Exploration of the Acquired Immunity PDF

PAGE 1 UNIVERSITÄTSMEDIZIN NEUMARKT A. M. https://edu.umch.de www.umfst.ro CAMPUS HAMBURG Assist prof Dr Andreea Cozac- 2024 May Szoke Exploration of the acquired immu...

PAGE 1 UNIVERSITÄTSMEDIZIN NEUMARKT A. M. https://edu.umch.de www.umfst.ro CAMPUS HAMBURG Assist prof Dr Andreea Cozac- 2024 May Szoke Exploration of the acquired immunity Assist prof Dr Raluca Niculescu The adaptive immune system PAGE 2 The adaptive immune system provides lasting immunity, specific to each pathogen that it encounters. It is made up of various cells and secreted molecules that contribute to its two main branches: 1. humoral-mediated immunity: involves the production of antibody 2. cell-mediated immunity: is the development of a cellular response against infected or cancerous body cells. The adaptive immune system PAGE 3 Difference between innate and adaptive immune system Robbins Basic Pathology, Kumar, Vinay, MBBS, MD, FRCPath; Abbas, Abul K., MBBS; Aster, Jon C., MD, PhD. © 2018. Cell-mediated immunity PAGE 4 Cell-mediated immunity is mediated by: T lymphocytes macrophages natural killer (NK) cells. The cell-mediated immune system is involved in the elimination of: Intracellular pathogens and infected cells (mainly viruses, mycobacteria and fungi) Tumor cells Foreign grafts The thymus plays an important role in cell-mediated immunity because it is the site of T cell maturation (hence they are called ‘T’ cells!). Cell-mediated immunity PAGE 5 Lymphocytes have multiple roles in the immune response and are divided into three main categories: T lymphocytes - responsible for cellular immunity B lymphocytes - responsible for humoral immunity NK lymphocytes Robbins Basic Pathology Kumar, Vinay, MBBS, MD, FRCPath; Abbas, Abul K., MBBS; Aster, Jon C., MD, PhD. © 2018. Cell-mediated immunity PAGE 6 T lymphocytes circulate in the blood, lymph, but the most important fraction is found quartered in lymphoid organs. They are found in traffic en route to the primary lymphoid organs or within. Lymphocytes can enter lymph nodes via specialized high endothelial venules or in lymph. They leave the node in lymph that is returned to the systemic circulation via the right lymphatic duct or thoracic duct. Crash Course Haematology and Immunology, Redhouse White, Gus, BSc (Hons); Vanbergen, Olivia, MA Oxon, MSc, MBBS (distinction). © 2019. Cell-mediated immunity PAGE 7 T lymphocytes are divided into several cell populations with specific functions. They do not differ morphologically: CD8 + or cytotoxic lymphocytes CD4 + or helper lymphocytes Cell-mediated immunity PAGE 8 Function of CD4 and CD8 CD4 and CD8 are ‘accessory’ molecules that play an important role in the T cell–antigen interaction. CD4 and CD8 have two important functions: They bind MHC class II and class I molecules, respectively, thereby strengthening the T cell–antigen interaction. Signal transduction. Robbins Basic Pathology, Kumar, Vinay, MBBS, MD, FRCPath; Abbas, Abul K., MBBS; Aster, Jon C., MD, PhD. © 2018. Cell-mediated immunity PAGE 9 CD4+ cell number decrease appears in:  Infections: viral infections, bacterial infections, parasitic infections, sepsis, tuberculosis, coccidioidomycosis  HIV  Steroids or immunosuppressive therapy  Burns  Trauma  Intravenous injections of foreign proteins  Malnutrition  Excessive physical effort  Surgery  Pregnancy  Psychological stress  Daily variation - CD4 decline towards noon and rise in the evening  PAGE 10 Cell-mediated immunity CD8+ cells growth is seen in:  Autoimmune diseases  Immune pathological reactions such as "Graft versus host disease”, transplant rejection  Congenital or acquired immune deficiency (AIDS) CD8+ cells decline is seen in:  After treatment with corticosteroids  Lupus erythematosus Normally, the CD4/CD8 ratio has a value between 1 and 4. In HIV infection the number of CD4 cells reduces and the ratio decreases below 1.  PAGE 11 Humoral immunity B lymphocytes originate initially in the fetal liver and subsequently in the bone marrow. In peripheral blood lymphocytes belong to about 10- 20% of line B. The main function of B lymphocytes is to produce antibodies (Ig). Immunology for Medical Students, Helbert, Matthew, MBChB, FRCP, FRCPath, PhD. © 2017.  