Hema MTAP Day 1 PDF

HEMATOLOGY 1 LEC [MTAP] Sir. Guanlao | Aug. 7, 2024 (Day 1/Part 1) | 1PM to 5PM Nutritive - deliver different substances from the one HEMATOLOGY part o...

HEMATOLOGY 1 LEC [MTAP] Sir. Guanlao | Aug. 7, 2024 (Day 1/Part 1) | 1PM to 5PM Nutritive - deliver different substances from the one HEMATOLOGY part of body (ex. intestines) pag kumain, yung nutrients From the Greek word, from the food iabsorb then dadaan sa liver then ideliver haima - "blood"; logia - "study of" sa iba’t ibang parts ng body. The study of the physiology of blood Excretory - mga tissues/organs as a result of normal Encompasses the analyses of: processes mag produce ng waste products and 1. Red and white blood cells mapupunta sa blood. Blood responsibility is to bring 2. Plasma components related to blood cell these waste products from this tissue to excretory structure and function such as cholesterol tissues (lungs for carbon dioxide, kidney for waste 3. Platelets and plasma proteins involved in products like urea nitrogen, creatinine, etc). coagulation Immunity - wbc (neutrophils, lymphocytes, BLOOD monocytes, eosinophils, and basophils) found in blood The vital, life-sustaining fluid circulating constantly in a in our body that contributes in immune system. closed system of blood vessels. Water balance - plasma protein (albumin) exerts Highly complex liquid connective tissue in which oncotic pressure so that mga fluids inside our blood cellular elements are suspended in a liquid supporting vessels will not leak out. substance in a form of plasma compared to other connective tissues na solid ang ground substance. Temperature control - blood carries heat, so pag Fluid connective tissues that constantly circulates mainit, namumula tayo kasi nag expand / vasodilate inside a closed system of our blood vessels. It carrys yung blood vessels in order to bring the heat papunta out many function particularly by delivering oxygen in sa surface yung heat, and para mag evaporate. Pag other substance throughout the body. malamig nag constrict mga blood vessels para ma Plasma - liquid part of the anticoagulated blood conserve yung heat. Serum - the liquid part of the clotted blood Transport of hormones - in CC 2, endocrine glands NOTES: are ductless glands which secretes hormone directly plasma - may fibrinogen / clotting factor 1; mas hazy. into the bloodstream and dadalhin sa target - Kapag kumuha ng blood sample then placed in a organ/tissue ng endocrine gland na yun. container with anticoagulant the liquid portion is still the CHARACTERISTICS OF BLOOD plasma. serum - negative for fibrinogen kasi converted na into solid fibrin clot; mas clear bc no fibrinogen na. - Pag nag clot yung blood then centrifuge tas kinuha natin tung liquid part, yung liquid part is yung serum. FUNCTIONS OF BLOOD NOTES: NOTES: volume - pag drained mo yung blood sa katawan ng Respiratory - because it delivers oxygen from lungs tao, mababawasan ng 6-8% ng total body weight. to tissues then from tissues to lungs. - 5-6L but still depends kasi sa female mas Homeostasis - maintain acid-base balance. onti yung blood kesa sa male. And sa infants In blood natin nandito buffer system (carbonic acid, 250-350mL lang. infants ‘di basta basta bicarbonate system, plasma proteins and kumukuha ng venous blood kasi onti lang hemoglobins) can participate in acid-base balance. ang kanilang blood volume. May mine-maintain din tayo na intravascular water viscosity - stickiness compared to the flow of distilled with the help of plasma proteins (albumin) it will help water. Relatively thicker than water because there are retain the fluid of the blood inside the blood vessels. dissolved components in plasma and formed elements Kaya pag nagkakaroon ng hypoproteinemia like patient like cells in blood. with kidney disease, if mababa protein, yung liquid fluid specific gravity - density of substance/fluid compared tumatagas na sya palabas ng blood vessels causing to water. Our blood has dissolved components so mas edema. dense talaga sya CAVADA & PUNZALAN 1  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM - whole blood: relatively lower ang whole CELLULAR PORTION blood ng male compared to female dahil sa - RBC also known as erythrocytes body frame. - erythro - red, cytes - cells - plasma & serum: mas mataas si plasma - WBC also known as leukocytes kesa serum kasi si fibrinogen wala na sa - leuko - white serum pero sa plasma meron pa, kaya mas - Platelets - thrombocytes magaan onti si serum. - thrombo means clot ph of blood - Platelets participate in clot formation - 7.35 - 7.45 LANG DAPAT. Pag tumaas RED BLOOD CELLS magkakaroon ng alkalosis/alkalemia. If lower Erythrocytes sa normal ph range magkakaroon ng Anucleate, biconcave cells that acidosis/acidemia. Strictly maintained by diff contains hemoglobin (disk shape) buffering system (carbonic acid bicarbonate Transports O2 and CO2 buffer system) Appear pink to red osmolality Diameter: 6 to 8 um - amount of dissolved components present in Zone of pallor / central pallor blood. - Specific gravity and osmolality - they both NOTES: measure kung gaano karami yung substance - Anucleate lang sila in their mature form (no nucleus) na present sa fluid na tine-test. during maturation stages of rbc may nucleus sila until - In measuring blood osmolality mine-measure orthrochromatic normoblast/metarubricytes, then after yung serum osmolality, kasi in order to get a that wala lang silang nucleus. plasma sample, your blood should contained - Main cytoplasmic component of rbc is hemoglobin. in a anticoagulant container. Yung Red ang hemoglobin bc of heme protein. anticoagulant may add up to the osmolality of - Heme is the color red - because it contains Fe2+ the sample. (ferrous iron). COMPOSITIONS OF BLOOD - Hemoglobin - protein na nag allow sa RBC to carry FLUID PORTION (55%) oxygen. H2O - Central pallor - need para matupi at mag pass through Macromolecules the blood vessels. Internal secretions WHITE BLOOD CELLS Dissolved gasses Leukocytes Inorganic compounds Protect the host against different infection - main function Types: CELLULAR PORTION (45%) 1. Granulocytes: neutrophil, eosinophil, basophil Red blood cells 2. Agranulocytes: lymphocyte, monocyte White blood cells NOTES: Platelets - End sa “phil” sila yung Granulocytes - End sa “cyte” sila yung Agranulocytes NOTES: - Presence of peroxidase containing granules FLUID PORTION - Lahat sila may granules mas obvious lang sa - mas kaonti yung fluid portion ng blood kesa cellular mga granulocytes. portion of our blood (rbc and wbc) - Main distinction nila is Granulocytes - has - Major component of plasma/serum is WATER! peroxidase yung granules. Macromolecules - Agranulocytes - no peroxidase yung - carbohydrates