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Lecture notes Microbiology, (BS236)
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Lecture notes from the BS236 Medical Microbiology Module at University of Essex
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Haemotology Introduction = The branch of medicine involving study and treatment of the blood and blood forming tissues. Study of: Blood, Haemopoiesis /Erythropoiesis (the process by which blood cells / red blood cells are produced), Homeostasis (maintaining a stable blood environment), Haemostasis (minimise blood
loss), Etiology (what causes illnesses related to blood), Diagnosis (how to detect illnesses related to blood). Tissue: part of an organism consisting of an aggregate of cells
having a similar structure and function. Blood is considered a connective tissue! Blood forming tissues: Bone marrow and lymphatic tissues: thymus, spleen and lymph
nodes. Blood Cell Types - 3 types: Red Blood Cells (Erythrocytes). Carry oxygen to tissues and CO2 to lungs. White Blood Cells (Leukocytes). Defence against infection.
Platelets (Thrombocytes). Combine with specialist proteins to form a clot. In 1 ml of blood there are (on average) 5 billion RBCs, 10 million WBCs and ~250 million
platelets. Phlebotomy and Anticoagulants - Blood is taken into a blood vacutainer by a phlebotomist. Tube contains anticoagulants. Common anticoagulants are:
Trisodium citrate (sodium citrate) = Binds metals, including calcium (required for clotting mechanism). Used when analysis of anticoagulant proteins needed. EDTA
(ethylenediaminetetraacetic acid) = Binds metals, including calcium (required for clotting mechanism). Used when full blood count needed. Heparin (sulfated
glycosaminoglycan) = Activates antithrombin, which in turn deactivates thrombin. Used when WBC analysis needed. Blood Preparation - Blood may be analysed whole or
separated into components. Basic separation achieved by centrifugation of blood into 3 constituents: Blood plasma is a Transport Medium for cells, dissolved nutrients
and plasma proteins. ‘Buffy Coat’ White Blood Cells (immune system) and Platelets (blood clotting). Red Blood Cells – Carry Oxygen and CO2. Haematocrit Levels -
Haematocrit, also known as the packed cell volume (PCV) or erythrocyte volume fraction (EVF), is the volume percentage (%) of red blood cells in blood. Normal levels
are ~45% for men and ~40% for women. Polycythemia: Primary-abnormality in bone marrow- usually by overstimulation of red cell precursors. Can also generate more
WBC and platelets. Secondary-elevated erythropoietin. Blood content statistics - 5x as viscous as water, Slightly alkaline, pH 7.4, Very well buffered. Haematocrit level:
40-45%, Venous blood (whole) contains 138 g/l of haemoglobin (13.8 g/dl),Mean cell volume is 92 femtolitres (92x10-15 litres ) Mean cell haemoglobin mass is 29.5
picograms (29.5 x10-12 g), Mean cell haemoglobin concentration is 330g/l. Full Blood Count - A full (or complete blood count) is most important and most commonly
requested test in haematology. Typically composed of 4 main diagnostics: Haemoglobin concentration = (Hgb) 120-174 g/L (12-17.4 g/dL), Haematocrit levels = (HCT)
31-53%, White blood cell count = (WBC) 3.5-11 x 109/L, Platelets = (PLT) 140-450x 109/L. But can contain many more: Mean Cell Volume (MCV) = 80-99 fl, Mean Cell
Haemoglobin (MCH) = 27-32 pg, Reticulocytes = (RCT) 20-80 x 109/L, Differentiated WBC = (e.g neutrophil count, typically in x109/L), Nucleated RBC = <0.1 x109/L.
Why so many ways to measure blood content? Some parameters may be in normal range of variation, but others may not. Gives information that may lead to further tests
to diagnose condition. FBC rarely leads to a diagnosis on its own. Used to inform on further specific tests for diagnosis or to monitor condition. Components of Blood - Red
Blood Cells = Erythrocytes (red blood cells). Generated in bone marrow (2.4 million every second), circulates for ~120 days until broken down and recycled by
macrophages in spleen and liver (mostly NOT in circulation). Contain large amount of oxygen carrying protein Haemoglobin. The main problem that arises with RBCs is
ANAEMIA. The main physiological consequence of anaemia is a decreased oxygen carrying capacity of blood (lethargy, weakness, dizziness).Common causes of
anaemia - Iron deficiency, Acute and chronic blood loss, Vitamin B12 / folate deficiency, Malignancy, renal/liver disease, lead poising, infection. Hereditary defects in
haemoglobin production: Sickle cell / Thalassaemia. Microcytes (MCV <80 fL) commonly caused by iron deficiency or sickle cell anaemia. Macrocytes (MCV >100 fL)
commonly caused by alcohol, pregnancy, vitamin B12 or folic acid deficiency. Both Micro- and Macro-cytosis often accompanied by anisocytosis (variable sizes).