PAGE 12 Humoral immunity B cells and antibody production B cells are activated by antigen in a T-cell-independent or dependent way. T helper (Th) cells are primed by APCs, which present antigen to Th cells in conjunction with MHC class II molecules. B cells are stimulated by antigen interacting with B cell receptors. Crash Course Haematology and Primed Th cells interact with B cells that also express antigen– Immunology MHC complexes. Redhouse White, Gus, BSc (Hons); Vanbergen, This interaction induces a sequence of surface receptor binding Olivia, MA Oxon, MSc, MBBS and cytokine production that results in B-cell activation, (distinction). © 2019. proliferation and differentiation.  PAGE 13 Immunophenotyping The lineage, maturation stage, or activation status of a cell often can be determined by analyzing the expression of different cell surface or intracellular molecules. In this technique, aliquots of 106 cells in liquid suspension are incubated with fluorochrome-conjugated monoclonal antibodies, rinsed to remove unbound antibody, and passed through a flow cytometry instrument. Cellular and Molecular Immunology Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. 2021.  PAGE 14 Immunophenotyping Monochromatic laser light (ultraviolet) impacts each cell, generating several light signals: FSC light is a measure of cell size SSC light is a measure of cell granularity. Fluorescent light emitted by fluorochrome-conjugated antibodies to cell-specific antigens is used to identify the cellular immunophenotype. Depending upon the number of fluorochrome-conjugated antibodies used, many different antigens can be simultaneously detected on each cell.  PAGE 15 Immunophenotyping Flow cytometry histograms of peripheral blood leukocytes. Hematology, Hudnall, S. David, MD. © 2012.  PAGE 16 Immunophenotyping For determination of lymphocyte subsets, which is the routine lymphogram, we use the following CD markers:  CD45, the leukocyte common antigen (used to investigate hematopoietic cells)  CD3, marker for T lymphocytes  CD4, marker for T helper lymphocytes (Ly Th)  CD8, marker for T cytotoxic lymphocytes (Ly Tc)  CD16 + 56, marker for NK lymphocytes (Ly NK)  CD19, marker for B lymphocytes (Ly B)  PAGE 17 Immunophenotyping Interpretation of CD markers Generally a CD-type marker is interpreted according Double positive CDX1 and to the figure below. Positive CDX2 CDX2 Negative Positive CDX1  PAGE 18 Immunophenotyping Counting the number of helper T-cells, the CD4+ lymphocytes, is a very important tool used to assess the immune system in people with HIV infection and to decide when to start anti-HIV treatment. It is also used to motorize the treatment in HIV infection. The normal CD4 count ranges between 500 and 1500 cells/mm3 and in adults, they represent 30% to 60% of the total number of lymphocytes. The CD8 count ranges between 150 and 1000 cells/mm3. Lymphocytes expressing CD45 high, are gated in R2  PAGE 19 Immunophenotyping Separation of lymphocyte populations: T (CD3 positive, CD4 or CD8), B (CD19 positive) NK (CD16+CD56 positive)  PAGE 20 Detection of specific antibodies 1. Determination of rheumatoid factor It is an IgM in serum of patients with rheumatoid polyarthritis. Basic and Clinical Immunology, Peakman, Mark, MBBS PhD FRCPath; Vergani, Diego, MD PhD FRCPath FRCP. © 2009.  PAGE 21 Detection of specific antibodies 2. Quantitative determination of IgE in serum The total IgE test may be used to screen for and detect allergic diseases (e.g. allergic asthma). IgE can be also increased in parasitic infections. Detection of immune complexes PAGE 22 1. Prick tests Prick test is performed for identifying the allergen responsible for various allergic manifestations. Skin prick testing detects immediate (type I) hypersensitivity. The reaction is mediated by the antigen-triggered IgE-mediated release of vasoactive substances from skin mast cells.  PAGE 23 Detection of immune complexes 1. Prick tests Small drops of commercially prepared antigen solutions are placed on marked areas on the forearm and lightly pricked into the skin using separate blunt lancets. The stratum corneum of the epidermis is punctured by gently pressing the blunt lancet perpendicular to the skin surface through the test solution. It is important to develop a prick testing technique that is reproducible. Food allergens are often tested by pricking the fresh food and then the skin (prick-to-prick testing). Cellular and Molecular Immunology, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. © 2021.  