in a form of glucose and other granules. carbohydrate products, protein in a form of plasma - Cytochemical staining in leukemia - one of proteins (albumin, globulins), and lipids (carried by thw wats to different cell lineage is through proteins). peroxidase. Ma differential yung may “blast” Internal secretions (myeloblast, monoblast, or lymphoblast) - Hormones & Immunoglobulins / Antibodies. sila yung mga myeloblast ang may Dissolved gasses peroxidase kasi sa kanila mang gagaling - Oxygen & Carbon Dioxide yung mga may “phil”. Inorganic compounds - sodium, calcium, potassium, etc. PUNZALAN & CAVADA 2  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM Neutrophils ‘di nataas kasi makikita lang nila is mga extracellular organism. Yung mga ‘di nag invade cell interior. Viruses are considered intracellular parasite, lymphocytes ang nataas. Monocytes - the premature / immature form of macrophage - nakikidaan lang sa dugo, actual site are sa tissues - Chronic infection / inflammatory diseases - kupffer cells, histiocyte in connective tissue, NOTES: langerhans cells kapag sa skin, - Lymphocytes pinakamarami sa infant/child, followed by neutrophil. - Pag nag form ng differential count sa patient normal lang na mas mataas ang lympho kesa neutrophil. Neutrophil - pinakamarami sa adult blood, then lympho, then monocyte, then eosinophils, then basophils. - very distinct sa structure sa peripheral blood bc of segmented nucleus. - Acute bacterial infection (early phase) - mataas total WBC, mataas neutrophil sa differential. Band neutrophils NOTES: - less segmented yung nucleus pero indentation is more neutrophil: segmented nucleus than ½ band neutrophil: indentation ng nucleus are MORE than ½. - this separates from metamyelocyte with less than ½ differentiates sa metamyelocyte (indentation NOT more than ½ indentation and band neutrophil with more than ½ kasi si band neutrophil ay more than ½) indentation eosinophil: bilobed nucleus against red orange granule bg - severe acute bacterial infection, demand for basophils: ‘di masyado makita nucleus because of its large, neutrophil increase kahit mga immature neutrophils nir- dark blue granules. release na ni BM para malabanan na yung infection. lymphocytes: para estimate normal size: one rbc inside the Eosinophil lymphocyte (normal size), pero pag mag insert ng rbc tas kasya - pinakaonti na mabibilang ito. is 2 rbc (atypical size) sya. - Bilobed nucleus against reddish orange background monocytes: kidney bean shaped / horse-shoe shaped. - parasitic infection Monocytes are characterized by vacuolation (macrophages - - allergic reaction phagocytes na materials). - Tumataass eosiophils if tissue invasive yung parasites PLATELETS (helminths) the more nag increase eosinophil count Thrombocytes - Among these: A. lumbricoides, A. duodenale, T. Involved in primary spiralis, and T. trichiura - T. spiralis ang pinaka tissue hemostasis (1-2 mins stop na invasive. Yung larvae ng T. spiralis napupunta sa bleeding) skeletal muscles. Ascaris, Duodenale, may phase Diameter: 2-4 um lang na nag enter sa body, nagkakaroon lang tayo ng Shape: round-oval, transient eosinophilia, then after umakyat sa lungs / anucleate, slightly granular trachea babalik sa small intestine then pagbalik Increased in inflammation, bababa ma ulit ang eosinophil count. trauma, or malignancy Basophils - Least makita sa peripheral blood. Dark blue nucleus. NOTES: - most of the time “0” ang report kasi wala tayong Thrombocytes nakikita / very rare sa normal peripheral blood. - not considered true cells; cytoplasmic fragments - seen in allergic reactions lang ito ng kanilang parent cell (megakaryocytes) - chronic myeloid leukemia (CLM) - basophilia - granularity ng platelets, hindi makikita sa bright field (sobrang daming mature basophils) microscope kasi maliit so gamit ng electron microscope Lymphocytes (EM) - T helper cells - sila yung pinaka in command sa entire system - Increase in cases of viral infections, kasi ONLY lymphocytes can recognize intracellular parasites. PUNZALAN & CAVADA 3  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM COMPLETE BLOOD COUNT NOTES: A panel of tests that includes enumeration of cellular - Osmotic fragility test: traditional test for hereditary elements, measurement of hematocrit, quantification of spirocytosis hemoglobin and statistical analyses that provide a - G6PD assay: to determine G6PD deficiency. "snapshot" of the cell appearances. - Sickle cell solubility screening: to screen for presence of sickle cell disease. 3 BASIC PARAMETERS: - Hemoglobin electrophoresis: ginagawa pag yung Red cell parameters: RBC count, Hgb, Hct, RBC parient is suspected to have a type of indices, RDW, Reticulocyte count. hemoglobinopathy. - Concern is the characteristics of rbc. - ESR: not actually a test for blood cells, it is a crude test - RBC Indices: MCV ay measure ng average for presence of inflammatory processes. Prone to size of RBC, MCH ay average content of error and variation kaya mas okay gamitin pang weight of hemoglobin, MCHC ay average measure/monitor ng inflammatory si CRP (C Reactive hemoglobin content of rbc in terms of Protein). proportion. - Bone marrow aspiration: mag collect niyan ay mga - RDW - measure of degree of anisocytosis physicians. To know bone marrow cellular/if may wherein it is the variation of RBC size. Pag cancerous cells sa bone marrow. more than 14, increase variation in rbc. - Flow cytometry: not a special hematological - Reticulocyte count: polychromatic rbc, procedure, ginagamit routinely sa hema. Analyzer sa indicate bone marrows ability to produce new hema like Sysmex usually ito yung mga analyzers that blood cells (particularly rbc). employs flow cytometry in counting rbc, wbc, and White cell parameters: WBC count, Differential count platelets. Specialized testing, gumagamit din ng flow Platelet parameters: usually Platelet count, pero sa cytometry together with florescent stains in order to mga tertiary MPV meron (synonymous sa MCV count special types of cells (T helper, T cytotoxic, etc). wherein it is an estimation of platelet size) - Cytogenetic analysis: usually sa practice nasa molecular lab na ito. NOTES: - Molecular diagnosis assays - Pinaka stable na test in hematology lab ang CBC. HEMATOPOIESIS - MCV - measures average size of rbc. A continuous, regulated process of blood cell PERIPHERAL BLOOD SMEAR production that includes cell renewal, proliferation, Microscopic examination of stained blood film to differentiation, and maturation. determine if there is presence of any abnormalities in Results in the formation, development, and blood cells in relation to shape, diameter, color, and specialization of functional blood cells. inclusions. Hematopoietic stem