Attention nDon´t manipulate the light microscopes, please. nThey are prepared for your work following presentation. nYou´ll receive an instruction how to use the LM and how to study blood smears. Blood Plasma & Blood cells blood Hematocrit: the volume of blood cells per unit volume of blood fig2 Blood cells (formed elements) nRed blood cells – erythrocytes nWhite blood cells – leukocytes nPlatelets – thrombocytes wbc_ident Granulocytes Agranulocytes Neutro- Eosino- Baso- Lymfo- Mono- artificial-blood-1 EM1bv20t REM TEM n n REMEMBER! n n nErythrocytes: 4 – 6 millions/ 1 ml of blood nLeukocytes: 5,000 – 9,000 / 1 ml nThrombocytes: 150,000 – 250,000/ 1 ml n n remember ERYTHROCYTES n4 – 6 million/μl nShape: biconcave disc, n dumble-shaped (cross section) nSize: 7.4 μm in diameter (= normocyte) nStructure: plasmalemma, cytoplasm + hemoglobin 33 % absence of the nucleus and cell organelles nLifespan: 120 days n blood_01 CRIB_blood_cells http://academic.pgcc.edu/~aimholtz/AandP/206_ONLINE/Blood/Blood_Images/rbc_biconcavedisk.jpg 0.8 μm wbc_ident 746px-A_red_blood_cell_in_a_capillary,_pancreatic_tissue_-_TEM nPolyglobulia – an increased number of ery nAnemia – a decreased number of ery n nPoikilocytosis – an occurrence of variously shaped ery (spherocytes, elliptocytes, drepanocytes = sickle cells, etc.) nAnisocytosis – an occurrence of variously sized ery (microcytes, macrocytes) poikilocytosis n drepanocytes n nspherocytes dacryosytes n (teardrop) n n n schystocytes elliptocytes n (keratocytes) stomatocytes spherocyte teardrop elliptocyte sicklecell schistocytes stomatocytes Anisocytosis n n n nMicrocytes Æ < 6.5 mm nNormocytes n Æ ± 7.4 mm nMacrocytes Æ > 8 mm n 4 Hemoglobin na conjugated protein: 4 polypeptide chains + heme groups = protoporphyrin ring with ferrous iron (Fe2+ ) nHb F (fetal) nHb A (adult) nnormochromatic ery: 32±2 picogramms (hyper-, hypo-) n hemoglobin nOsmotic resistance and hemolysis (osmotic pressure has a great effect on living cells, because their walls are semipermeable membrane) nIsotonic conditions nin hypertonic solution – ery shrink irregularly and become crenated nin hypotonic solution – ery swell, their plasmalemma come to the rupture, Hb is released – hemolysis – (the rest of ery = ghost) crenation (a) crenation is caused by water movement out of a cell in a hypertonic solution. (b) hemolysis is caused by water movement into a cell in a hypotonic solution. hemolysis%5B1%5D FG13_022 Reticulocytes nImmature ery are released from the bone marrow into the peripheral blood (0.5 – 1.5 %) nthe rests of organelles – ribosomes, mitochondria nmaturation into ery during 24 hours n Polychromatic_erythrocyte Reticulocytes%20Special%20Stain brilantcresyl blue substantia reticulofilamentosa Functions of ery n ntransport of oxygen from the lungs ntransport of carbon dioxide from the tissues LEUKOCYTES nGranulocytes: n - neutrophils n - eosinophils n - basophils nGeneral characteristic: nPolymorphonuclears with nacidophilic cytoplasm and nSpecific + azurophilic granules nAgranulocytes n - lymphocytes n - monocytes n nGeneral characteristic: nMononuclears with nbasophilic cytoplasm and nazurophilic granules n Granulocytes2 Lymphocyte Monocyte bloodCells Erythrocytes (A) Thrombocytes (G) Granulocytes: (pink cytoplasm, specific granules) neutrophil segment (E,C) neutrophil band (I) eosinophil (D) basophil (J) Agranulocytes: (blue cytoplasm, azurophilic granules) lymphocyte (H, B) monocyte (F) Granulocytes nGeneral charcteristic: -polymorphonuclears – different shape of nuclei - - n band segmented with chromatin bridges -acidophilic cytoplasm – bright-pink -specific granules – with special enzymes -azurophilic granules – with lysosomal enzymes -all granulocytes are able to migrate from the vessels and by diapedesis invade a site of inflamation Granulocytes neutrophils – eosinophils - basophils n band n n 12-14 mm n n10-12 mm