PAGE 24 Detection of immune complexes 1. Prick tests Procedure Patients with allergies to an antigen will have antigen-specific IgE already bound to mast cells in the skin, and the mast cells will be activated. In response to antigen-stimulated release of mast cell mediators, local blood vessels first dilate and then become leaky to fluid and macromolecules, which produces redness and local swelling (a wheal). Subsequent dilation of vessels on the edge of the swelling produces the appearance of a red rim (the flare). Basic and Clinical Immunology, Peakman, Mark, MBBS PhD FRCPath; Vergani, Diego, MD PhD FRCPath FRCP. 2009.  PAGE 25 Detection of immune complexes 1. Prick tests Immunology for Medical Students, Helbert, Matthew, MBChB, FRCP, FRCPath, PhD. © 2017.  PAGE 26 Detection of immune complexes 1. Prick tests Interpretation In case of a positive reaction, a wheal occurs at the application site (elevation) surrounded by erythema (redness) of various sizes (in average 3-5 mm) and associated with itching. Maximum diameter occurs in 15-20 minutes and disappears in about an hour. If the patient has a type III hypersensitivity reaction to the allergen, in 2-3 hours we observe the occurrence of a papule with a diameter of 1-5 cm. Patients should have stopped antihistamines 48 hours before the test. Prick tests are used to demonstrate allergy to: aeroallergens (e.g. house dust mite) foods (e.g. hen’s egg or peanut) contact urticaria to latex.  PAGE 27 Detection of immune complexes 1. Prick tests Immunology for Medical Students, Helbert, Matthew, MBChB, FRCP, FRCPath, PhD. © 2017. Dermatology: An Illustrated Colour Text, Gawkrodger, David J., DSc MD FRCP FRCPE; Ardern-Jones, Michael R., BSc MB BS DPhil FRCP. © 2021.  PAGE 28 Detection of immune complexes 1. Prick tests A positive test correlates well with a positive allergen-specific IgE test (usually an enzyme-linked immunosorbent assay [ELISA]; the radioallergosorbent test [RAST] is no longer used). In most cases (i.e. ≈ 80%) where the skin prick test is positive, IgE antibody will be detectable in the serum. The risk of anaphylaxis is very small, but resuscitation facilities, including adrenaline (epinephrine) for intramuscular injection, antihistamines and oxygen are recommended, especially for testing of food allergy and high-risk subjects (asthmatics).  PAGE 29 Detection of immune complexes 1. Prick tests Advantages Disadvantages Simple test A little uncomfortable but not painful Rapid test – the result is provided in 15-12 The test for food allergens is less reliable – minutes possible false negative reactions Inexpensive test The size of the wheal does not correlate with Can give you the allergen – allergens being the severity of symptoms tested are selected in accordance with the patient's history Safe test – it uses small amount of allergen The test can be performed at any age  PAGE 30 Detection of immune complexes 2. The patch test Patch testing remains the gold standard in the diagnosis of ACD. U.S. Food and Drug Administration–approved ready-to-use-skin patch tests are used for the diagnosis of contact dermatitis. The TRUE (Allerderm) and TROLAB (Hermal) patch test kits are two commonly used commercial products that allow testing for many allergens simultaneously. There are also specific allergen panels when occupational exposure is suspected, including a hairdressing tray or a florist tray. Chemicals that patients bring in should not be used because the substances may cause severe skin damage and all ingredients may not be known.  PAGE 31 Detection of immune complexes 2. The patch test A ‘standard series’ of about 45 substances is applied to every patient, with additional allergens as necessary. Dermatology: An Illustrated Colour Text, Gawkrodger, David J., DSc MD FRCP FRCPE; Ardern-Jones, Michael R., BSc MB BS DPhil FRCP. Published January 1, 2021. Pages 48-49. © 2021.  