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BASIC HAEMATOLOGY BLOOD CONSTITUENTS RBC’S WBC’S PLT’S PLASMA BONE MARROW STEM CELLS WBC’S Late normoblasts Reticulocytes RBC’S Megakaryocytes Granulocytes Non-granulocytes Neutrophils Lymphocytes Eosinophils Monocytes PLT’S Basophils Notes RBC’s – Live ~ 120 days Spleen; Hb broken down in Liver to Bile Acids & Bilirubin. NB. Decrease in size as they get older. Lymphocytes B-Lymphocytes (Bone Marrow) – Produce Ab’s against invading antigens eg. Viruses. T-Lymphocytes (Thymus) – Ab’s are bound to the cell membrane (“Killer” T cells). Monocytes Phagocytic - “eat foreign bodies, antigens, debris, etc. Neutrophils & Eosinophils Contain proteolytic enzymes to break down cell walls. Basophils contain Histamine & Heparin (Inflammatory processes) and are also thought to be involved in the metabolism of Fat. Erythropoietin – produced by Kidneys in response to low O2 levels in blood gives increase in RBC’s. RED BLOOD CELLS – ERYTHROCYTES ANAEMIA Decrease in Hb, HCT, & RBC’s Blood Loss (Internal/External Bleeding) Haemolysis (destruction) Bone Marrow Disease Iron Deficiency (Haemoglobin) Bone Marrow Disease Non-regenerative Anaemia – No Retics despite Anaemia Regenerative Anaemia – Bone Marrow Retics RDW RDW RBC No’s RBC Size Retics present Retics Present (Larger RDW) Increased MCV Decreased MCHC Iron Deficiency Lack of Hb Smaller RBC’S with less Hb (MCV Decreased) (MCHC Decreased) NB. Decreased MCV also results from Porto-systemic shunts due to Iron assimilation in the Liver. POLYCYTHAEMIA Increase in RBC’s Dehydration Bone Marrow disease Rarely – Cardiovascular Disease; Kidney malfunction; Polycythaemia vera Megakaryocytes PLATELETS – THROMBOCYTES Platelets Blood Clots Thrombocytopenia (increase) – If MPV unchanged evaluate bone marrow. Thrombocytosis (decrease) – Blood loss; Iron deficiency; Bone Marrow Cancer. WHITE CELLS – LEUCOCYTES LYMPHOCYTES Produce Ab’s against non-bacterial diseases ie. Viruses Lymphocytosis (Increase) Viral diseases Stress (Cats & Horses) due to Adrenaline. Leukemia Lymphopenia (decrease) Steroid therapy Steroid release from Adrenals (Cushings) Distemper or Parvo MONOCYTES 2nd line of defence against Micro-organisms/Bacteria (after Neutrophils) Monocytosis – Chronic disease; Stress; Steroid diseases Monocytopenia – Irrelevant in Vet. Medicine NEUTROPHILS 1st line of defence against Micro-organisms/Bacteria or inflammatory diseases. Neutrophilia – Band Neutrophils (immature forms) released form Bone Marrow in response to inflammation. (Left – shift). - Chronic stress – due to steroid release (eg. Cushings) but no bands. Neutropenia – Invader is winning EOSINOPHILS Eosinophilia – Hypersensitivity (Allergic Reactions) - Parasites (Fleas, Ticks, worms in horses) Eosinopenia – Steroid mediated stress. BASOPHILS Normal to have none. Basophilia – Very rare in Vet. Medicine - Hypersensitivity (as above) - Very rare cancers in dogs. Basopenia – Does not exist. HAEMATOLOGY ANALYSERS 12ul sample ~6 x 106 /mm RBC’s PLT’s (+WBC’s very small nos) ~10 x 103 /mm WBC’s Hb (RBC’s lysed Hb) 1/20,000 Dilution 1/300 Dilution REQUIRE A FLOW OF SINGLE CELLS Two commonly used methods: Laminar flow Cytology (Hydrodynamic Focusing) 0 0 0 0 Small aperture aspiration o 0 24 hour maintenance. CDC – WBC’s & RBC’s Sysmex – WBC’s Celldyne – WBC’s & RBC’s Partial vacuum ABC – WBC’s & RBC’s HMT – WBC’s & RBC’s (MS5) Sysmex – RBC’s Celldyne – WBC’s & RBC’s Cellyvet – Vetlab Medonic – Menarini COUNTING METHODS a) Impedance To achieve diff use “lyse” reagent that shrinks white cells. Eg. ABC: Lympho’s>Mono’s>Neutro’s>Eosino’s b) Laser Scatter (eg. Sysmex, Celldyne, Lazercyte) Sensor Source Side Scatter Front Scatter Monocytes Sensor Lymphocytes Front Scatter Side Scatter OTHER TYPES OF HAEMATOLOGY ANALYSERS a) VS2000 – Hematek OHP’s b) QBC – Quantitative Buffy – Coat Haematocrit tubes coated with fluorescent dye which results in differential staining of DNA & RNA in the White cells, Reticulocytes & Platelets. Uses small plastic “float” to separate white cells in Buffy Coat. Buffy Coat P BENM L R PLASMA Detector RBC’s UV Source NB. Floating discriminators in Impedance/ Laser Scatter Counters simply refer to the ability to change cut-off points between different types of cells in different species. Viz a viz Smart Cards in the ABC.