cells - foundation of adult Requires visual acuity and integrity hematopoietic system. Purpose: 1. Estimation of WBC and platelet count NOTES: 2. Review of WBC differential - Process of production of blood cells. NOTES: - Blood cells ancestors nila ang HSCs. Sila yung stem - Isa sa mga routine na ginagawa. Ginagawa ito pag cells dedicated to become nay type of blood cells. Ang may flagged / abnormal. HSCs nanggagaling sa more primitive form of stem - Purpose: tignan yung RBC morphology if mababa cells. MCV. STEM CELLS - PBS to confirm the result of the analyzers. Types: - Maraming blast wbc = leukemia 1. Undifferentiated or uncommitted stem cells SPECIAL HEMATOLOGICAL PROCEDURES - stem cells na dumadami lang pero stem cells pa rin Osmotic fragility test 2. Multipotential or committed stem cells Bone marrow aspiration - kapag committed stem cells na, they are now destined G6PD assay to become one of our bodies. Pwede pa rin magiba iba Flow cytometry ang patutunguhan based on the environment in the Sickle cell solubility screening bone marrow. Cytogenetic analysis Hemoglobin electrophoresis Origins of hematopoietic stem cells: Molecular diagnosis assays 1. Monophyletic theory - most widely accepted Erythrocyte sedimentation rate 2. Polyphyletic theory PUNZALAN & CAVADA 4  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM Characteristics: NOTES: 1. Capable of self-renemal - Starting with Hematopoietic Stem Cell/Pluripotent - can propagate / undergo cell division Stem Cell. Pag nag undergo ng differentiation 2. Can reconstitute a hematopoietic system of lethally irradiated pwedeng maging myeloid stem cell/lymphoid stem cell. host - Myeloid stem cell nandito yung granulocytes, - basis / principle ng BM transplantation monocytes, rbc, and platelets. - Ex: bone marrow myeloma / disease - before sya - Lymphoid stem cell nandito yung mga lymphocytes. bigyan ng bone marrow tissues coming from a donor FETAL HEMATOPOIESIS yung recipient papatayin yung existing BM ng patient Mesoblastic phase -> Hepatic phase -> tsaka sya bibigyan ng new BM coming from the donor Medullary phase na. Kahit na lethally irradiated / namatay na original NOTES: BM, yung BM ng donor sya na magpaparami / fill ng - Mesoblastic / yolk sac - liver - bone marrow nawalang BM ng patient. - During our fetal development, nagsisimula sa 3. Give rise to different progenies mesoblastic (yolk sac) dito nag originate yung ating - Stem cells jan nanggagaling yung iba’t ibang cells primitive erythroblast (dito nanggaling certain types of rbc na makikita lang sa fetal development), after few Fates of stem cells: months, mag enter na yung hepatic / liver 1. Self-renewal hematopoiesis, yolk sac mawawala na tas liver na mag - if ‘di pa committed they can repopulate the stem cells take over sa hematopoietic activity. Kapag towards the population until such time that these stem cells become end na ng pregnancy mag assume na ng commited. Once they become committed, mag hematopoietic yung bone marrow dito na mangyayare undergo na sila ng differentiation process in order to yung Medullary / Bone marrow hematopoiesis (until produce specific type of cells. adult). 2. Differentiation - kapag sobrang tagal ng ‘di committed ng stem cells, need mamatay / cell death para ‘di mapuno / overpopulate yung bone marrow. 3. Apoptosis LINEAGE-SPECIFIC PROGENITOR CELLS Common myeloid progenitor cell ○ dito lahat manggaling yung lahat ng ating blog cells with exemptions of lymphocytes. ○ pag nakaroon ng Myeloid leukemia mas mesoblastic malala / maraming affected na cell lines. - dito nag produce mga primitive erythroblasts, that Common lymphoid progenitor cell carries embryonic hemoglobin (gower 1, gower 2, and ○ dito nanggagaling yung lymphocytes. portland hemoglobin) ○ lesser affected kasi isang cell line lang yung - this embryonic hemoglobin carries different globin affected kesa sa myeloid progenitor cells. chains that are far different from the composition of NOTES: adult hemoglobin. - yung ating hematopoietic stem cell / multi potential hepatic / liver stem cell once it undergoes maturation and - mark the beginning of definitive hematopoiesis and differentiation pwede siyang mag undergo ng 2 path: decline of primitive hematopoiesis. common myeloid / common lymphoid progenitor cells. - mag start na mag produce yung cells that are seen in adulthood like; HbA2, HbA, and Fetal hemoglobin LINEAGE-SPECIFIC PROGENITOR CELLS medullary / bone marrow - site of hematopoiesis until adulthood. HEMATOPOIETIC SITES IN ADULTS Bone marrow Liver Thymus Spleen Lymph Nodes NOTES: - Bone marrow nanggaling ang mga blood cells PUNZALAN & CAVADA 5  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM - Thymus, spleen, and lymph nodes pwedeng maging ○ Is bluish, as it mature nawawala pagka-blue site of hematopoiesis pero particularly ito ay applicable napapalitan color. Like pronormoblast it start for lymphocytes. as a huge cell with basophilic cytoplasm - Thymus primary site of T cell development. (blue), as it matures, nag iiba color until such - Spleen and Lymph nodes - nandito nagkakaroon time na maging mature rbc (red na sya). differentiation stages ang mga lymphocytes particularly 8. Possible appearance of granules in cytoplasm once activated by an antigen. ○ such as in the case of granulocytes. - So in rare cases, pag mag fails yung BM to produce blood cells, the liver may take over again as the primary site / site of hematopoiesis. BONE MARROW Tissue confined within the cavities of the cortical bones. Two types: 1. Yellow marrow - RED BLOOD CELLS composed of adipocytes Production | Clearance | Energy Metabolism | Membrane 2. Red marrow - site of Physiology | Hemoglobin Synthesis | Iron Metabolism hematopoiesis NOMENCLATURE OF RBC R - ribs S - skull, sternum, scapula V - vertebra P - pelvic bones, proximal end of long bones NOTES: - Bone marrow nasa loob ng long bones. Yellow marrow - primarily composed of adipocytes. NOTES: Red marrow - normoblast and erythroblast: almost the same ang - ‘Di makikita kung saan-saan, sa RSVP lang! difference lang is yung term na “normoblast” like - Primary site of hematopoiesis ang red marrow. earliest stage is “pronormoblast” & “proerythroblast”. MATURATION OF HEMATOPOIETIC STEM CELLS - normoblast nomenclature ang gagamitin natin. pero 1. Decrease in cell size and N:C ratio remember pa rin yung rubriblast. ○ malalaki ang mga immature cells, paliit ng MATURATION SERIES paliit as it matures. N:C ratio lumiliit din (start as 1:1 tas then mas dumadami cytoplasm compared sa nucleus. Nawawala nuclei, and nuclear size lumiliit din.) 2. Loss of nucleoli 3. Decrease in nuclear size 4. Chromatin condensation ○ Yung condensation ng nucleus is pag mas immature, mas lighter yung staining. As it matures, pa dark ng pa dark until very condense yung itsura ng nucleus. 