

    • Blood- Blood is sometimes considered to be a fluidconnective tissue because of the mesenchymalorigin of its cells and a low ratio of cells to liquidintercellular substance, the blood plasma.- In human adults about 5 liter of blood contribute7-8 % to the body weight of the individual.- The contribution of red blood cells (erythrocytes)to the total volume of the blood (haematocrit) isabout 43%.- Erythrocytes are the dominant (99%) but not theonly type of cells in the blood.
    • - We also find leukocytes and, in addition, bloodplatelets.- Erythrocytes, leukocytes and blood platelets arealso being referred to as the formed elements ofthe blood..- Erythrocytes and blood platelets perform theirfunctions exclusively in the blood stream.- In contrast, leukocytes reside only temporarily inthe blood.- Leukocytes can leave the blood stream throughthe walls of capillaries and venules and entereither connective or lymphoid tissues.
    • Whole Blood Centrifuged with AnticoagulantPlasmaBuffy Coat(54%)(1%)Red Cells (45%)H2OProteinElectrolytesNutrientsBlood GasesHormones-91-92%-7-8%-7-8%(white cells and platelets)(erythrocytes)Serum - plasma minus fibrinogenRed Cells6/ mm3White Cells - 6-9 X 103/ mm3Hematocrit (volume RBC’s/unit plasma)males - 40-50%females- 35-40%
    • Red Blood Cells (erythrocytes)7.5-8.5 um dia. Biconcave Discsstagnant blood- rouleauxrapidly-flowing bloodErythrocyte Shape Varies with Tonicityhypertonic isotonic hypotonic very hypotonic(spheres) (ghosts)Sickle Cells(valine-glutamine subst.b-hemoglobin chain)Spherocytosis(spectrin alteration)
    • Erythrocytes- Erythrocytes do not contain a nucleus.- They do contain haemoglobin, which fills almost theentire cytoplasm- Erythrocytes are unable to move actively, but they areremarkably elastic and can withstand deformation.- They are typically biconcave disks although their shape isinfluenced by osmotic forces.- The average diameter of the disk is ~7 µm. Sinceerythrocytes can be found in the vast majority ofhistological sections in small numbers even in perfusedtissues , they will often allow us to estimate the size ofother structures or cells.
    • - Mature erythrocytes do not contain organelles,and their cytoplasm looks fairly homogenouseven in the EM- At high magnification some granularity may bevisible in EM images.- The granular appearance is caused byhaemoglobin molecules- Foetal erythrocytes (up to the 4th month ofgestation) are larger than "adult" erythrocytes,and they are nucleated.- The later feature they share with erythrocytes ofother animal classes (e.g. amphibia and birds).
    • Functions of Erythrocytes :- Erythrocytes function in the transport of oxygen.- Haemoglobin ; the oxygen binding protein inerythrocytes, contributes about 30% of the weightof an erythrocyte.- The lifespan of an erythrocyte in the bloodstreamis 100-120 days- About 5×1011 erythrocytes are formed/destroyedeach day.
    • 1- Respiratory function2- Nutritive3- ExcretoryUrea4- Regulation of body temperature .
    • 5- Regulation of metabolism .6- Defence .Antibodies.7- Carriage and regulation of hormone secretion8- Water balance .9- Blood coagulation .
    • Microscopy of ErythrocytesBlood Smear- erythrocytes SEM- erythrocytesRED BLOODCELLS
    • Leukocytes :Leukocytes can be further subdivided into :1) granular leukocytes, i.e. neutrophils, basophils andeosinophils,2) and non-granular leukocytes, i.e. monocytes andlymphocytes- In healthy individuals the relative numbers ofcirculating leukocyte types are quite stable.- A differential leukocyte count would typicallyproduce the following cell frequencies (numbers inparentheses are the range of normal frequenciesreported in different texts) :
    • • ~ 60% neutrophils (50% - 70%)• ~ 3% eosinophils (>0% - 5%)• ~ 0.5% basophils (>0% - 2%)• ~ 5% monocytes (1% - 9%)• ~ 30% lymphocytes (20% - 40%)- Changes in their relative numbers indicate thatsomething abnormal is happening in the organism.- A larger than usual number of neutrophils(neutrophilia) would indicate e.g. an acute or chronicinfection.- The number of basophils and eosinophils may increase(eosinophilia or basophilia) as a consequence of e.g.allergic disorders.