Classifications (MCV, MCH numbers vary between sources) Microcytic (hypochromic, small RBCs ) – MCV < 80fl - MCH <27pg. Example causes: Iron deficiency,
Thalassaemia, Lead poisoning. Normocytic (normochromic) - MCV 80-95 fl - MCH > 27pg. Example causes: Haemolytic anaemia, Acute blood loss, Renal disease, Bone
marrow failure (e.g. post-chemotherapy). Macrocytic - MCV > 95fl Example causes: Vitamin deficiency (B12, foliate), alcohol, liver disease, aplastic anaemias. White
Blood Cells - Leukocytes (white blood cells). Part of immune system, protecting against infection (bacteria, viruses, parasites). Five different types of cells in 2 groups:
Neutrophils: Granulocyte. Polymorphonuclear (irregular 3-5 lobed nucleus). Part of innate immune system – attacks invading organisms like bacteria, yeast and debris by
phagocytosis. Some may contain vacuoles. Eosinophil: Granulocyte. Polymorphonuclear (linked 2-lobed nucleus). Part of immune system combating parasites. Involved
in allergic responses. Releases histamine. Basophil: Granulocyte. Polymorphonuclear (numerous granules usually obscure nucleus). Participates in hypersensitivity
reactions. Releases histamine and heparin. Granulocytes release or produce toxic materials by ‘degranulation’ on the ingestion of microorganism. These include:
Enzymes (Lysozymes to dissolve bacteria cell wall, acid hydrolases to further digest bacteria). Low pH vesicles (pH 3.5-4.0) to assist acid hydrolase activity. Toxins
(reactive nitrogen species (e.g. nitric oxide) and reactive oxygen species (e.g. hydrogen peroxide, singlet oxygen)). Antimicrobial Agents (e.g. defensins). Monocyte:
Agranular. Mononuclear (often kidney shaped). May have vacuoles in cytoplasm (right hand image) similar role to neutrophils. Releases cytokines (e.g. interleukins),
expresses tissue factor (for haemostasis) and cooperates with lymphocytes to generate antibodies. Lymphocyte: Agranular. Mononuclear (often occupies 95% of cell).
, Separated into B cells (generation of antibodies) and T cells (helps B cells to make antibodies and destroy cells infected with viruses). Natural Killer (NK) cells are also
type of lymphocyte-attacks virus infected cells and tumour cells by releasing cytokines to trigger cell lysis or apoptosis. Most common serious disease of white blood cells
is leukaemia. number of white blood cells change: Bacterial, viral and fungal infections raise numbers of WBCs to deal with microorganisms. Raised immature WBCs can
be found in many leukaemias (Uncontrolled proliferation of malignant clones). Some medications lower WBC count or in diseases such as aplastic anaemia. Haemoglobin
has a chromophore (part of a molecule responsible for colour), use Visible spectrometry. Problem: Some haemoglobin is oxy (bright red) some is deoxy (deep red) and
some (~1%) met/ferric (brown). This gives different answers. How would you account for these? Get haemoglobin into same oxidation and ligation state. Add Drabkins
reagent- mixture of potassium ferricyanide and cyanide. The ferricyanide makes all the haemoglobin met (ferric) and the cyanide then binds to the ferric iron to make
cyanomethaemoglobin. How to get MCV & RBCC? Mean cell volume (MCV) Automated: By Coulter counter. In this type of apparatus, the red cells pass one-by-one
through a small aperture and generate a signal directly proportional to their volume. . Red blood cell count (RBCC) =Manual: Dilute blood, make blood film, count cells
using haemocytometer. Automated: Flow cytometry. Uses laser and electric impedance to count and identify types of cells. Flow Cytometry. Lasers shone through a
moving flow of blood cells. Light is scattered forward and to the side. The forward scattering informs on the size of the cell the side scattering depends on the shape of the
nucleus, the amount and type of cytoplasmic granules etc.