PAGE 32 Detection of immune complexes 2. The patch test Preparing patch tests Small amounts of the test substances are applied to the 8- mm-diameter aluminium discs on adhesive tape (Finn chambers) that are used for patch testing. The exact selection of test substances depends on taking a detailed history including the clinical problem, the site of the dermatitis, the environmental contacts (noting ingredients labels on any cosmetics or workplace products used) and the patient’s occupation. Dermatology: An Illustrated Colour Text, Gawkrodger, David J., DSc MD FRCP FRCPE; Ardern-Jones, Michael R., BSc MB BS DPhil FRCP. Published January 1, 2021. Pages 48- 49. © 2021.  PAGE 33 Detection of immune complexes 2. The patch test The most common site for application is the upper back. The patient should not have a sunburn or other significant rash in the test area and should not have used topical corticosteroids on the patch test site for at least 1 week and oral corticosteroids at a dose greater than 15 mg for 1 month before patch testing. Both topical and systemic steroids decrease the patient's ability to produce a positive response. Once patch testing is planned, the TRUE test strips are applied to the upper back and then reinforced with paper tape. The patient should keep the area dry for at least 48 hours and refrain from activities that cause excessive sweating. Netter's Internal Medicine, Schwarz, Emily J.; Riggs Runge, Whether oral antihistamines will affect the test results remains Susan. © 2009. controversial.  PAGE 34 Detection of immune complexes 2. The patch test After 48 to 72 hours, the patient should return, the outline of the tests marked, and the patches removed for the first reading. After the initial reading, the patient should ideally return for a second reading 5 to 7 days after the initial patch test placement. The second reading is necessary because some allergens, including gold and disperse blue dye, take a longer time to induce a response. Macleod's Clinical Examination, Tidman, Michael J. Published January 1, 2018. Pages 283-293. © 2018.  PAGE 35 Detection of immune complexes 2. The patch test In this case allergens 3 and 19 reacted. Basic and Clinical Immunology, Peakman, Mark, MBBS PhD FRCPath; Vergani, Diego, MD PhD FRCPath FRCP. © 2009. Detection of immune complexes PAGE 36 2. The patch test When interpreting the patch test, clinical correlation is recommended: false-positive reactions may occur secondary to excited skin syndrome, because of a low concentration of the suspected substance, failure to perform a later reading, wet patches, or the influence of corticosteroids. true allergic reactions tend to itch more than irritant ones. The second reading is particularly important in elderly patients, who may take longer to react.  PAGE 37 Detection of immune complexes 3. Intradermal tests The allergen is injected into the dermis, and these tests are mainly used for assessing cellular immune response. The Mantoux test (Tuberculin Sensitivity Test or PPD test for Purified Protein Derivative) is used for diagnosis of tuberculosis. With a syringe it is injected in the forearm and intradermal 0.1 ml of 5 tuberculin units of liquid tuberculin. The patient's arm is examined after 48 to 72 hours - type IV immune reaction. Netter's Internal Medicine, Weber, David J.; Leone, Peter A.; Rutala, William A. Published January 1, 2009. Pages 725-734. © 2009. Detection of immune complexes PAGE 38 3. Intradermal tests In case of a positive reaction an induration area occurs around the site of the injection. The diameter of the indurated area is measured and not the erythema (redness). Negative results occur in people being immunologically compromised, like those having HIV infection and low CD4 T cell counts. False positive occur in case of nontuberculous mycobacteria or previous administration of BCG vaccine. Prior vaccination with BCG may cause a false-positive result for many years afterwards. Also the touching and scratching of the area can give a false Underwood's Pathology, Sewell, W.A. Carrock. © 2019. positive result.  PAGE 39 Histocompatibility tests Red cell antigens are molecules capable of provoking an immune response. The surface membrane of the red cell is studded with numerous different molecules, and these can all potentially act as antigens. They do not provoke an immune response in the individual that has synthesized them, because they are ‘self’ antigens in their native physiologic host. However, if transfused into an individual that does not possess the same array of antigens on their native red cells, an immune response occurs. This can be fatal. As one would expect, there are different red cell antigens; the most clinically important are: the ABO system the Rhesus antigens.  