    • Granular Leukocytes :- Granular leukocytes are all approximately the same- size about 12-15 µm in diameter.- Their nuclei form lobes, and nucleoli cannot be seen.- The number of nuclear lobes varies according to cell type.- All granulocytes are motile.- The term granulocytes refers to the presence of granules inthe cytoplasm of these cells.- The granules correspond to secretory vesicles andlysosomes- Specific granules are the granules which are only found inone particular type of granulocytes.
    • GRANULAR LEUKOCYTESSpecific Granules(lysosomes & phagocytins)Azurophil Granules(lysosomes)Neutrophil (PMN) (55-65%)(polymorphonuclear leukocyte)EctoplasmicLayerLobesofNucleusSpecificGranulesPseudopodiaNeutrophil
    • Neutrophil Granulocytes ( or neutrophils)- Have a very characteristic nucleus.-It is divided into 3-5 lobes which areconnected by thin strands of chromatin.-The number of lobes increases with cell age.Up to 7 lobes can be found in very oldneutrophils (hypersegmented cells).-Neutrophils (like all other granulocytes,monocytes and lymphocytes) contain all theorganelles that make up a typical cell.
    • -In addition to the usual complement of organelles,they also contain two types of granules :I) Primary granules (or A granules) containlysosomal enzymes and are likely to be primarylysosomes, although they are larger (0.4 µm) thanthe "ordinary" primary lysosome.II) Secondary granules (or B granules), thespecific granules of the neutrophils, containenzymes with strong bactericidal actions.-The specific granules of neutrophils stain onlyweakly if they are at all visible they are"neutral", hence the term neutrophil.
    • Functions of Neutrophils- Neutrophils play a central role in inflammatoryprocesses.- Large numbers invade sites of infection in response tofactors (e.g. cytokines) released by cells which reside atan infection site.- Neutrophils are the first wave of cells invading infectionsires- Receptors in their plasma membrane allow them torecognise foreign bodies, e.g. bacteria, and tissuedebris, which they begin to phagocytose and destroy.- The phagocytotic activity of neurophils is furtherstimulated if invading microorganisms are "tagged" withantibodies (or opsonised).
    • -Neutrophils cannot replenish their store of granules.-The cells die once their supply of granules has beenexhausted.-Dead neutrophils and tissue debris are the majorcomponents of pus-Their lifespan is only about one week.-Lost neutrophils are quickly replenished from a reservepopulation in the bone marrow.-Because they are younger, their nuclei have fewer lobesthan the "average" neutrophil.- A high proportion of neutrophils, with few nuclear lobesindicates a recent surge in their release from the bonemarrow.
    • Shown in the following photo are red blood cells in asmear, small platelets (p) and a neutrophil with a multilobednucleus. The drumstick like projection from the nucleus iscalled a "Barr Body" and represents the condensed Xchromosome. This signifies that the blood was taken from afemale
    • GRANULAR LEUKOCYTESEosinophils (1-3%)Eosinophilic (acidophilic)granules(destroy antigen-antibodycomplexes)EosinophilCrystalsSpecificGranules
    • Eosinophil granulocytes ( or eosinophils)- Their nucleus usually has only two lobes.- Almost all of the cytoplasm appears filled with thespecific granules of the eosinophils. As the term"eosinophil" indicates, these granules are not neutral butstain red or pink when eosin or a similar dye is used inthe staining process.- Aside from the usual complement of organelleseosinophils contain some large rounded vesicles (up to 1µm) in their cytoplasm.- These granules correspond to the eosinophilic grains thatwe see in the light microscope.- The specific granules contain, in addition to enzymesthat otherwise are found in lysosomes, an electron-dense,proteinaceous crystal.- This crystal is composed of major basic protein (MBP).
