Pa 7 - Exploration of the Nonspecific/Innate Immunity PDF 2024

PAGE 1 https://www.umfst.ro PA 7 - Exploration of the https://edu.umch.de nonspecific/innate immunity. Assist. prof. Andreea Cozac-Szoke, MD, Ph 2024 May Assist. Prof. Irina-Bianca Kosovski, MD, Ph...

PAGE 1 https://www.umfst.ro PA 7 - Exploration of the https://edu.umch.de nonspecific/innate immunity. Assist. prof. Andreea Cozac-Szoke, MD, Ph 2024 May Assist. Prof. Irina-Bianca Kosovski, MD, Ph Assist. Prof. Raluca Niculescu, MD, Ph Introduction PAGE 2 The defense system Nelson Essentials of Pediatrics Marcdante, Karen J., MD; Kliegman, Robert M., MD; Schuh, Abigail M., MD, MMHPE. Published January 1, 2022. Pages 301-306. © 2022. Innate Immunity - the early defense against infections PAGE 3 1. Definition The survival of multicellular organisms requires mechanisms for defense against microorganisms infections and the elimination of damaged and necrotic cells. This type of host defense is known as innate immunity, also called natural/nonspecific immunity or native immunity. The cells and molecules that are responsible for innate immunity make up the innate immune system. Innate Immunity- the early defense against infections PAGE 4 2. Components of Innate Immunity The components of the innate immune system include: sentinel cells in tissues (resident macrophages, dendritic cells, mast cells, etc) circulating and recruited phagocytes (monocytes and neutrophils) NK cells plasma proteins (complement, cytokines). Innate Immunity PAGE 5 4. Mechanism The two principal types of reactions of the innate immune system are: 1. inflammation: the accumulation and activation of leukocytes and plasma proteins at sites of infection or tissue injury-> act together to kill mainly extracellular microbes and to eliminate damaged tissues. Crash Course Haematology and Immunology, Redhouse White, Gus, BSc (Hons); Vanbergen, Olivia, MA Oxon, MSc, MBBS (distinction). 2019. Innate Immunity PAGE 6 4. Mechanism 2. antiviral and tumour defense: defense against intracellular viruses and tumour cells, even in the absence of inflammation, is mediated by natural killer (NK) cells, which kill virus-infected cells by: the exocytosis of lytic proteins such as perforin and granzymes -> direct lysis of abnormal cell cytokines production (IFNγ, TNF): mediate cytotoxic effects (by inducing up-regulation of MHC molecules on target cells) and also activate other components of the innate and adaptive immune system (T cells) which block viral replication within host cells. Basic and Clinical Immunology, Peakman, Mark, MBBS PhD FRCPath; Vergani, Diego, MD PhD FRCPath FRCP. 2009. 1. The nonspecific immune cells PAGE 7 The phased arrival of different populations of leukocytes into a site of infection. The nonspecific immune cells are: Phagocytes: o Neutrophils – bacteria o Eosinophils – enzymes that kills parasites o Macrophages - "big eaters" Non phagocytic leukocytes: o Basophiles – role in allergic response o Natural killer lymphocytes – antiviral and anti-tumor activity Immunology, Male, David, BA, MA, PhD; Peebles, R. Stokes, MD; Male, Victoria, BA, MA, PhD. © 2021. 1. The nonspecific immune cells PAGE 8 Neutrophils/Polymorphonuclear cells (PMN) PMN-> can be identified using the surface marker CD66. They have two types of granules containing important antimicrobial substances: the primary granules (azurophilic) predominate in young forms and contain cationic proteins and defensins, proteolytic enzymes (elastase and cathepsin G), lysozyme (attacks the cell walls of bacteria). Typically, the myeloperoxidase is involved in the generation of compounds with bactericidal effect. the second types of granules are found in mature neutrophils. These include lysozyme, NADPH oxidase system components involved in the production of toxic oxygen species, lactoferrin protein (iron chelate), and vitamin B12-binding protein. 