PAGE 40 Histocompatibility tests 1. The ABO antigen system Among the 33 systems (representing over 300 antigens are listed by the International Society of Blood Transfusion), ABO remains the most important in transfusion and transplantation since any person above the age of 6 months possess clinically significant anti-A and/or anti-B antibodies in their serum. The ABO system refers to various forms of the ‘H’ antigen= an oligosaccharide molecule expressed at the surface of red cells. Its physiologic function is unknown.  PAGE 41 Histocompatibility tests 1. The ABO antigen system ‘O’ antigen If the H antigen is unmodified, the antigen is known as the ‘O’ antigen. ‘A’ antigen If the H antigen has an N-acetylgalactosamine moiety added, it becomes the ‘A’ antigen. If the person possesses the gene for the relevant galatosaminyltransferase enzyme (the A antigen gene), the native H antigen is modified in this way, forming the A antigen. Red cells therefore express A antigens at their surface. Basic Immunology: Functions and Disorders of the Immune System, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. 2020.  PAGE 42 Histocompatibility tests 1. The ABO antigen system ‘B’ antigen If the H antigen has an added galactose moiety, the resulting antigen is the ‘B’ antigen. If the person possesses the gene for the relevant galatosyltransferase enzyme (the B antigen gene), the native H antigen is modified in this way, forming the B antigen. The red cells therefore express B antigens at their surface. Basic Immunology: Functions and Disorders of the Immune System, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. 2020.  PAGE 43 Histocompatibility tests 1. The ABO antigen system Antibodies Blood group A contains antibody against blood group B in serum and vice-versa, while blood group O contains no A/B antigen but both their antibodies in serum. Basic Immunology: Functions and Disorders of the Immune System, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. 2020.  PAGE 44 Histocompatibility tests 1. The ABO antigen system ABO blood group Frequency in UK Antigens on red Agglutinated by Genotypes (phenotype) Caucasians (%) cells O 44 O None OO A 45 A Anti-A AO or AA B 8 B Anti-B BO or BB AB 3 AB Anti-A and Anti-B AB Medical Sciences, Newland, Adrian C.; MacCallum, Peter; Davies, Jeff. © 2019.  PAGE 45 Histocompatibility tests 1. The ABO antigen system ABO group determination is defined by: the demonstration of antigens A and/or B on the surface of human red blood cells (Forward Grouping) the presence or absence or anti-A and/or anti-B antibodies in the plasma (Reverse Grouping). It is therefore appropriate to identify the erythrocytic antigens using known anti-A, anti-B and anti-A,B reagents (red blood cell test), then to confirm the preceding results by verifying the presence of the corresponding antibodies in the plasma from the test blood using known red blood cells A1, B and, possibly, A2 and O (plasma test). To establish ABO blood and Rh type: the principle of hemagglutination.  PAGE 46 Histocompatibility tests Forward Grouping Reverse Grouping 1. The ABO antigen system In the left half is shown the interaction between red cells from a person of blood groups A, B, AB and O (shown down the left side of the tile) and serum containing the anti-A, anti-B, and anti-AB antibodies. In the right half is shown the interaction between serum or plasma from a person of the various blood groups, with group A, group B and group O red cells. Medical Sciences, Newland, Adrian C.; MacCallum, Peter; Davies, Jeff. © 2019.  PAGE 47 Histocompatibility tests 2. The Rhesus antigens Currently, the Rh-system consists of 50 defined blood group antigens out of which only five are important expressed on an individual’s red cells: C, c, D, E, and e. D is the most clinically significant. RBC surface of an individual may or may not have a Rh factor or immunogenic D-antigen: Rh-positive (D-antigen present) Rh-negative (D-antigen absent). Presence of a single ‘D’ allele confers Rh positive status (it is dominant). Rh positive individuals are therefore DD or Dd, and Rh-negative individuals dd. Predominance of Rh negativity is strongly determined by race. Less than 1% of Africans and Asians are Rh negative, whilst up to 15% of Caucasians are Rh negative.  