5. Possible change in shape of nucleus ○ Granulocytes - ang ating mga myeloblast pag nag m-mature mag change yung itsura ng nucleus from round magkaka-indentation then until such time magiging segmented. 6. Possible loss of nucleus ○ particularly sa ating mga rbc. Kasi magiging nucleated lang sila until a certain stage (metarubricyte / orthochromatic normoblast) NOTES: 7. Decrease in basophilia Pronormoblast / Rubriblasts: ○ Particularly yung staining ng cytoplasm, yung - youngest; can undergo cell mitosis mga immature blood cells kapag immature - loose chromatin kaya medyo light staining pa. mapapansin yung cytoplasm - cell that is recognizable to be of red cell lineage PUNZALAN & CAVADA 6  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM - Open chromatin - medyo light pa staining - Stays 1-3 days in circulation then plitted (polish) - Cytoplasm: basophilic, blue sya. Ang ating basic stain before it becomes erythrocytes. which is Methylene blue, color blue sya. Sya yung nag - Paano malalaman kung polychromatophilic attract ng basic stains kasi yung contents nila is erythrocytes sya or reticulocytes? Based on stain use acidic. Kapag ang cell content is acidic, it will attract - Wright’s stain (Romanowsky stain) - kapag the basic stains (magiging basophilic) polychromatic erythrocytes - Kapag ang cell content is basic, it will attract the acid - Supravital stain (New Methylene blue (NMB) stains (magiging eosinophilic). / Brilliant Crescent Blue (BCB) - reticulocyte - acidic -> basophilic & basic -> eosinophilic ang tawag. - Blue sya kasi basophilic (naging acid kasi mataas ang Erythrocytes: content ng ribosomes & RNA) kasi acidic in nature - no nucleus. sya. Basophilic normoblast / Prorubricytes: - Staining characteristics mas magiging intense, magiging darker na sya kasi mas nagiging condense yung chromatin. - Basophilic kasi ang cytoplasm nya is mas deep blue kesa sa pronormoblasts bc of the increase amount of ribosomes & RNA. - Ribosomes & RNA - for protein synthesis (particularly NOTES: hemoglobin ang gagawin). Pronormoblast - Blue kasi yung raw materials for producing hemoglobin - malaki in relation to mature RBC naiipon na sya, pero hindi pa na fform si hemoglobin - nucleus ay bilog kaya wala pang pinch of red yung cytoplasm ng - loose chromatin kaya light staining basophilic normoblast. - N/C ratio almost 1 kasi halos occupy ng nucleus yung Polychromatic normoblast / Rubricytes: cytoplasm - No nucleolus, can undergo mitosis (last stage) Basophilic normoblast - Nucleus mas dark staining kesa sa basophilic. - yung cytoplasm nito sa pronormoblast ay mas blue or - Mas nagiging intense staining kasi mas condensed deep blue because of increase amounts of ribosomes yung chromatin. and RNA in prep to produce hemoglobin - Cytoplasm unique appearance: Murky gray-blue Polychromatic normoblast cytoplasm because more than 1 color “poly” - color of cytoplasm ay murky gray blue appearance kasi nagkakaroon ng red discoloration nag-aagaw na yung nag aagaw na red and blue bc of increase amounts of red and blue. Murky gray-blue due to increasing hemoglobin sa cytoplasm amount of hemoglobin. Nag start na mag produce - nucleus is dark stained yung immature cells ng hemoglobin. Orthochromatic normoblast - Medyo detected na in staining. - nucleus is condensed or maliit - Last stage of mitosis - cytoplasm resembles mature red blood cells kaya Orthochromatic normoblast / Metarubricyte: orthochromatic tawag - nucleus is completely condensed (black - if yung nucleus ng orthochromatic normoblast ay appearance), meaning patay na nucleus. The more ejected magiging polychromatophilic na siya nag ccondensed, the more na nawawalan ng function. Polychromatophilic erythrocytes - Ejected - itatapon na ng cells - last nucleated stage ito. - wala na nucleus pero mas malaki compared sa mature - Cytoplasm - parang mature rbc tignan appears pink to RBCs orange cytoplasm “ortho” meaning similar, almost - will stay 1-3 days and i-polish ng splenic macrophages similar na sya sa immature rbc kasi pink to red. to become mature RBC - Not capable of mitosis kasi patay na nucleus. ERYTHROCYTES - Last stage with nucleus. Shape: biconcave Diameter: 7-8 um Polychromatophilic erythrocytes / Polychromatic Thickness: 1.5-2.5 um erythrocytes / Reticulocytes: Cytoplasm: salmon pink with central zone of pallor - No nucleus kasi ejected na. No mitosis. (similar sa pagka-pink ng salmon yung cytoplasm) - Cytoplasm appears similar with orthochromatic Lifespan: ~120 days (3-4 months) normoblast, the only difference lang is yung fully NOTES: condensed nucleus in ortho. - Bakit limited lifespan ng RBC? kasi wala ng nucleus - Mas malaki kesa sa matured rbc. so yun yung limiting factor - PUNZALAN & CAVADA 7  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM OVERVIEW OF RBC MATURATION SERIES - in mechanical or traumatic causes: may fat deposits in endothelial lining, this fat deposits or arterosclerotic pluck(?) medyo rough surface so pag RBC dumaan sa rough area na surface, RBC membrane will become ruptured. - cellular elements: Nagkakaroon ng free hemoglobin sa plasma as a result of intravascular hemolysis. Extravascular: - Occurs in the spleen, ano meron sa spleen pano na- NOTES: eliminate ni spleen ang dying RBC ? through - basophilic -> eosinophilic reticuloendothelial system. - N/C ratio - paliit nang paliit - Reticuloendothelial system is a system of different - chromatin condensation - it condense as it matures macrophages in many parts of body especially spleen STIMULI FOR ERYTHROPOIESIS - Splenic macrophages: responsible for eliminating HYPOXIA senescent RBCs Decreased oxygen in cells and tissues - Spleen (2 parts are red pulp and white pulp) Detected by the kidneys - Red pulp: a lymphoid tissue wherein you can find Induces production of EPO splenic macrophages and madalas na sequester mga dying/old RBCs. Ang kukuha sa kanila is mga splenic ERYTHROPOIETIN macrophages. Produced in the kidneys EXTRAVASCULAR HEMOLYSIS Mechanism of action: Removal of senescent RBCs 1. Early release of reticulocytes occurs in two ways: 2. 2. Inhibition of apoptosis 1. Culling - entire cell is 3. 