1. The nonspecific immune cells PAGE 9 Monocytes/Macrophages Macrophages are big phagocytes that can be identified using both morphology and membrane marker CD14. Contain a large number of lysosomes having a similar content with the PMNs granules. Phagocytosis is an active process, determined by receptor mediated binding of pathogen agent. The process is nonspecific at first contact with the antigen, via CD36 receptor, irrespective of the pathogen. 1. The nonspecific immune cells PAGE 10 Monocytes/Macrophages Membranes of micro-and macrophages contain specific receptors for the Fc portions of IgG (CD16, CD64), complement factor C3b (CD11b). In the case of a previous encounter with the pathogen prior it takes place its combination with specific antibodies and / or complement factor C3b. Immunology, Male, David, BA, MA, PhD; Peebles, R. Stokes, MD; Male, Victoria, BA, MA, PhD. © 2021. 1. The nonspecific immune cells PAGE 11 Natural Killler (NK) Cells Natural killer cells (NK) cells are large lymphocytes and usually they contain some azurophilic granules. They cannot be identified morphologically, only using CD56, CD16 and CD3 surface markers (CD56 + CD16 + / CD3-). NK cells recognize and remove viral infected cells and malignant transformed cells. 1. The nonspecific immune cells PAGE 12 Functions of NK cells. A, NK cells recognize ligands on infected cells or cells undergoing other types of stress and kill the host cells. In this way, NK cells eliminate reservoirs of infection as well as dysfunctional cells. B, NK cells respond to IL-12 produced by macrophages and secrete IFN-γ, which activates the macrophages to kill phagocytosed microbes. Cellular and Molecular Immunology, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. © 2018. 2. The nonspecific immune humoral system PAGE 13 The complement system The complement system, the main nonspecific humoral defense mechanism, activates, and its main effects are increased vascular permeability, chemotaxis, opsonization and lysis of bacteria. Complement C3 represents 70% of total protein complement system and is involved in complement activation via both the classical and the alternative pathways. C3 is synthesized in the liver, macrophages, fibroblasts, lymphoid cells and skin. Together with other components of the complement system, C3 can be consumed in various antigen-antibody immunological reactions. Immunology, Male, David, BA, MA, PhD; Peebles, R. Stokes, MD; Male, Victoria, BA, MA, PhD. © 2021. 2. The nonspecific immune humoral system PAGE 14 Cellular and Molecular Immunology, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. © 2018. 2. The nonspecific immune humoral system PAGE 15 It is recommended to determine C3 when we suspect: a hereditary or acquired deficiency immunologic diseases associated with an increased rate of complement consumption: systemic lupus erythematosus (SLE), chronic active hepatitis, certain chronic infections, membrane proliferative glomerulonephritis and poststreptococal glomerulonephritis It is also used for evaluation of disease activity in SLE where C3 levels correlate with the severity of nephritis.  2. The nonspecific immune humoral system PAGE 16 Coagulation system Clotting system contributes to nonspecific defense by participating in the inflammatory response by increasing vascular permeability and stimulating chemotaxis for phagocytes. It can also release substances with direct antimicrobial effect. For example, beta-lysine is released by platelets during coagulation process and its effect is the lysis of Gram positive bacteria. 2. The nonspecific immune humoral system PAGE 17 Others a) Lactoferrin and transferrin: they limit the bacterial growth, by binding iron, an essential nutrient for bacteria. b) Interferon: can limit viral replication inside the cells. c) Lysozyme: breaks down the cell wall of bacteria (e.g. saliva – Gram + bacteria). d) Interleukin-1: is an inflammatory interleukin responsible for fever and generation of other acute phase proteins, some being able to opsonize bacteria. e) Derivatives from arachidonic acid – e.g. prostaglandins, leukotrienes are important inflammatory mediators. The investigation of the inflammatory syndrome PAGE 18 The inflammatory syndrome is actually a response mechanism of the