    • Functions :- The presence of antibody-antigen complexes stimulatesthe immune system.- Eosinophils phagocytose these complexes and this mayprevent the immune system from "overreacting".- Their granules also contain the enzymes histaminaseand arylsulfatase.- These enzymes break down histamine andleukotrienes, which again may dampen the effects oftheir release by basophils or mast cells.- MBP, which can also function as a cytotoxin, and itsrelease by eosinophils may be involved in the responseof the body against parasitic infections, which areaccompanied by an increase in the number ofeosinophils.
    • Granular LeukocytesBasophils (less than 1%)Basophilic GranulesheparinhistamineserotoninBasophil
    • Basophil Granulocytes ( or basophils)- Basophilic granulocytes have a 2 or 3 lobed nucleus- The lobes are usually not as well defined as inneutrophilic granulocytes and the nucleus may appear S-shaped.- The specific granules of basophils are stained deeplybluish or reddish-violet.- Their color corresponds closely to the color of thenucleus which sometimes is difficult to see amongst orbehind the granules.- The granules are not as numerous as those ineosinophils.- The specific granules of basophils (about 0.5 µm) appearquite dark in EM pictures.- They contain heparin, histamine lysosomal enzymesand leukotrienes (the later correspond to the slow-reacting substance of anaphylaxis or SRS-A).
    • Eosinophil Neutrophil Basophil
    • AGRANULAR LEUKOCYTESMonocytes (3-8%)MononuclearPhagocytic SystemLymphocytes (20-25%)B Lymphocytes(humoral immunity)T-Lymphocytes(cell mediated immunity)Monocytes Lymphocytes
    • Non-granular LeukocytesMonocytes- These cells can be slightly larger than granulocytes (about12-18 µm in diameter).- Their cytoplasm stains usually somewhat stronger thanthat of granulocytes, but it does not contain any structureswhich would be visible in the light microscope using mosttraditional stains (a few very fine bluish grains may bevisible in some monocytes).- The "textbook" monocyte has a C-shaped nucleus.- Monocytes contain granules (visible in the EM) which inappearance and content correspond to the primary granulesof neutrophils, i.e. the granules correspond to lysosomes.
    • Functions :- Once monocytes enter the connective tissue theydifferentiate into macrophages.- At sites of infection macrophages are thedominant cell type after the death of the invadingneutrophils.- They phagocytose microorganisms, tissue debrisand the dead neutrophils.- Monocytes also give rise to osteoclasts, whichare able to dissolve bone.- They are of importance in bone remodelling
    • Lymphocytes- These cells are very variable in size.- The smallest may be smaller than erythrocytes(down to ~5 µm in diameter) while the largest mayreach the size of large granulocytes (up to 15 µmin diameter).- How much cytoplasm is discernible depends verymuch on the size of the lymphocyte.- In small ones, which are the majority oflymphocytes in the blood, the nucleus may appearto fill the entire cell.
    • - Large lymphocytes have a wider rim ofcytoplasm which surrounds the nucleus.- Both the nucleus and the cytoplasm stainblue (and darker than most other cell typesin the blood).- The typical lymphocyte only contains theusual complement of cellular organelles.- The appearance of lymphocytes maychange drastically when they are activated .
    • Functions- Most lymphocytes in the blood stream belong to eitherthe group of B-lymphocytes (~5%) or the group of T-lymphocytes (~90%).- Unless they become activated, the two groups can noteasily be distinguished using routine light or electronmicroscopy.- Upon exposure to antigens by antigen-presenting cells(e.g. macrophages) and T-helper cells (one special groupof T-lymphocytes) B-lymphocytes differentiate intoantibody producing plasma cells.- The amount of cytoplasm increases and RER fills a largeportion of the cytoplasm of plasma cells.
    • - T-lymphocytes represent the "cellular arm" of theimmune response (cytotoxic T cells) and mayattack foreign cells, cancer cells and cells infectedby e.g. a virus.- T-lymphocytes and B-lymphocytes form the vastmajority of lymphocytes in the blood stream, butthey do not add up to 100%, and they usually aresmall lymphocytes.- The much less frequent medium-sized or largelymphocytes may represent e.g. natural killer (Nk -) cells which belong to the group of largegranular lymphocytes, or haemopoietic stemcells of which a few will be circulating in theblood stream.