PAGE 48 Histocompatibility tests 2. The Rhesus antigens Antibodies against Rh D are usually IgG, as opposed to the IgM antibodies commonly targeted against A and B antigens. Clinical implications of the IgG nature of anti-D antigens include that RhD incompatible transfusion reactions are usually inconsequential. IgG are able to cross the placenta (unlike the larger IgM). The clinical significance of this is that Rh disparity between mother and fetus (specifically between a Rh − ve mother and a Rh + ve fetus) can lead to placental transfer of anti-Rh D maternal antibodies. This is in contrast to the large, bulky IgM anti-A and anti-B antibodies, accounting for why ABO mother-fetus incompatibility is of no consequence. Prophylaxis is given against Rh immunization using anti-D Ig for pregnant Rh-negative mothers who have given birth to Rh-positive child. Also in contrast to the ABO system, antibodies to Rh D (‘anti-D’) are only present in RhD−ve individuals that have experienced a sensitizing event. A ‘sensitizing event,’ refers to an event which leads to development of anti-D antibodies. Transfusion with Rh D + ve cells to a Rh − ve recipient is a sensitizing event.  PAGE 49 Histocompatibility tests 2. The Rhesus antigens Basic and Clinical Immunology, Peakman, Mark, MBBS PhD FRCPath; Vergani, Diego, MD PhD FRCPath FRCP. Published January 1, 2009. Pages 293-305. © 2009.  PAGE 50 Blood transfusion The term ‘blood transfusion’ refers to the therapeutic use of any of the particular components or ‘blood products’ obtained from donation of whole blood, e.g., red cells, platelets, fresh frozen plasma (FFP) and its derivatives. Nowadays, whole blood transfusion has become extremely uncommon and is only rarely used. Davidson's Principles and Practice of Medicine, Watson, HG; Culligan, DJ; Manson, LM. 2018.  PAGE 51 Blood transfusion A unit of fresh frozen plasma An apheresis platelet unit A unit of red cell concentrate Davidson's 100 Clinical Cases, Turner, Cecil Essentials of Medicine, Parnes,, Aric.2021. Cecil Essentials of Medicine, Parnes, Aric. 2021. M.L. 2012.  PAGE 52 Blood transfusion Emergency cross-matching The full cross-matching process takes time. In emergencies, where ABO group is unknown, red cells that are known to be group O and Rh D negative can be transfused immediately. Once blood group status is known, ‘group specific’ blood can then be issued (taking ~ 25 minutes), whilst full serologic cross-matching is performed (taking ~ 45 minutes, assuming no antibodies in the planned recipient).  PAGE 53 Blood transfusion ABO incompatible transfusion IgM antibodies in the recipient’s plasma bind to antigens on the donor red cells. This activates the complement pathway leading to intravascular destruction (haemolysis) of red cells and a huge release of inflammatory cytokines. These cause shock, renal impairment and disseminated intravascular coagulopathy. This scenario is often fatal and most commonly arises because of human error leading to accidental transfusion of an ABO incompatible blood type.  PAGE 54 Experiment- Blood grouping and Rh typing Principle of the test: the demonstration of antigens A and/or B, D on the surface of human red blood cells= hemagglutination. Materials Reagents: ABO system reagents (antiA reagent, antiB reagent, antiAB reagent), antiD reagent or ABOTest Cards Whole venous/capillary blood Sticks Plates/slides Pipettes Gloves Disinfectant wipes Sterile needles  PAGE 55 Experiment- Blood grouping and Rh typing 1. The slide/plate method Principle of the test: Reagents that contain Anti A, Anti B, Anti AB, and Anti D are used to demonstrate the present of antigens A and/or B, D on the surface of human red blood cells= hemagglutination.  PAGE 56 Experiment- Blood grouping and Rh typing 1. The slide/plate method Steps 1. Mark 4 wells of a clean plate/slide as A-B-AB-D. 2. Place 1 drop of anti A (blue) in the well A, 1 drop of anti B (yellow) reagent in the well B, 1 drop of anti AB (colorless) in the well AB and 1 drop of D reagent in the well D. 3. Add 1 drop of blood to each well. 4. Mixed the blood and reagent using a clean stick. Spread each mixture in the well into a smooth round circle with an area of 10-15mm diameter. 