body to different harmful stimuli or pathological processes, in which the immune system tries to: limit the injury repair the subsequent damages affecting the tissues eliminate the offending agent. It suggests a viable immune system, which tries to eliminate the trigger causing tissue damage. !!! When suspecting an inflammatory syndrome, there are some tests that can confirm it, but all of these tests must be interpreted correlating their results with the clinical status of the patient and, if needed, other paraclinical investigations. The investigation of the inflammatory syndrome PAGE 19 A. Erythrocyte sedimentation rate (ESR) The reduced logistic requirements and low cost make this test one of the most common ordered blood tests. Indications: high sensitivity-> used as a screening test for an inflammatory process low specificity -> a non-specific test for inflammation-> a systemic inflammatory process (whatever the etiology) is associated with an increased ESR a monitoring tool of some chronic inflammatory diseases’ response to treatment. The ESR denotes merely the presence of tissue damage or disease, but not its severity; it may be used to follow the progress of the diseased state, or monitor the effectiveness of treatment. This is not a diagnostic test! The investigation of the inflammatory syndrome PAGE 20 A. Erythrocyte sedimentation rate (ESR) Principle of the test Is the result of the interaction between two opposing forces: Crash Course Haematology and Immunology, 1. one that promotes the erythrocytes’ sedimentation (increased if Redhouse White, Gus, BSc (Hons); Vanbergen, Olivia, MA Oxon, MSc, MBBS (distinction). © 2019. the erythrocytes are organized in aggregates or rouleaux structures) 2. the other one inhibits the erythrocyte’s sedimentation- this appears due to the electronegative coat that the red cells present on their surface; a reciprocal repelling of the RBCs stopping to create aggregates or rouleaux structures The investigation of the inflammatory syndrome PAGE 21 A. Erythrocyte sedimentation rate (ESR) Principle of the test The RBCs aggregates or rouleaux structures -> masses of erythrocyte with an increased weight reported to their surface -> sinking faster in a plasma suspension -> increasing the ESR value. Factors that interfere with negative charges found on the RBCs surface will promote erythrocytes’ sedimentation: some molecules such as plasmatic proteins and especially fibrinogen have the ability of binding to the RBCs surface -> inactivate the negative electrical charges -> indirectly promoting rouleaux formation. The investigation of the inflammatory syndrome PAGE 22 A. Erythrocyte sedimentation rate (ESR) Principle of the test The test is performed by placing an anticoagulated sample of the patient’s blood into a test tube (special for ESR measurement) placed and held unmoved, vertically for an hour. At the end of one hour, the ESR value is read and recorded. Medical Sciences, Newland, Adrian C.; MacCallum, Peter; Davies, Jeff. © 2019. The investigation of the inflammatory syndrome PAGE 23 A. Erythrocyte sedimentation rate (ESR) Normal values are age-dependent tend to increase with aging gender and racial variations: female patients often have a higher ESR than male patients do, in absence of any pathology; the Afro-American population is found to have an increased ESR. The investigation of the inflammatory syndrome PAGE 24 A. Erythrocyte sedimentation rate (ESR) Age ESR’s normal value Comments Newborn (0-28 days of life) 0-2 mm/h - 1 month-puberty 3-13 mm/h - Women 100 mm/h are usually associated with severe diseases such as autoimmune disorders, infectious diseases or neoplasia. The investigation of the inflammatory syndrome PAGE 26 A. Erythrocyte sedimentation rate (ESR) Conditions associated with a decreased ESR More rarely seen: RBCs’ shape or size abnormalities= anizo/poikilocytosis: sickle cell disease (sickle-shaped erythrocytes; the patient’s RBCs tend to polymerize together and suffer hemolysis, along with numerous microvascular occlusions), hereditary spherocytosis, etc; Polycythemia (↑RBC number-> ↑HCT->↓ESR); Severe leukocytosis; Hypofibrinogenemia. The investigation of the inflammatory syndrome PAGE 27 A. Erythrocyte sedimentation rate (ESR) Method of detection 1. Manual: The Westerngren method: The Westergren method is the gold standard. The Wintrobe method: The Wintrobe method uses tubes of only 100 mm long with a smaller diameter than standard Westergren tubes. Because the Wintrobe tubes are shorter than the Westergren tubes, the method is less sensitive than the Westergren method. EDTA blood without extra diluent is added to the tube and allowed to sediment for 60 minutes. After 60 minutes the distance that the blood cells have fallen is registered in mm. 2. Semiautomatic analyzers. The investigation of the inflammatory The Westerngren method PAGE 28 syndrome A. Erythrocyte sedimentation rate (ESR) Semiautomatic analyzers Dr Irina-Bianca Kosovski, personal collection Dr Irina-Bianca Kosovski, personal collection The investigation of the inflammatory syndrome PAGE 29 A. Erythrocyte sedimentation rate (ESR) Factors which influence the ESR include: 1. Number of cells available—anemia, polycythemia. 2. Tendency toward aggregation. 3. Physical or chemical composition of plasma—increased fibrinogen or globulin increases sedimentation rates, increased albumin decreases it. 4. Presence of macrocytes, which fall more rapidly than microcytes. The investigation of the inflammatory syndrome PAGE 30 B. Acute-phase proteins determination Are molecules synthesized by the liver as a result to the inflammatory syndrome. The process begins at the site of the inflammatory process where neutrophils and macrophages try to react to this process, limiting the inflammatory reaction; these inflammatory cells have the property of secreting cytokines into the bloodstream: interleukins IL-1, IL-6, IL-8 Tumor Necrosis Factor-α (TNFα). Their presence into the bloodstream will have as a result the genesis of an acute-phase reaction, the liver will: amplify the synthesis of some proteins (known as positive acute-phase proteins/reactants) lower the synthesis of other proteins, whose serum concentrations will obviously decline (these are known as negative acute-phase proteins/reactants). The investigation of the inflammatory syndrome PAGE 31 B. Acute-phase proteins determination POSITIVE ACUTE-PHASE PROTEINS NEGATIVE ACUTE-PHASE PROTEINS Name Short description Name Short description C-reactive protein A more detailed overview Transferrin An iron-binding and (CRP) is available further later in transport protein. this chapter. Ferritin It is an organic complex Transcortin A transport protein that formed by apoferritin binds cortisol and (protein) and iron deposits; corticosterone. it is one of the ways the iron can be stored. The investigation of the inflammatory syndrome PAGE 32 B. Acute-phase proteins determination POSITIVE ACUTE-PHASE PROTEINS NEGATIVE ACUTE-PHASE PROTEINS Name Short description Name Short description Ceruloplasmin The transport protein for Albumin The most important copper ions; its deficiency it is plasmatic protein, with responsible for Wilson disease. various functions: transport, α-1 antitrypsin A protein that inhibits the creating and maintaining the trypsin’s action. oncotic pressure etc. α-1 A substance that inhibits the antichymotrypsin chymotrypsin’s action. The investigation of the inflammatory syndrome PAGE 33 B. Acute-phase proteins determination POSITIVE ACUTE-PHASE PROTEINS NEGATIVE ACUTE-PHASE PROTEINS Name Short description Name Short description Haptoglobin A protein capable to bind the C3 complement’s A protein involving in the free-hemoglobin, for factor activation of the complement subsequent use. Its cascade; its deficiency makes concentration increases in patients prone to severe inflammatory disorders and bacterial infections. decreases in hemolytic During the inflammatory anemia. process the value of C3 Fibrinogen A more detailed overview is complement’s factor is low available further later in this because it is consumed, not chapter. because it’s synthesis decreases. The