    • BLOOD PLATELETS(thrombocytes)Hyalomere(purple granules)(pale blue)GranulomerePlatelet- light micrographmicrotubulesglycogenmicrotubulesmicrotubulesglycogengranulesmicro-tubules
    • Blood Platelets ( or Thrombocytes)- Blood platelets do not contain a nucleus- Unlike erythrocytes, which also lack anucleus, the blood platelets of mammals havenever been nucleated cells.- Instead, blood platelets are fragments of thecytoplasm of very large thrombocyte precursorcells, megakaryocytes.- Like other cells involved in the formation inblood cells, megakaryocytes are found in thebone marrow.- Platelets are about 3 µm long but appearsomewhat smaller in the microscope.
    • - This is because their cytoplasm is divided into two zones:an outer hyalomere, which hardly stains, and an innergranulomere, which contains bluish staining granules.- These granules are usually not individually visible withthe highest magnification on your microscope, and thegranulomere appears more or less homogeneously blue.- In addition to different types of vesicles (i.e. thegranules), mitochondria, ribosomes, lysosomes and alittle ER are present in the thrombocyte granulomere.- Different types of vesicles contain either serotonin(electron-dense delta granules; few) or compoundsimportant for blood coagulation (alpha granules - theyalso contain platelet-derived growth factor (PDGF)which may play a role in the repair of damaged tissue).- The hyalomere contains cytoskeletal fibers, whichinclude actin and myosin.
    • Functions of Blood Platelets- Platelets assist in haemostasis, the arrest ofbleeding- Serotonin is a potent vasoconstrictor- The release of serotonin from thrombocytes,which adhere to the walls of a damaged vessels,is sufficient to close even small arteries- Platelets, which come into contact withcollagenous fibers in the walls of the vessel(which are not usually exposed to the bloodstream), swell, become "sticky" and activate otherplatelets to undergo the same transformation.- This cascade of events results in the formation ofa platelet plug (or platelet thrombus).
    • - Finally, activating substances are released fromthe damaged vessel walls and from the platelets.- These substances mediate the conversion of theplasma protein prothrombin into thrombin.- Thrombin catalyzes the conversion of fibrinogeninto fibrin, which polymerizes into fibrils andforms a fibrous net in the arising blood clot.- Platelets captured in the fibrin net contract leadingto clot retraction, which further assists inhaemostasis.- Blood coagulation is a fairly complexprocess, which involves a large number of otherproteins and messenger substances.- Deficiencies in any one of them, either inherited oracquired, will lead to an impairment ofhaemostasis.
    • Erythrocytes, Lymphocytes, Neutrophils and PlateletsBar Body
    • Blood ClottingProthrombin Thromboplastin (released from platelets and injuredvessel wall)ThrombinFibrinogen Fibrin (clot)(feltwork of fibrils, plateletsand blood cells)thrombuslesion platelets
    • Blood PlasmaPlasma- transparent, yellow liquid vehicle for cellsSerum- plasma without fibrinogenSerum Proteins: Albumin colloid osmotic pressure and provide forsolubility of hydrophobic components (e.i. lipids)Globulinsgamma globulins- e.i. IgG (immune globulins-antibodies)beta globulins - hormone transport (e.i. thyrogloblin)Transferrin- iron transportCerruloplasmin- copper transportSerum Lipoproteins:-chylomicrons- fatty acid transport-Very Low Density Lipoproteins (VLDL))-Low Density Lipoproteins (LDL)-High Density Lipoproteins (HDL)
    • - Haemopoietic cells (those which produceblood) first appear in the yolk sac of the 2-week embryo.- By 8 weeks, blood making has becomeestablished in the liver of the embryo, and- by 12-16 weeks the liver has become themajor site of blood cell formation. It remainsan active haemopoietic site until a few weeksbefore birth. The spleen is also active duringthis period, particularly in the production oflymphoid cells, and the foetal thymus is atransient site for some lymphocytes.Haemopoiesis Where blood is made
    • The highly cellular bone marrow becomes an active bloodmaking site from about 20 weeks gestation and graduallyincreases its activity until it becomes the major site ofproduction about 10 weeks later.- At birth, active blood making red marrow occupies theentire capacity of the bones and continues to do so for thefirst 2-3 years after birth.The red marrow is then very gradually replaced byinactive, fatty, yellow, lymphoid marrow.The latter begins to develop in the shafts of the long bonesand continues until, by 20-22 years, red marrow is presentonly in the upper ends of the femur and humerus and inthe flat bones of the sternum, ribs, cranium, pelvis andvertebrae.However, because of the growth in body and bone sizethat has occurred during this period, the total amount ofactive red marrow (approximately 1000-1500 g) is nearlyidentical in the child and the adult.