3. Reduced bone marrow transit time phagocytosed NOTES: 2. Pitting - removal of - If you experience tissue hypoxia in any part of the inclusions or damaged body, if nagkaroon decrease oxygen in cells and membrane tissues, it is called hypoxia. This will become a primary NOTES: triggering event for stimulation of erythropoiesis or - Culling - buong RBC kakainin RBC production niya - Hypoxia is detected by our kidneys so pag na-detect - Pitting - kinukuha lang / kakagatin ng splenic ng kidney, it will produce a hormone called macrophage yung mga abnormal parts or damage erythropoietin wherein it is an hormone produced by parts (hollow-jolly bodies, heinz bodies, etc.) the kidney that will induce the formation of rbc. - damaged na yung RBCs sa pitting or also - Mechanism of action of erythropoietin: called bite cells which occurs in G6PD - inhibits apoptosis of stem cells, so pag onti patients. if may heinz bodies sa RBC as a ang mag-apoptosis na stem cells so marami result of desaturation of hemoglobin, pag maging committed to become RBCs dumaan RBC na may heinz bodies sa spleen, HEMOLYSIS so kakainin ng splenic macrophage yung INTRAVASCULAR HEMOLYSIS heinz bodies resulting to bite cells and Mechanical or traumatic causes eventually these bite cells will be called by the Cellular elements are released in the plasma spleen so they will be eliminated METABOLIC PATHWAYS EXTRAVASCULAR HEMOLYSIS Occurs in the spleen Spleen - Indiscriminate filter of circulating blood - Parts: 1. Red pulp 2. White pulp 3. Marginal zone NOTES: NOTES: - Embden - pinaka central pathways ng metabolism - Senescent / old / dying / rupture / destruction of rbc depends on this embden pathway Intravascular: - Hexose / Pentose phosphate - detoxifies peroxides - due to pathologic cause or a disease process through G6PD. this G6PD have central role in detoxification of oxygen radicals in RBCs PUNZALAN & CAVADA 8  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM - Methemoglobin reductase - reverting your - Maintenance of extracellular and intracellular methemoglobin (abnormal hgb that carries iron in cations - Naka depende yung overall charge ng cells ferric state) so it reverts back into functional and amount of fluid na nasa loob and labas. hemoglobin (carries ferrous state) only ferrous state - Cytoskeletal - pinaka frame ng ating cell membrane. can perform oxygen HEMOGLOBIN - Rapoport - 2,3-DPG reduces affinity of hemoglobin for Conjugated globular protein oxygen or pampadulas sa oxygen or acts as lubricant. composed of 4 heme groups Pag mataas 2,3-DPG dudulas agad si oxygen papunta and 2 pairs of polypeptide sa tissues. Yung efficient oxygen transport is dapat chains. need meron 2,3-DPG. Main cytoplasmic content of PLASMA MEMBRANE RBCs. Thickness: ~5 um; elastic and deformable. Transport of O2 Composition: 8% carbohydrates, 54% proteins, 40% NOTES: fats - hemoglobin: major protein Phospholipids: component or main cytoplasmic content of RBCs - Inner: phosphatidylinositol, phosphatidylserine, - color red ang hemoglobin because of this particular phosphatidylethanolamine protein - Outer: phosphatidylcholine, sphingomyelin - 2 pairs of polypeptide = 4 heme groups - Heme groups - allow to transport oxygen - why nagiging capable ang hemoglobin in carrying oxygen? because of smaller protein called heme HEME Protoporphyrin IX with ferrous iron (Fe2+). Four (4) heme groups per molecule of hemoglobin. One (1) heme molecule reversibly binds one (1) O2 molecule. NOTES: NOTES - Heme also known as - Inner phospholipid: polar heads and non-polar tails, “Ferroprotoporphyrin IX” cell interior yung (nasa loob) yung mga nasa ilalim - Heme protein is tetrapyrrole - there are 4 pyrrole najan yung mga inner phospholipids. rings and in the middle is ferrous iron - Extracellular space: nandito yung mga outer - One heme molecule reversibly binds 1 oxygen phospholipids molecule or yung O2 molecule. - If may 1 heme that binds to 1 oxygen molecule, ang 1 Glycocalyx - produces zeta potential hemoglobin molecule carries 4 oxygen molecules kasi Trans-membranous proteins may 4 heme groups. 1. Aquaporin-1 - creates an inward flow of H,O HEME SYNTHESIS 2. Glut-1 - energy-free glucose transport Occurs in the mitochondria and cytoplasm of 3. ATPase-dependent cation pumps - developing RBCs. maintains IC-to-EC ratio of Na* (1:12) and K* Begins in pronormoblasts and ends in (25:1) polychromatophilic RBCs. Cytoskeletal proteins - spectrin (a and B), actin, Mature RBCs do not produce heme. tropomyosin, adducin, tropomodulin, dematin, protein 4.1 NOTES: - Zeta potential - net negative charge produced by plasma membrane. Para saan ang net negative charge? if all RBCs have zeta potential, may similar charge like negative to negative so they will repel each other. Zeta potential is crucial so RBCs will not bump into each other para maiwasan ma-rupture ang membrane. - Trans-membranous - nag ccross ng buong span ng cell membrane. Yung red sa pic sya yung example nito. PUNZALAN & CAVADA 9  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM NOTES: NOTES: - Pano na-synthesize ang heme? your heme is - majority ng Hb F ang makikita pag nasa fetal stage, synthesized in cytoplasm and mitochondria of maturing kasi si gower 1, gower 2, and Portland makikita lang cells. sila sa primitive erythroblast. - Kahit mga reticulocytes wala ng nucleus pero may - At birth, Hb F pa rin pinaka marami have remnants that can still manufacture small - After birth until adulthood, Hb A1 ang predominant then amounts of heme molecules. Hb A2, meron pa rin Hb F pero small amounts na lang - Mature rbc cannot produce heme kasi wala ng RNA - Common na globin chains ay alpha remnants and ribosomes. HEMOGLOBIN FUNCTION Two (2) molecules of d-ALA condense to form a Binds, transports, and unloads O2 to tissues pyrrole. 1 g of Hgb = 1.34 mL of O2, and 3.47 mg of Fe2+ Four (4) molecules of PBG condense to form UPG. O2 affinity depends on partial pressure of O2 (PO2). O2 saturation curve - describes the relationship of pO2 and O2 affinity of Hgb. 27 mmHg - P50 value; partial pressure required to saturate 50% of Hgb with O2 - 2 molecules of d-ALA: prior the formation of d-ALA - to form ALA -> start as glycine + Succinyl CoA -> ALA which happens in mitochondria in developing RBC - with ALA, it forms d-ALA (delta-ALA) then 2 molecules will condense to form Porphobilinogen which happens NOTES: in cytoplasm - Partial pressure of oxygen is an indirect measure of - Coproporphyrinogen will enter again the mitochondria how much oxygen is present. Gases na naka dissolved - Heme synthase or ferrochelatase: this enzyme will sa blood mahirap imeasure using routine analytic incorporate iron or ferrous iron to protoporphyrin IX to procedures like spectrophotometer. Ang minea- form Protoporphyrin IX or also known as heme protein. measure is amount of pressure exerted from the gas. GLOBIN SYNTHESIS - The relationship between partial pressure of oxygen Produced in the normoblast cytoplasm. and oxygen affinity of hemoglobin can be detected Two (2) pairs of unlike polypeptide chains. using oxygen saturation curve Chromosome 11: beta, delta, epsilon, and gamma - P50 value - pressure in which hemoglobin will be chains saturated with 50% of hemoglobin with oxygen. Chromosome 