investigation of the inflammatory syndrome PAGE 34 B. Acute-phase proteins determination Most common used for determining the presence of an inflammatory syndrome are: CRP fibrinogen Comparison of the change in C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR) following an inflammatory stimulus. Crash Course Haematology and Immunology, Redhouse White, Gus, BSc (Hons); Vanbergen, Olivia, MA Oxon, MSc, MBBS (distinction). © 2019. The investigation of the inflammatory syndrome PAGE 35 B. Acute-phase proteins determination CRP C-reactive protein (CRP) is a positive acute-phase protein, synthesized by the liver as a response to IL-6, released especially by macrophages from the inflamed tissue. The role: activating the complement system, by binding to molecules of phosphocholine found on the surface of some bacteria and dead cells. During an inflammatory process, it undergoes a dynamic transformation regarding its concentration - its level rises above the normal range interval in about 6 hours-> the peak at about 48 hours. CRP results are not influenced by anaemia. The investigation of the inflammatory syndrome PAGE 36 B. Acute-phase proteins determination CRP Its normal value it is considered to be under 2.5 mg/l. It can increase as much as 1,000-fold in response to injury or infection. Though commonly used as a marker for inflammatory processes, it has no predictive value. Its concentration increases in: – infectious diseases – autoimmune disorders – malignancies – metabolic diseases – myocardial infarction and other pathologies involving extensive areas of necrotic tissue. Its concentration decreased in: liver failure, case in which all the hepatic functions are affected. The investigation of the inflammatory syndrome PAGE 37 B. Acute-phase proteins determination CRP Principle of the test: latex agglutination test. A suspension of polystyrene latex particles of uniform size is coated with the IgG fraction of an antihuman CRP-specific serum. Latex particles coated with antibody to CRP are reacted with patient serum. In this case, the CRP is acting as the antigen. If CRP is present above normal threshold levels, the antigen– antibody combination will result in a visible agglutination Immunology &Serology in Laboratory Medicine, MARY LOUISE reaction. TURGEON, Elsevier, 2014. The investigation of the inflammatory syndrome PAGE 38 B. Acute-phase proteins determination CRP Interpretation visible no agglutination agglutination reaction Mims' Medical Microbiology and Immunology, Goering, Richard V., BA MSc PhD; Dockrell, Hazel M., BA (Mod) PhD; Zuckerman, Mark, BSc (Hons) MBBS MRCP MSc FRCPath; Chiodini, Peter L., BSc MBBS PhD FRCP FRCPath FFTM RCPS (Glas). Published January 1, 2019. Pages 129-143. © 2019. The investigation of the inflammatory syndrome PAGE 39 B. Acute-phase proteins determination Fibrinogen It is a positive acute-phase glycoprotein synthesized by the liver, with an important role in the blood coagulation cascade; it is eventually converted into fibrin, process catalyzed by thrombin. Fibrinogen also serves to promote aggregation of red blood cells, and increased levels contribute to an increased risk for developing coronary artery disease. Normal values are situated between 200-400 mg%. Higher values are associated with inflammation. Lower values denote a high consumption of fibrinogen (e.g. disseminated intravascular coagulation, DIC). The investigation of the inflammatory syndrome PAGE 40 C. CBC- WBC + formula An inflammatory syndrome which has an infectious etiology will have as a result leukocytosis - an increase of the leukocyte number above the maximal value accepted. By analyzing the leukocytes’ subpopulations, it is possible to be more precise regarding the type of infection: neutrophilia (higher than normal neutrophils’ concentration) is suggestive for a bacterial infection; eosinophilia (higher than normal eosinophils’ concentration) points to either a parasitic infestation, or allergic states (such as asthma, hay fever or other types of allergies). The investigation of the inflammatory syndrome PAGE 41 