    • - Adult red marrow- has a large reserve capacity for cell production. In childhood andadulthood, it is possible for blood making sites outside marrow, suchas the liver, to become active if there is excessive demand as, forexample, in severe haemolytic anemia or following haemorrhage.- In old age- red marrow sites are slowly replaced with yellow, inactivemarrow.- Red marrow forms all types of blood cell and is also active in thedestruction of red blood cells.- Red marrow is, therefore, one of the largest and most active organsof the human body, approaching the size of the liver in overall massalthough as mentioned it is distributed in various parts of the body.- About two-thirds of its mass functions in white cell production(leucopoiesis), and one-third in red cell production (erythropoiesis).However as we have already seen there are approximately 700 timesas many red cells as white cells in peripheral blood.- This apparent anomaly reflects the shorter life span and hencegreater turnover of the white blood cells in comparison with the redblood cells.
    • Bone and bone marrow• Bone marrow is a spongy material that is foundinside the bones (particularly the pelvic bones).• Stem cells are blood cells at the earliest stage ofdevelopment in the bone marrow.• Within the bone marrow, stem cells develop intothe different blood cells described below.• When the cells are fully mature they are releasedinto the bloodstream.• Like a factory, bone marrow produces the cellswhich develop into the three different types ofblood cells:• red blood cells, white blood cells, platelets, .• Normally, most of the stem cells in the body arein the bone marrow and there are only very smallnumbers in the bloodstream. However, it ispossible to stimulate the stem cells to move intothe bloodstream, by using low doses of certainchemotherapy drugs or injections of growthfactors. Stem cells can be collected from thebloodstream or from the bone marrow.• A transplant using stem cells collected from thebone marrow is sometimes called a bone marrowtransplant, when in fact it is really a transplant ofstem cells.
    • -Haemopoietic cells surround the vascular sinusoids and are supported by reticularconnective tissue , -In addition to the endothelial cells of the sinusoids and thereticulocytes of the connective tissue, macrophages are frequent in red bone marrow.Endothelial CellsMegakaryocyteDiapedesisAdipocyteEndothelialDiscontinuityReticularFibersHemopoeticCompartmentBloodSinusoid
    • Light microscopic picture of bone marrow
    • Haemopoietic Cells-The basis of haemopoiesis is a small population of self-replicating stem cells, which ultimately can generate alltypes of blood cells.-Their progeny may develop into either lymphocyticstem cells or pluripotent haemal stem cells (colony-forming unit - stem cell - CFU-S).-The latter type gives rise to stem cells which can formthe major groups of blood cells other than lymphocytes.- Depending on their progeny it is possible to differentiateburst-forming unit of the erythroid line (BFU-E),colony-forming unit - granulocytes and macrophages (CFU-G/M), andcolony-forming unit - megakaryocytes (CFU-Mk).
    • ERYTHROPOEISIS-The first identifiable stage of erythropoiesis is the proerythroblast -a large, slightly basophilic cell, which contains a large, lightlystained nucleus .-Proerythroblasts proliferate to generate a sequence of cells whichshow a gradual decrease in size and condensation of their chromatin.-They are named after changes in the staining characteristic of theircytoplasm (basophilic erythroblast, polychromatophilic andorthochromic normoblasts).-The nucleus is finally extruded from the normoblast-The cell enters circulation as a reticulocyte, which still containssome organelles.-Reticulocytes remain for a few days in either the bone marrow orthe spleen to mature to erythrocytes.