16: alpha and zeta chains - X-axis is horizontal (partial pressure) NOTES: - Y-axis is vertical is P50 (oxyhemoglobin saturation/ - Hemoglobin (heme & globin) partial saturation) - Heme is attached in globin chains - Those 3 types of curve represents different types of - Peptide chains are produced in Chromosome 11 & 16. hemoglobin - Chromosome 16: A and Z (alpha & zeta) - The more the partial pressure increases the more na - Chromosome 11: lahat ng nasa gitna (beta, delta, and mataas yung concentration ng oxygen sa hemoglobin gamma) - Yung nasa middle na curve (27 mm mercury), sa y axis HEMOGLOBIN ASSEMBLY yun ay 50, normal hemoglobin is saturated at its 50% capacity. - 2-3,DPG - pampadulas so the more na maraming 2- 3,DPG - the more na mabilis mawawala si oxygen kay hemoglobin. Left shift: - at 27mmHg, mas mataas na yung content ng kanyang oxygen. Nagkakaroon ng left shift if may abnormal hemoglobin, alkalosis, bumaba DPG, bumaba temp. Kapag nagkaroon left shift, remember that affinity of hemoglobin for oxygen ay tumataas so the more na nagshift sa left, mas love nya si hemoglobin, ‘di nya agad bibitawan si hemoglobin. PUNZALAN & CAVADA 10  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM - Pag nag increase affinity ng hemoglobin for oxygen, ‘di agad mabibitawan si oxygen. Magkakaroon ng abnormality ng delivery of oxygen kasi ‘di nya agad binibitawan. - Level ng 2,3DPG mas magkakaroon ng higher affinity of oxygen Right shift: - mas bumababa affinity nung hemoglobin for oxygen. pag may acidosis, mataas ang 2,3-DPG, fever: mabilis binibitawan ni hemoglobin si oxygen. At 27mmHg, ABNORMAL HEMOGLOBIN VARIANTS yung saturation ng hemoglobin is less than 40% na Starting from methemoglobin -> sulfhemoglobin -> lang (graph), samantalang pag normal 50% dapat. carboxyhemoglobin (based on the table above) - Bakit mas mababa yung amount of oxygen dun sa hemoglobin? kasi nga binibitawan nya agad si oxygen. NOTES: - Normally at 27mmHg, 50% capacity dapat. - sa normal, hemoglobin ay may ferrous iron. valence is 2+ and kapag oxidized, magiging 3+ HEMOGLOBIN FUNCTIONS: - Carboxyhemoglobin: carries carbon monoxide (has Left shift thousand folds of affinity compared to oxygen). Decreased body temperature (e.g., hypothermia) Reversible pero mahirap tanggalin kasi masyado Alkalosis (increase body pH) mataas affinity ng hemoglobin for carbon monoxide Hemoglobin variants with increased oxygen affinity compared to oxygen. Multiple transfusions with 2,3-BPG depleted red blood - Hyperbaric O2: patient ilalagay sa chamber tas sa cells chamber ay i-increase pressure ng oxygen to displace - donated blood ay walang substrate for production of carbon monoxide 2,3-BPG, yung level nito sa blood bags ay mauubos. HEMOGLOBIN MEASUREMENTS pag tinransfuse sa patient, yung circulated RBC will Co-oximetry have higher affinity for oxygen so magkakaroon Spectral problem in oxygen carrying capacity absorption Right shift measurement of Increased body temperature hemoglobin Acidosis (decrease body pH) variants Hemoglobin variants Increased 2,3-BPG Cyanmethemoglobin method HEMOGLOBIN VARIANTS Reference method Hemoglobin with modified structure due to chemicals Measures all forms of Hgb except sulf-Hgb. or drugs. Read at 540 nm. Normal variants: Drabkin's reagent: 1. Oxyhemoglobin 1. Lysing agent 2. Deoxyhemoglobin and 2. Potassium ferricyanide Carbaminohemoglobin 3. Potassium cyanide Abnormal variants: NOTES: 1. Methemoglobin - Lysing agent: sinisira rbc para makuha yung 2. Sulfhemoglobin hemoglobin na immeasure, once Hgb is released, it will 3. Carboxyhemoglobin react with potassium ferricyanide (oxidizing agent) to form methemoglobin then yung potassium cyanide ay NOTES: magiging cyanide molecule donor to form - oxyhemoglobin - bright red kasi merong oxygen and cyanmethemoglobin makikita ito sa arterial blood kasi oxygenated blood - cyanmethemoglobin is stable compound and not easily ang nasa arteries natin deteriorates kaya reference method - Deoxyhemoglobin - walang oxygen so appearance - CO-oximetry: based on absorption of light of variance ay dark red or purplish. venous blood ang color ng hemoglobin at different wavelengths blood at medyo dark kasi nabawasan oxygen and it is IRON delivered in tissues Bivalent (Fe2+) and trivalent forms (Fe3+). - Carbaminohemoglobin - may nakakabit na carbon Compartments: dioxide kasi dinadala rin ng carbon dioxide ang ating 1. Functional: hemoglobin and myoglobin (Fe2+) RBC pabalik sa ating lungs 2. Transport: transferrin (Fe3+) PUNZALAN & CAVADA 11  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM 3. Storage: ferritin and hemosiderin (Fe3+) NOTES: - iron is the principal element responsible in red color of heme - functional form (bivalent -> Fe2+) - transport form (trivalent -> Fe3+) - Transferrin - major plasma protein that carries ferric iron WHITE BLOOD CELLS Protects the host from infections and are relatively colorless. Differentiated by light microscopy or by flow cytometry. Named after their acid- base staining and nuclear appearance. Granulocytes: NOTES: - With oxidase-positive granules Myeloblast - Neutrophil, eosinophil, basophil - largest and youngest Agranulocytes: - light staining - With oxidase-negative granules - Type 1 (no visible granules) - Lymphocyte, monocyte - Type 2 (20 azurophilic granules) Promyelocytes NOTES: - largest sabi sa table pero sa reality is mag start sa - Neutrophils (attracts acid and basic stains) malaki papaliit - Eosinophils (attracts acid stains - eosin) - Eccentric location (nasa gilid) and full of azurophilic - Basophils (attracts basic stains - methylene blue) granules, promyelocytes na agad! OVERVIEW OF ADULT HEMATOPOIESIS - promyelocyte exerts procoagulant activity: nag exert ng clotting NOTES: - Acute promyelocytic leukemia (a medical emergency) - most of WBC because it exerts procoagulant activity. patient is at risk comes from myeloid cell of DIC and can cause death due to excessive bleeding. line while lymphocytes Myelocyte manggagaling sa - “dawn of neutrophilia” lymphoid cell lines - Blue to purplish / Lilac appearance of cytoplasm - No nucleoli pero oval pa yung nucleus - in RBC, last stage capable of cell division is polychromatic normoblast GRANULOCYTE MATURATION SERIES - in granulocyte, last stage capable of cell decision is myelocyte Metamyelocyte - less than ½ yung indentation of nucleus Band Neutrophil / Stab form - hindi pa segmented yung nucleus pero indentation is more than ½ ng diameter ng nucleus GRANULOCYTE MATURATION SERIES PUNZALAN & CAVADA 12  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM LYMPHOCYTE MATURATION SERIES NOTES: - neutrophil - attracts basic and acid stains - eosinophils - granules of eosinophilic attract eosin stains which is