n 10 mm n n n n segment Differential white cell count (DWCC) nTotal number of leukocytes: normal values n Neutrophils - bands 4 % - segments 68 % Eosinophils 3 % Basophils 1 % Lymphocytes 20 % Monocytes 4 % ∑ = 100 % remember Neutrophil granulocytes (neutrophils) n71 % of all white blood cells (DWCC) nÆ 10 – 12 mm nCytoplasm: bright pink (eosinophilic = acidophilic ) nSpecific granules: neutrophilic (Æ 0.3 mm) (enzymes:alcaline phosphatase, kolagenase, lysozyme, …) nNucleus: band-shaped (4 %) or segmented (67 %) (2-5 segments) n S96538-163-f004 neutrophil350 band350 HEME001 Neutrophil-EM_copy_small Functions of Neu na central role in inflammatory processes – Neu invade, by diapedesis from the vessels into sites of infection and release some factors (e.g. cytokines) ncell membrane receptors allow Neu to recognise foreign bodies (bacteria, tissue debris), which they begin to phagocytose and destroy. n n The Neu die once their supply of granules has been n exhausted. Their lifespan is only about one week. Dead neutrophils and tissue debris are the major components of pus. Eosinophil granulocytes (eosinophils) n1– 4 % of all white blood cells (DWCC) nÆ 12 – 14 mm nCytoplasm: bright pink (eosinophilic = acidophilic ) nSpecific granules: eosinophilic (Æ 0.5 – 1 mm) (enzymes: acid phosphatase, peroxidase,histaminase, arylsufatase …) nNucleus: dumb-belt, (2 segments) chromatin bridge Fig1 7290350f8 eo104le Eosinophil_small eosinophil400 Functions of Eos nphagocytosis of antibody-antigen complexes and prevention of the immune system from "overreacting„ neos are involved in the response of the body against parasitic infections, which are accompanied by an increase in the number of eosinophils. Basophil granulocytes (basophils) nup to 1 % of all white blood cells (DWCC) nÆ up to 10 mm nCytoplasm: bright violet-pink (lightly basophilic ) nSpecific granules: (Æ 2 mm) (heparin, histamin, …) nNucleus: „shape of dick S“ ba103le HEME005 basophil_small Functions of Baso nheparin and histamine are vasoactive substances. They dilate the blood vessels, make vessel walls more permeable and prevent blood coagulation. They facilitate the access of heparinocyte in a site of infection. nantibodies produced by plasma cells (activated B-lymphocytes) bind to the receptors on the plasma membrane of basophils. If these antibodies come into contact with antigens, they induce the release of the contents of the basophil granules. Agranulocytes nGeneral charcteristic: -mononuclears – shape of nuclei is spherical (in Ly), oval or bean-shaped (in Mono) n n -basophilic cytoplasm – blue -NO specific granules -azurophilic granules – with lysosomal enzymes n ly106le LYMPHOCYTES nClassification: n - according to origin – T-Ly (thymus), B-Ly n (bone marrow @ bursa of Fabricius in birds) n - according to the size – small (Æ 8 mm), n medium (Æ 10-12 mm), large (Æ 16-18 mm), n - according to the function – natural killer cells, n helper cells, memmory cells, supressor cells, n - according to life-span (long, short) Lymphocytes - structure n20 % of all white blood cells (DWCC) n n n nsmall, medium-sized, large Ly ncytoplasm – dark blue, contains non-specific azurophilic granules with lysosomal enzymes (hydrolases) and numerous ribosomes nnucleus – round, hyperchromatic – coarse grains of heterochromatin (dark violet colour) n lymphocyte_small blood5 smalllymph350 Functions of Ly nB-lymphocytes differentiate into antibody producing plasma cells and so they represent "humoral immunity" nT-lymphocytes represent the "cellular immunity" and may attack foreign cells, cancer cells and cells infected by e.g. a virus MONOCYTES n5 % (DWC), Æ 15 – 20 mm ncytoplasm – voluminous, bright blue, contains non-specific azurophilic granules with lysosomal enzymes (hydrolases) and numerous ribosomes nnucleus – oval to bean-shaped, finely dispersed chromatin monocyte_small monocyte370 Functions