C. CBC- WBC + formula Leukopenia - meaning a lower than normal leukocytes’ concentration: some viral infections; infection with bacteria from the Rickettsia order; some types of malignancies; tuberculosis. The investigation of the inflammatory syndrome PAGE 42 D. Protein electrophoresis Electrophoresis represents a separating process of electrically charged molecules, under the influence of an external electric field; these molecules will travel across a membrane with different speed (according to their molecular weight and structure), travelling to an electric pole (either anode or cathode, depending on their electrical charge). When analyzing the serum proteins, the pattern resulted can have different shapes, depending on the pathology type: acute inflammatory process: a slightly increased protein level, along with a hypoalbuminemia, associated with an increase of α1 and α2-globulins (most positive acute-phase reactants migrate in the α1 and α2 globulin fractions). The investigation of the inflammatory syndrome PAGE 43 E. Other investigations For establishing a final diagnosis, a good management of the case and ultimately in order to prescribe the adequate treatment, the etiology and site of the inflammation process must be localized: Ultrasonography X-rays Computer-tomography scanning (CT) Magnetic resonance imaging (MRI) They are all based on the fact that an inflammatory process usually affects an organ structure, leading to modifications visible with these methods. The process of ordering examinations for a patient presenting an inflammatory syndrome must be a selective one, depending on the patient’s clinical status (which will guide the physician to which organs are most likely affected) and the case’s complexity. Experiment no 1 PAGE 44 Latex agglutination test for C-Reactive Protein Principle of the test: Principle The C-reactive protein rapid latex agglutination test is based on the reaction between patient serum containing CRP as the antigen and the corresponding antihuman (CRP) antibody coated to the treated surface of latex particles. The coated particles enhance the detection of an agglutination reaction when antigen is present in the serum being tested. If CRP is present above normal threshold levels, the antigen–antibody combination will result in a visible agglutination reaction. Experiment no 1 PAGE 45 Latex agglutination test for C-Reactive Protein Materials CRP latex reagent, which contains a 1% Test slides suspension of polystyrene latex particles Timer coated with anti–human CRP produced in goats or rabbits Disposable stirrers Positive human serum control Serological pipettes Negative human serum control Plasma/ Serum Sampling pipettes Experiment no 1 PAGE 46 Latex agglutination test for C-Reactive Protein Procedure 1. Using one of the pipettes provided, fill it about two thirds full with undiluted serum. While holding the pipette perpendicular to the slide, deliver one free-falling drop to the center of one oval on the slide. Using semiautomatic pipette: put 50 µl. 2. Using the squeeze-dropper vials provided, add one drop of positive control and one drop of negative control to separate ovals on the slide. 3. Resuspend the latex reagent by gently mixing the vial until the suspension is homogeneous. Place one drop of CRP latex reagent next to each serum specimen and to each control. Experiment no 1 PAGE 47 Latex agglutination test for C-Reactive Protein Procedure 4. Using separate stirrers, mix each specimen and control until the entire area of each oval is filled. 5. Tilt the slide back and forth, slowly and evenly, for 2 minutes. Place the slide on a flat surface and observe for agglutination using a direct light source. 