orange (ph of granule content ng eosinophils? basic stains) kasi content nya is basic - basophils - granules is blue DAY 2 (DAY 1 CONT.): ROUTINE HEMATOLOGICAL TESTS (acidic) kaya attracts basic ANTICOAGULANTS USED IN HEMA LAB stains kasi content nito ay acid MONOCYTE MATURATION SERIES NOTES: EDTA - Pinaka common is EDTA (purple top) NOTES - Bind to remove calcium from the plasma to - monocyte: we use cell markers in a form of coagulations monoclonal antibodies for flow cytometry - Recommended anticoagulant by the ICSH MACROPHAGES (International Council for Standardization in Vacuolated cytoplasm Hematology) Functions: - CLSI (Clinical and Laboratory Standards Institute) 1. Phagocytosis - Bakit sya yung recommended anticoagulant for CBC? 2. Antigen Kasi yung anticoagulant that preserves well your blood presentation cell morphology yung konting variations sa proportion Examples: Kupffer cells, sa ating anticoagulant to blood ratio, hindi agad agad Langerhans cells, sisirain yung morphology ng cells but of course pag microglia, alveolar nagkaroon ng excessive na variations doon sa macrophages, recommended na ratio, that would lead to cell osteoclast disruption morphology kaya ‘di tayo nag aaccept ng NOTES underfilled and overfilled samples. - Phagosomes: contain engulf materials - Underfilled - causes shrinkage - Kupffer cells - liver - Overfilled - causes clot of the sample - Langerhan cells- skin Sodium citrate - Microglia - CNS - common: 3.2% (light blue top, coagulation tests PT and - Alveolar - lungs PTT) - Osteoclast - bone - (Black top) for ESR - Histiocytes - Connective tissues - Similar with the action of EDTA, your sodium citrate forms an insoluble salt with calcium then it removes calcium from plasma therefore inhibiting the coagulation cascade. - 9:1 (9 for blood and 1 for anticoagulant) PUNZALAN & CAVADA 13  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM - If onting underfilled / overfilled reject kasi it can cause - Kada square may 1 mm^2 erroneous result - Yung may nakalagay na W, ginagamit for manual wbc Heparin count - light green stoppered tube - Yung large central square ang ginagamit sa rbc - Mechanism of action differs from EDTA & Sodium counting. Citrate because Heparin, instead of removing calcium, - Large central square has a total of 25 smaller it inhibits the enzyme thrombin which is the active form squares. 5 lang binibilangan natin (center and 4 outer of prothrombin. squares) - usually found in capillary tubes - Yung distance between counting chamber and glass - hindi sya gingamit sa hema because yung slide / coverslip, may 0.1mm space. Dito nilalagay anticoagulant nito usually ginagamit sa plasma liquid. So, total volume ng specimen / suspension is chemistry 0.9mm^3 - ginagamit for manual hematocrit -.1 x.9 =.9mm^3 - bakit sya inappropriate for most test? Yung ating PIPETTES heparin this is an Acid Mucopolysaccharide (medyo acidic) ‘di ginagamit kapag mapperform blood smear - Kapag kumuha tayo ng blood sample from heparinized tube, then we will make a blood smear, pahg nagstain tayo, it will stain excessively blue (basic dye), then if medyo acidic yung pH ng blood as a result of heparin, it will atrract the basic stain. COMPLETE BLOOD COUNT Panel of tests that includes cellular enumeration, hemoglobin and hematocrit determination, and NOTES: statistical analyses to provide an overview of blood cell - Yung bulb ng RBC thoma pipette mas malaki morphology. compared sa WBC thoma pipette RBC parameters: RBC count, Hgb, Hct, RBC indices - Wbc thoma pipette mas konti dilution (1:20) dilution (MCV, MCH, MCHC), RDW, reticulocyte count factor is 20 WBC parameters: WBC count, WBC differential count - Unopette: usually ginagamit for manual platelet count (percentage or absolute) - Sahli: usually ginagamit sa hemoglobin estimation Platelet parameters: Platelet count, MPV GENERAL FORMULA FOR CELL COUNTING HEMOCYTOMETRY Levy chamber with improved Neubauer ruling. Two raised surfaces (3 mm x 3 mm) separated by an H-shape moat. Large squares: nine (9) 1 mm2 Large corner squares: 16 smaller squares; for WBC counting Large central square: 25 smaller squares (0.04 mm2 NOTES: each); for RBC counting - Hindi lang ‘to sa ginagamit sa hema, this also applies Distance between counting surface and cover slip: 0.1 to body fluid cell count. mm - May iba’t ibang variation pero ito ang generic form ng Total volume: 0.9 mm3 formula. - Saan nanggaling yung 10? Ito yung depth natin which is 0.1mm yung layo ng coverslip sa ating counting chamber. Pag inakyat mo sya nilagay sa numerator mag move ka ng 2 so magiging 10 na sya. - Mas madaling tandaan yung 2nd formula. MANUAL BLOOD CELL COUNT SAMPLE PROBLEM Total cell count on side 1: 92 NOTES: Total cell count on side 2: 100 - 9mm square yung area Dilution: 1:20 - Yung surface na binibilugan ni sir najan yung Find: Total WBC count newbauer ruling, makikita pag viniew under microscope, separated by H-shape moat. PUNZALAN & CAVADA 14  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM SOURCES OF ERRORS NOTES: NOTES: - The dilution is 1:20 kasi yun dilution ng wbc thoma WBC count pipette. - Fingerprints: it interferes yung pag tingin sa chamber - Cell count = cell counted x df x 10 / area in square pwede nyang I-obscure yung chamber / glasslide mm^2 magiging malabo pag tingin sa chamber - First step: Substitute the variables - Dusts: Pwede pagkamalang cells - Kunin average ng dalawa (100 + 92 / 2 = 96) - Pag na contaminate ng acids / hypertonic solution ma - 96 x 20 (df) / area in mm^2 kasi 4 na large corners rupture yung cells square ang binilangan each large square corners has - Dahan dahan dapat pag charge ng fluid sa counting mm^2 which is 4 x.1 (yun yung distance ng coverslip chamber para ‘di umapaw. and chamber) = 4.8x10^9 wbc per liter of whole blood. - Pag nag overflow yung dilutions, yung mga cells sasama doon sa umapaw, ‘di na mabibilang ng maayos yung mga cells. - Cells pag chinarge na doon (by inertia) iikot ikot pa yung mga cells, need muna magsettle yung mga cells before bilangin. - Pag may severe anemia (usually nagpapakita na yung mga metarubricytes/orthochromatic normoblasts) This nucleated RBCs are also resistant to lysing action of acids. Kapag nag apply ng WBC diluting fluid, ‘di sila ma-lyse so pwedeng ma count as wbc that can lead to erroneous WBC count. Manual RBC count Platelet count - isotonic kasi syempre pag naging hypotonic - dirty pipette: dusts pwedeng pagkamalang platelets marurupture ang rbc ang iniingatan natin dito yung - Platelets satellitosis: Neutrophils surrounded by morphology ng rbc during dilution platelets (can cause falsely decreased platelets kasi - Yung rbc 5 small square of the large central square instead of counting platelets, hindi