of Mono nmonocytes enter the connective tissue they differentiate into macrophages. At sites of infection macrophages are the dominant cell type after the death of the invading neutrophils. nmacrophages phagocyte microorganisms, tissue debris and the dead neutrophils. n nmono also give rise to osteoclasts, which are able to distroy bone. They are of importance in bone remodelling. THROMBOCYTES (blood platelets) n150,000 – 300,000 / 1 ml of blood nthrombocytosis X thrombocytopenia n- are not cells, but cytoplasmic fragments of large cell (megakaryocyte) in bone marrow nshape: spindle-shaped discoid plate nsize: 2 – 4 mm ncytoplasm – basophilic (bright violet-blue), contains microtubules and a,d and l granules: n alpha granules – fibrinogen, … n delta granules – serotonin, Ca ions, ATP and ADP,… n lambda granules – are small lysosomes Platelet structure nHyaloplasm contains microtubules (on the periphery of platelet) n nGranuloplasm contains granules n n slide0013_image037 23281325 Platel1 cornell_platelets pltclmp th100le loadBinary loadBinary Functions of thrombocytes nPlatelets assist in haemostasis, the arrest of bleeding. nSerotonin is a vasoconstrictor. Its release from thrombocytes, adhering to the walls of a damaged vessels, is sufficient to close even small arteries. Platelets, which come into contact with collagenous fibers in the walls of the vessel, swell, become "sticky" and activate other platelets to undergo the same transformation. This cascade of events results in the formation of a platelet plug (or platelet thrombus). Finally, activating substances are released from the damaged vessel walls and from the platelets. These substances mediate the conversion of the plasma protein prothrombin into thrombin. Thrombin catalyzes the conversion of fibrinogen into fibrin, which polymerizes into fibrils and forms a fibrous net in the arising blood clot. Platelets captured in the fibrin net contract leading to clot retraction, which further assists in haemostasis. RedBloodCells1 you too can be wealthy Please, note the following instructions How to prepare blood smear? n Blood type test Friendly Doctor.gif (4339 bytes) How to prepare blood smear - I nSmears of peripheral blood must be made immediately. nStep 1: Place drop of blood about 1cm from the frosted end of a clean slide. bloodsmear1 How to prepare blood smear - II nStep 2: hold the end of a second slide (“spreader”) against the surface of the first slide at an angle of 30-45 degrees. bloodsmear3 How to prepare blood smear - III nStep 3: draw it back to contact the drop of blood. Allow the blood to spread and fill the angle between the two slides. n bloodsmear4 How to prepare blood smear - IV nStep 4: Push the “spreader” slide at a moderate speed forward until all of the blood has been spread into a moderately thin film. bloodsmear5 How to prepare blood smear - V nSmear is prepared for fixation (methyl alcohol, 3-5 minutes) and staining (special panoptic method according to Pappenheim can be used) wcd%20PBS%20slide How to study blood smear in the light microscope (LM)? nthe lense of immersion objective (magnif. 100x) is immersed into a drop of oil and blood smear is prepared for study in the LM; nswitch on your LM and see into the eypice: blood cells should be visible in the light field nif not, try to focuse picture – you may use ONLY the fine adjustment knob! nif you are not succesfull, ask for help your teacher, do NOT use the coarse adjustment knob! exclamation_mark_boing_md_wht Light microscope manipulation Olympus04 Course adjustment knob don‘t use today Fine adjustment knob only that can be used to focuse the image animation_exclamation Stage with slide holder (3) Lever of holder (1) Focusing knobes (2). wbc_ident Monocyte Ø 10-12 μm lymphocyte Ø 8-10 μm neutrophil – band Ø 10-12 μm neutrophil – segment Ø 10-12 μm eosinophil Ø 12-14 μm basophil Ø 10-12 μm thrombocyte