    • ERYTHROPOEISISHemocytoblast ProerythroblastErythroblastBasophilic Polychroma-tophilicErythroblastOrthochroma-tophilicErythroblastNuclearExtrusionReticulocyteRBCPluripotentialStem Cell1. dispersedchromatin2. nucleoli3. cytoplasmic1. clumpedchromatin2. nonucleoli3. maximum1. condensedchromatin2. grey-greencytoplasm(hemoglobinsynthesis)1. condensedeccentricnucleus2. pinkcytoplasm1. anucleate2. spherical3. slightbasophiliabasophiliabasophilia(free ribosomes)
    • MYELOPOEISIS-Myeloblast appear by L/M similar toproerythroblast.-They proliferate to generate promyelocytes.-Promyelocytes begin to accumulatenonspecific granules, but they are still ableto divide.-The maturation of their progeny, themyelocytes, is characterised by theaccumulation of specific granules andchanges in nuclear morphology.-Metamyelocytes have a C-shaped nucleus.
    • MYELOPOEISIS - ( granulopoeisis )Hemocytoblast(multipotential)Myeloblast(multipotential)Promyelocyte(multipotential)1. loss of cytoplasmicbasophilia2. dispersed chromatinEosinophilopoeisis Neutrophilopoeisis Basophilopoeisis
    • EosinophilicMyelocyteEosinophilicMetamyelocyte Eosinophil(band cell)Specific andMetachromatic GranulesEOSINOPHILOPOEISIS
    • NEUTROPHILICMYELOCYTE NEUTROPHILICMETAMYELOCYTENEUTROPHILOPOEISISNeutrophil
    • BasophilicMyelocyteBasophilicMetamyelocyteBasophilBASOPHILOPOEISIS
    • THROMBOPOEISIS• -Are, as mentioned above, fragments of thecytoplasm of megakaryocytes.• -Megakaryocytes are very large cells (up to160 µm in diameter), which contain verylarge, highly lobulated, polyploid nuclei.• -Megakaryocytes are in turn the product ofthe differentiation of basophilicmegakaryoblasts.
    • THROMBOPOEISISHemocytoblastEndomitosis(w/o karyokinesis(w/o cytokinesis)Reserve Megakaryocyte (megakaryoblast) Platelet-Forming MegakaryocyteResidual Megakaryocyte PlateletsPlatelet-Shedding(fusion ofplasma membrane)
    • This diagram shows the formation of the different types of blood cellsfrom a common source the stem cell
    • Connective tissue typeand characteristicsFunctions LocationsAreolar (loose) connective tissue.Loose array of random fibers with awide variety of cell typesNourishes and cushions epithelia,provides arena for immune defenseagainst infection, binds organstogether, allows passage for nervesand blood vessels through othertissuesUnder all epithelia; outer coverings ofblood vessels, nerves, esophagus,and other organs; fascia betweenmuscles; pleural and pericardial sacsAdipose tissue (fat). Large fat-filledadipocytes and scanty extracellularmatrix.Stores energy, conserves body heat,cushions and protects many organs,fills space, shapes bodyBeneath skin; around kidneys, heart,and eyes; breast; abdominalmembranes (mesenteries)Dense irregular connective tissue.Densely spaced, randomly arrangedfibers and fibroblasts.Toughness; protects organs frominjury; provides protective capsulesaround many organsDermis of skin; capsules around liver,spleen, and other organs; fibroussheath around bonesDense regular connective tissue.Densely spaced, parallel collagenfibers and fibroblasts.Binds bones together and attachesmuscle to bone; transfers force frommuscle to boneTendons and ligamentsCartilage (gristle). Widely spacedcells in small cavities (lacunae);rubbery matrix.Eases joint movements; resistscompression at joints; holds airwayopen; shapes outer ear; moves vocalcords; forerunner of fetal skeleton;growth zone of childrens bonesExternal ear, larynx, rings aroundtrachea, joint surfaces and growthzones of bones, between ribs andsternum, intervertebral discsBone (osseous tissue). Widelyspaced cells in lacunae; much ofmatrix in concentric onionlike layers;hard mineralized matrix.Physically supports body, providesmovement, encloses and protectssoft organs, stores and releasescalcium and phosphorusSkeletonBlood. Erythrocytes, leukocytes, andplatelets inTransports nutrients, gases, wastes,hormones,Circulates in cardiovascular system