6. The CRP positive control serum must show distinct agglutination, and the negative control must be nonreactive. If the reagent fails to agglutinate with the positive control, or does agglutinate with the negative control, it should be discarded. Experiment no 1 PAGE 48 Latex agglutination test for C-Reactive Protein Results 1. A positive reaction is reported when the specimen shows agglutination, indicating the presence of CRP in the serum at a level equal to or greater than 0.6 mg/dL= 6mg/l. 2. A negative reaction is characterized by a lack of visible agglutination in the undiluted specimen. visible agglutination reaction= no agglutination= POSITIVE TEST NEGATIV TEST Experiment no 2 PAGE 49 Erythrocytes sedimentation rate- Westergren method Principle of the test When anticoagulated whole blood is allowed to stand in a narrow vertical tube for a period of time, the RBCs – under the influence of gravity - settle out from the plasma. Because some alteration exist in the distribution of charges on the surface of the RBC (which normally keeps them separate form each other), their coming together to form large aggregates known as rouleaux. Note that the ESR denotes merely the presence of inflammation, but not its severity. Experiment no 2 PAGE 50 Erythrocytes sedimentation rate- Westergren method Materials Vacutainer (purple/blue cap) of 2 ml with patient anticoagulated whole blood ESR Quality Control (pathologic and normal whole blood) Small vacutainer (orange cap) with anticoagulant solution Westergren tube Westergren rack Pasteur pipette Latex gloves Disinfectant (alcohol) solution https://www.lpitaliana.com/en/groups/3250/sediplast-the-safe- system-for-esr-test.html Experiment no 2 PAGE 51 Erythrocytes sedimentation rate- Westergren method Steps 1. Transfer the venous blood from the big vacutainer tube (purple/blue cap) in the smaller one (orange cap) until the sign (0.8 ml). 2. Mix the small vacutainer (orange cap) gently. 3. Be attentive: there should be no clots and air bubbles in the blood!!! 4. Insert a Westergren tube in the small vacutainer until the blood level reaches 200 mm. Experiment no 2 PAGE 52 Erythrocytes sedimentation rate- Westergren method Steps Erythrocyte Sedimentation Rate (ESR) – Sediplast- Affiliates, https://www.gundersenhealth.org/ Experiment no 2 PAGE 53 Erythrocytes sedimentation rate- Westergren method Steps 5. Put the tube vertically in the rack in an area free from vibrations, drafts and direct sunlight at the room temperature. 6. Wait one hour. 7. Read the upper level of RBC column exactly after one hour: the distance from the bottom of the plasma meniscus to the top of the descended erythrocytes is read and recorded in mm. The buffy coat that is made up of leukocytes should not be included in the erythrocyte column. Experiment no 2 PAGE 54 Erythrocytes sedimentation rate - Westergren method Reference Range The distance of fall of erythrocytes, expressed as millimeters/hour. Normal values for the erythrocyte sedimentation rate (ESR), as derived using the Westergren method, are as follows: Male: ≤ 15 mm/hr Female: ≤ 20 mm/hr Child: ≤ 10 mm/hr Newborn: 0-2 mm/hr References PAGE 55 1. Basic Immunology: Functions and Disorders of the Immune System, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. Published © 2020. 2. Erythrocyte Sedimentation Rate (ESR) – Sediplast- Affiliates, https://www.gundersenhealth.org/ 3. https://www.lpitaliana.com/en/groups/3250/sediplast-the-safe-system-for-esr-test.html 4. Mims' Medical Microbiology and Immunology, Goering, Richard V., BA MSc PhD; Dockrell, Hazel M., BA (Mod) PhD; Zuckerman, Mark, BSc (Hons) MBBS MRCP MSc FRCPath; Chiodini, Peter L., BSc MBBS PhD FRCP FRCPath FFTM RCPS (Glas). Published January 1, 2019. Pages 129-143. © 2019. 5. Immunology &Serology in Laboratory Medicine, MARY LOUISE TURGEON, Elsevier, 2014. 6. Crash Course Haematology and Immunology, Redhouse White, Gus, BSc (Hons); Vanbergen, Olivia, MA Oxon, MSc, MBBS (distinction). © 2019. 7. Medical Sciences, Newland, Adrian C.; MacCallum, Peter; Davies, Jeff. © 2019. 8. Cellular and Molecular Immunology, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. © 2018. 9. Immunology, Male, David, BA, MA, PhD; Peebles, R. Stokes, MD; Male, Victoria, BA, MA, PhD. © 2021. 10. Cellular and Molecular Immunology, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. © 2018. 11. Guyton and Hall Textbook of Medical Physiology, Hall, John E., PhD; Hall, Michael E., MD, MS. 2021.