Ø 2-4 μm erytrocytes Ø 7.4 μm Erythrocytes (Ø 7,4 μm) Thrombocytes (2-4 μm) Granulocytes: neutrophilic eosinophilic basophilic 10-12 μm 12-14 μm 10 μm Agranulocytes: Lymfocytes (8-10 μm) Monocyte (15-20 μm) P R O T O C O L How to count leukocytes in blood smear? ndifferential white cell count (DWCC) is an important hematologic screening which helps to diagnose nleukocytes percentage is the result of this investigation n100 white cells must be count and registered in the table prepared for all types of leukocytes (Neu-bands, Neu-segments, Eos, Baso, Ly, Mono) narithmetic sum of each type of leukocytes represents their percentage (%) How to count leukocytes in blood smear? nblood smear have to be systematicaly viewed (it avoids repeatedly count the same cells) n n n or n n n vertical browsing horizontal browsing DWCC Table 1 2 Neu bands / Neu segments //// // /// Eos / Baso Ly // //// Mono // 10 cells 10 cells 9 10 results norm // 4 % //// / /// 68 % / // 3 % / 1 % / //// 20 % 4 % 10 cells 10 cells 100% 100 % Differential white cell count (DWCC) nTotal number of leukocytes: normal values n Neutrophils - bands 4 % - segments 68 % Eosinophils 3 % Basophils 1 % Lymphocytes 20 % Monocytes 4 % ∑ = 100 % remember Anomalies of DWCC n * sum total of bands and segments has to be compared with norm; n normal value is 71 % (4 % bans + 68 % segments) é Increased number ê Decreased number Neutrophils* neutrophilic granulocytosis neutrophilic granulocytopenia Eosinophils eosinophilic granulocytosis eosinophilic granulocytopenia Basophils basoophilic granulocytosis basoophilic granulocytopenia Lymphocytes lymphocytosis lymphocytopenia Monocytes monocytosis monocytopenia Normal ratio of neutrophil bands and segments n nTurn to the left – bands are increased nTurn to the right – segments are increased in peripheral blood n n n n n n n n n n n n Bands : Segments ratio is 4 % : 68 % = 1 : 17 Turn to the right Turn to the left n blood-cell ... thank you for attention ... Normal-Peripheral-Blood-50x-website Mature-basophil-100x-website-arrow Mature-basophils-100x-website-arrow Normal-mature-monocyte-100x-website-arrow Monocyte-100x-website-arrow Monocyte--lymphocyte--neutrophil-50x-website-arrow Normal-peripheral-blood-lymphocyte-100x-website-arrow Neutrophil-filaments-100x-website-arrow blood4.jpg (40969 bytes) basophil3.JPG (40518 bytes) blood1.jpg (23030 bytes) ? Basophil_Wright_Giemsa_5 blood5.jpg (31442 bytes) blood3.jpg (23187 bytes) ? hemtest1 Ery 7.4 mm Neu – band Neu – segment Eos 12-14 mm Baso 10 mm 10 – 12 mm Platelet 2 – 4 mm Lymfo 8 mm Mono 15 – 20 mm f21-1_whole_blood_separ_c f21-3_preparing_a_blood_c Blood investigation Blood smear Light microscope nEyepieces nObjective lense nStage with slide holder nCondenser nOn/off switch nLight intensity control nSource of light beam nCourse adjustment knob nFine adjustment knob Olympus01 Light microscope manipulation n n non/off switch knob (1) (rocker or wheel SW) npivoted potenciometer (2) regulate intensity of emited light n Olympus03 Light microscope manipulation n nStage with slide holder (3) nLever of holder (1) nFocusing knobes (2). n Olympus04 Light microscope manipulation n nUse mechanism of cross shift for shift of the slide on the stage n n Ç n Å Æ n È n Olympus05 Light microscope manipulation nfocuse a picture in LM and look at it with both eyes nregulate a distance between the eyepices so, you can see one focused circular field n n n bad correct n Olympus06.gif (5474 bytes) Light microscope manipulation nLook at the slide only through the right eyepiece and focuse some point in the picture. nWithout refocusing, look at the left eyepiece. nIn doing so, screw the ring below the left eyepiece to focuse the same point. nSo, the dioptric correction is set up. n n n n n n n n n nNow, you can start to study blood nsmear in your LM J Olympus07.gif (4519 bytes) Basophil 00007604 eosinophil400