Erythropoeisis and Blood Grouping = What is Haemopoiesis? - Haemopoiesis (Haematopoiesis) is the process of the development of blood cells. Ontogeny of
Haemopoiesis –where does it take place? Foetus = 0-2 months - yolk sac, 2-7 months – liver, spleen, 5-9 months – bone morrow. Infants = Bone marrow (most bones).
Adults = Bone marrow (mainly in sternum, femurs, pelvis). However, in adults blood cells can also be generate in spleen, liver and lymph node, usually under pathological
conditions (e.g. myelofibrosis, chronic bleeding, haemolytic anaemias. For adults, intramedullary haemopoiesis is in bone marrow, extramedullary haemopoiesis in liver
and spleen. Haemopoiesis has many steps and types of cells. Blood stem cell can be differentiated into either a myeloid stem cell or a lymphoid stem cell. Lymphoid stem
cells then differentiate into lymphocytes. Myeloid stem cells differentiate into platelets, myeloblasts and Red Blood Cells. RBC regulation - If the concentration of RBCs is
low, the body has a cell signalling pathway in order to upregulate the production of RBCs via erythropoeisis. Hypoxaemia (blood hypoxia) is sensed by liver and kidneys.
Liver and kidneys secrete Erythropoietin. Erythropoietin stimulates bone marrow to increase erythropoiesis. Increased production of RBCs. Increased Oxygen transport.
High altitudes cause the production of more RBCs which is why athletes train at high altitudes. Erythropoiesis is the process of the development of red blood cells. How
may cells are produced in bone marrow each day? 5x 1011 to 1x 1012 cells each day (500 billion - 1 trillion). This is regulated by many factors including cytokines. Major
cytokine involved in regulating erythropoiesis is glycosylated protein erythropoietin. Erythropoietin produced mainly (~90%) in kidneys, Responds to O2 concentration
(gene regulated by HIF-Hypoxic Inducing Factor), Erythropoietin stimulates erythropoiesis, so increases O2 delivery, an alpha helices structure that has sugar groups
(glycosylated) attached to it. Erythropoiesis - Progenitor phase: Starts with Haemopoietic stem cell which differentiate to various colony forming units (CFUs), early stage
differentiation stimulated by stem cell growth factors. Later CFUs have erythropoietin receptors that form antigens on the surface and are thus stimulated by erythropoietin.
Erythroblast precursor phase (development phase): 3 main events – production of ribosomes in preparation for globin synthesis, haemoglobin accumulation, including
import of iron by transferrin, ejection of nucleus and breakdown of mitochondria to form reticulocyte. Final maturation. Reticulocytes - found in blood stream, however can
be seen in the bone marrow (immature RBCs) have ribosomal RNA which is removed. Erythropoietin (EPO) and iron are both vital to erythropoiesis, but they are involved
at different stages of the process of differentiation and maturation from pluripotent (haemopoietic) stem cell to erythrocyte. EPO is crucial over a 10-13 day period when
burst-forming unit–erythroid (BFU-E) cells are transforming into colony-forming units–erythroid (CFU-E) that differentiate into proerythroblasts (pronormoblasts). Iron
incorporation into Hb synthesis is evident during the second, shorter (3–4 days) stage as erythroblasts develop into reticulocytes. At this time, a lack of iron can impair full
hemoglobinisation of the RBCs, leading to functional iron deficiency. In absence of EPO, apoptosis within the CFU-E and BFU-E stages occurs. Reticulocyte Maturation -
Some reticulocytes stay in bone marrow to mature into RBCs before passing into blood stream. Some reticulocytes mature in blood stream. ~1% of RBCs in blood are
reticulocytes, as seen by mRNA stain. Increased levels of reticulocytes (reticulocytosis) generally after blood loss. Decreased levels of reticulocytes could indicate:
Anaemias, bone marrow malignancies, vitamin/mineral deficiencies (B9 B12, iron), chemotherapy. Reticulocytes are defined as immature RBCs containing ribosomal
RNA. ABO blood system - Discovered by Karl Landsteiner in 1901 (Nobel Prize 1930). 3 Allelic genes, A, B and O (ABO gene on chromosome 9) 6 possible
combinations, but only 4 main phenotype: A, B, AB, O. Blood Type A corresponds to A antigens on your RBCs, and B antibodies in your blood plasma. Blood type B has
B antigens on the RBCs and A antibodies within the blood plasma. Blood type AB has both A and B antigens on the RBCs and no antibodies within the blood plasma.
Blood type O has no antigens on RBCs but A and B antibodies in blood plasma. This is important when recieving blood or giving blood. Blood type determination -
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