5. Rotate the plate/slide for 2 min and look for agglutination with naked eye. 6. Record the reactions and interpret the results.  PAGE 57 Experiment- Blood grouping and Rh typing 1. The slide/plate method Result Positive (+): Little clumps of red cells are seen floating in a clear liquid. Negative (–): Red cells are floating homogeneously in a uniform suspension. Interpretation If agglutination is not observed with A and neither with B cells, then the patient’s blood group is O. If agglutination is observed with A and AB, then the patient’s blood group is A. If agglutination is observed with B and AB, then the patient’s blood group is B. If agglutination is observed with A, B and AB, then the patient’s blood group is AB. If agglutination is observed with D cells, then the patient’s Rh is positive. If agglutination is not observed with D cells, then the patient’s Rh is negative.  PAGE 58 Experiment- Blood grouping and Rh typing 1. The card method Principle of the test ABTest Card device includes three deposits of dried reagents on a membrane: - 1 spot of Anti-A, 1 deposit of Anti-B and 1 spot for Anti-D for the patient - 1 spot of Anti-A and 1 deposit of Anti-B for the blood bag. Because we use them just in didactical scope, each part of the card will correspond to one student. The reagents areas are prepared from monoclonal antibodies coming from in vitro culture supernatants of murine hybridomas.  PAGE 59 Experiment- Blood grouping and Rh typing 2. The card method Steps 1. Select the card: take one part of the card. 2. Hydration of reactive areas: add one drop of distilled water on each reaction zone. 3. Add the blood: disinfect the finger using the lancet/needles prick the finger of the patient and place 1 drop of blood on each reactive area the drop is indicated to have a minimum size of 3 mm. 4. Mixed the blood and reagent using a clean stick. Spread each mixture in the well into a smooth round circle with an area of 10-15mm diameter. 5. Rotate the plate/slide for 2 min and look for agglutination with naked eye. 6. Record the reactions and interpret the results.  PAGE 60 Experiment- Blood grouping and Rh typing 2. The card method Result Positive (+): Little clumps of red cells are seen floating in a clear liquid. Negative (–): Red cells are floating homogeneously in a uniform suspension. Interpretation If agglutination is not observed with A and neither with B cells, then the patient’s blood group is O. If agglutination is observed with A, then the patient’s blood group is A. If agglutination is observed with B, then the patient’s blood group is B. If agglutination is observed with A and B, then the patient’s blood group is AB. If agglutination is observed with D cells, then the patient’s Rh is positive. If agglutination is not observed with D cells, then the patient’s Rh is negative.  PAGE 61 References Basic and Clinical Immunology, Peakman, Mark, MBBS PhD FRCPath; Vergani, Diego, MD PhD FRCPath FRCP. 2009. Robbins Basic Pathology, Kumar, Vinay, MBBS, MD, FRCPath; Abbas, Abul K., MBBS; Aster, Jon C., MD, PhD. © 2018. Cecil Essentials of Medicine, Parnes,, Aric.2021. Medical Sciences, Newland, Adrian C.; MacCallum, Peter; Davies, Jeff. © 2019. Basic Immunology: Functions and Disorders of the Immune System, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. 2020. Underwood's Pathology, Sewell, W.A. Carrock. © 2019. Netter's Internal Medicine, Weber, David J.; Leone, Peter A.; Rutala, William A. Published January 1, 2009. Pages 725- 734. © 2009. Basic and Clinical Immunology, Peakman, Mark, MBBS PhD FRCPath; Vergani, Diego, MD PhD FRCPath FRCP. © 2009. Macleod's Clinical Examination, Tidman, Michael J. Published January 1, 2018. Pages 283-293. © 2018. Netter's Internal Medicine, Schwarz, Emily J.; Riggs Runge, Susan. © 2009. Dermatology: An Illustrated Colour Text, Gawkrodger, David J., DSc MD FRCP FRCPE; Ardern-Jones, Michael R., BSc MB BS DPhil FRCP. Published January 1, 2021. Pages 48-49. © 2021. Immunology for Medical Students, Helbert, Matthew, MBChB, FRCP, FRCPath, PhD. © 2017. Cellular and Molecular Immunology, Abbas, Abul K., MBBS; Lichtman, Andrew H., MD, PhD; Pillai, Shiv, MBBS, PhD. © 2021. Crash Course Haematology and Immunology, Redhouse White, Gus, BSc (Hons); Vanbergen, Olivia, MA Oxon, MSc, MBBS (distinction). © 2019. Hematology, Hudnall, S. David, MD. © 2012.