mo makikita yung - Magkaiba male and female (mas mataas ang male mga nakapalibot sa mga neutrophils) since mas maraming RBCs due to their body size) - To correct: Use sodium citrate (mas maraming sodium Manual WBC count citrate. Compared sa EDTA, you have to perform - Yung capillary yung galing sa capillary puncture na correction din sa computing kasi na dilute din ang ating cinollect sa microtainer with EDTA. blood) - Common ginagamit for manual wbc not sa blood but sa CORRECTED WBC COUNT body fluids is yung TURK’s solution kasi advantage Nucleated RBCs are falsely counted as WBCs. may gentian violet na somehow instain yung mga WBC Used when nucleated RBCs are > 5 per 100 WBCs in so that it is easily identified under the microscope. PBS - What is the characteristic of the diluting fluid for WBC Formula: count? It contains ACIDS. - Para saan ang acid? To in order to lyse RBCs, so ang maiiwan lang ay mga WBCs. - dito sa wbc count para mas mapadali yung counting, inaalis na yung rbc (counting area 4 large area squares NOTES: of the whole chamber). - pag nagkaroon ng flagging due to nucleated RBCs, - This ranges nagbabago bago slightly per laboratory. gagawa peripheral blood smear to confirm nucleated Platelet count RBCs then perform corrected WBC count upon - 150,000 or 450,000 platelets confirmation in order to account for this nucleated RBCs mistaken as WBC. PUNZALAN & CAVADA 15  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM - Differential count: bibilang 100 wbc then titignan in order for your Drabkins reagent to react with the distribution ng different types of wbc carboxyhemoglobin - Isasama natin yung number ng nucleated rbcs. HEMATOCRIT DETERMINATION Bibilang 100 wbc but bibilang ka separated nucleated Aka packed cell volume. rbc para makakuha ka ng ratio ng nucleated RBCs per Volume of packed RBCs that occupies a given volume 100 WBCs of WB. - According to rodaks: pag > or equal 5 tsaka perform Reported either in percentage (%) or liters per liter (L/L) corrected wbc count Reference range: - This much nucleated RBCs can cause erroneous WBC Male: 40-54% or 0.40-0.54 L/L counting. Female: 35-49% or 0.35-0.49 L/L SAMPLE PROBLEM Sources of errors: 1. Improper sealing of capillary tube 2. Increased AC concentration 3. Improperly mixed specimen 4. Insufficient centrifugation time 5. Delay in reading results 6. Including buffy coat in reading 7. Improper use of microhematocrit reader 8. Trapped plasma 9. Introduction of interstitial fluid NOTES: NOTES: - 17.8 x 100 / 15 + 100 = 15,478/mm^3 or 15.5x10^9/L 1. Yung manipis, at one end nandoon yung band (di - 17,800 per mm^3 = 15,478/mm^3 or 15.5x10^9/L tatakpan) yung other end kung saan pinapasok yung - Yung excess na nabilang is due to nucleated rbc blood, tinatakpan ng clay / paraffin wax. Pag HEMOGLOBIN DETERMINATION cinentrifuge na natin sya, pag hinila na yung rbc Reference method: cyanmethemoglobin method pababa maiipon lang sila sa seal, pag improper sealing Reagent: Drabkin's reagent (lysing agent)+potassium technique with the shear force of centrifugation ferricyanide + potassium cyanide) matatanggal yung blood sample Read at 540 nm; does not measure sulf-Hgb (co- 2. Pwede mag shrink mga rbc (falsely decrease) kasi oximeter) lumiit mga rbc Reference range: 3. Pwede mag clot, kaya pwedeng ‘di mag separate Male: 13.5 - 18 g/dL (135-180 g/L) entirely Female: 12 - 15 g/dL (120-150 g/L) 4. Pag kulang sa prescribe time, ‘di mag separate Sources of errors: completely yung RBCs from the plasma/serum 1. Exposure of reagent to light 5. Mag resuspend rbc pag ‘di agad binasa kaya aangat 2. Leukocytosis ulit sa plasma yung rbc pag ‘di binasa agad 3. Lipemia 6. Edge ng rbc with the plasma dapat doon tatama yung 4. Hemolysis-resistant RBCs ating reader hindi sa intermediate layer which is buffy 5. Abnormal globulins coat 6. Carboxyhemoglobin 7. False reading pag di marunong gumamit NOTES: 8. Hemoglobinopathies ‘di complete mag separate yung - cyanmethemoglobin: stable compound formed from plasma with rbc mas mag mag mumukhang mataas oxidation and transfer of cyanide molecule to our yung hematocrit hemoglobin, then measured spectrophotometrically. 9. Pag inisqueeze ng sobra sobra yung puncture site, - Drabkin’s reagent (lysing agent ginagamit to release mag iintroduce tayo sa capillary tube ng interstitial fluid hemoglobin molecules from rbc; usually stored in so ma dilute lalo rbc kaya mangyayare magiging amber bottle since its photosensitive) falsely decreased. - Potassium ferricyanide (acts as oxidizer; converts hemoglobin into methemoglobin) - Potassium cyanide acts as cyanide molecule donor para maging cyanmethemoglobin - CO-oximeter ginagamit when suspecting sulf-Hgb - Amber bottle: light sensitive - Leukocytosis: turbidity (abnormally higher) - Hemolysis-resistant RBC: if may hemoglobinopathy - Carboxyhemoglobin: na measure naman siya with Drabkins but u have to wait for longer incubation period PUNZALAN & CAVADA 16  Aug. 7, 2024 (DAY1 & 2 / PART 1) | 1PM to 5PM - sa first mark, yung junction / clay kung saan tumapat - pag ‘di nakikita yung discrepancy sa blood baka nasa 2nd line doon sa 1st junction ng plasma doon sa rbc machine ang problem need irecalibrate / pa check. without including the buffy coat (0.45 sa pic) - 15 x 3 = 45 +/- 3 so the acceptable range is 42-48 - Buffy coat contains platelets and wbc - Hct is 36 so mababa rin siya, ‘di siya pasok sa range - Nakatutok dapat sa 100 kasi it represents the entire natin so therefore magccheck ulit tayo ng mga possible volume of blood. sources of discrepancies nito RULE OF THREE - Unless hindi talaga makita yung source of this Quick visual check of hemoglobin and hematocrit discrepancy in patient sample, it could be an indication results. that the machine is encountering some troubles. Need Only applies to specimens with normocytic and ipa-calibrate/maintenance normochromic red blood cells. - Pag normal rbc indication of machine failure. Values discrepant to this rule may indicate abnormal RBC INDICES RBCs or instrument error. Should NOT be used to predict results of RBC count, Hgb, and Hct. NOTES: - ginagamit for quality control check NOTES: - Normocytic and normochromic rbc but of course ‘di - Machine na nag ccompute nito usually malalaman pag ‘di nag perform actual cbc (manual / - Most of the time ginagamjt ng physician is MCV and automated) MCHC. - Ginagamit rule of three before i-release result - MCH is equally useful as MCV and MCHC in identifying - Pag ‘di pumasok sa parameters ng rule if three, most different types of anemia. of the time hindi normocytic / normochromic yung rbc MCV: titignan sa blood smear, pag may abnormality talaga - average of rbc volume in fento liters (fL) EXAMPLES - estimation of rbc size