Overview of the Immune System PDF

Republic of Iraq Ministry of Higher Education & Scientific Research Middle Technical University Baquba Medical Technical institute Medical laboratories techniques Dept. Overview of the Immune System By...

Republic of Iraq Ministry of Higher Education & Scientific Research Middle Technical University Baquba Medical Technical institute Medical laboratories techniques Dept. Overview of the Immune System By Msc. Saif A. Mohammed October, 2021 Immunity Immunity is body's ability to resist or eliminate potentially harmful foreign materials or abnormal cells  Pasteur Observing Rabies Vaccination Serological test Serologic reactions that are in vitro Antigen-antibody reactions provide methods for the diagnosis of disease and for the identification and quantitation of antigens and antibodies. Simple serological techniques are called simple, because, these procedures involving direct demonstration and observation of reactions, they do not require the participation of accessory factors such as; indicator system, or specialized equipment. Some examples of these tests are the agglutination reactions, and precipitation reactions.  Immunodiagnostic methods A laboratory techniques that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample. Types of Serologic Tests Primary binding test - directly measure the binding of antigen to antibody e.g. RIA, IF, ELISA. Secondary binding test - measure the results of antigen – antibody interaction in vitro, e.g. precipitation, complement fixation. In vivo test - measures the actual protective effect of antibodies in a host, e.g. passive cutaneous anaphylaxis.  Agglutination test Perception test Specimen Collection & Processing Serum and plasma Specimens :- Specimen must not be grossly hemolyzed , lipemic or bacterially contaminated or it will be rejected. Specimens should be frozen if not received in the lab within 24 hours unless specifically addressed below.  Bibliography 1. MLAB 1235 Immunology/Serology and Lab. Panola college , USA 2. Jawetz, Melnick, & Adelberg’s, Medical Microbiology. Twenty-Eighth Edition. by McGraw-Hill Education. Chapter five. 3. Nptel university, Biotechnology – Cellular and Molecular Immunology Republic of Iraq Ministry of Higher Education & Scientific Research Middle Technical University Baquba Medical Technical institute Medical laboratories techniques Dept. Practical Immunology Lec 3 :- - How to collect blood samples By Msc. Saif A. Mohammed October, 2022  Three popular methods of blood collection are: 1. Arterial Sampling. 2. Venipuncture Sampling. 3. Fingerstick Sampling. Blood Specimen Collection and Processing The first step in acquiring a quality lab test result for any patient is the specimen collection procedure. The venipuncture procedure is complex , requiring both knowledge and skill to perform.  Venipuncture Procedure 1. Label the tube with the patient's. particulars. 2. Put tourniquet on the patient about. 3-4' above the venipuncture site. 3. Ask patient to form a fist so. veins are more prominent. 4. After finding the vein, clean the position 5. Assemble needle and vacuum. 6. Insert the collection tube into the vacum 7. Remove cap from needle. 8. Use thumb to draw skin tight. Venipuncture Procedure  How do you troubleshoot venipuncture? TROUBLESHOOTING GUIDELINES: 1. Change the position of the needle. or move it backward (it may have penetrated too far). 2. Adjust the angle (the bevel may be against the vein wall). 3. Loosen the tourniquet. 4. Re-anchor the onother vein.  Dilution and Titration Dilution : - is the process of making a solution weaker from stronger ones. Titer: is measure the amount of antibody or antigen in the body fluid. Or the concentration (maximal dilution) of specimen (antibodies or antigens) that gives visible agglutination. Serial Dilutions - A serial dilution is any dilution where the concentration decreases by the same quantity in each successive step by a constant amount = N. Application of Dilution 1. Dilution of Antigens or Antibodies. 2. Calculate the Titer. 3. For diagnosis of Microorganisms. 4. Counting of Bacteria.  To prepare a simple dilution of a substance we need: 1. Dilute (the concentrate), is added to a fixed volume of diluent. Frequently, the concentrate consists of a serum specimen. 2. Diluent, Distal water, Normal saline or a buffered solution. 3. Sets of tubes. Serial double dilution (Two fold dilution) Procedure 1. Preparation of serial doubling dilutions of serum and control. 2. Place sets of 10 tubes in a rack; number them Al-Al0. 3. Place 0.9 ml of buffered solution or normal saline in the first tube and 0.5 ml in each succeeding tube. 4. Place 0.1 ml of test serum in the first tube in row A. 5. Mix thoroughly and transfer 0.5 ml of the mixture to the second tube. Mix the contents and transfer 0.5 ml of the mixture to the third tube. This process is continued until tube 8, after mixing, 0.5 ml of the serum dilution is discarded. 6. To each tube is then added 0.5 ml of antigen. The contents of the tube are thoroughly mixed, thus giving final serum dilutions of 1:10, 1:20, 1:40 etc. 7. Tube 9 is a positive control (positive serum plus antigen); tube 10 is a negative control, tube containing the diluent and the antigen only.  Serial double dilution (Two fold dilution) Serial decimal dilution (Tenfold dilution) Procedure: 1. Preparation of serial decimal dilutions of serum and control. 2. Place sets of 10 tubes in a rack; number them Al-Al0. 3. Place 0.9 ml of buffered solution or normal saline in the first tube and 0.9 ml in each succeeding tube. 4. Place 0.1 ml of test serum in the first tube in row A. 5. Mix thoroughly and transfer 0.1 ml of the mixture to the second tube. Mix the contents and transfer 0.1 ml of the mixture to the third tube and so. This process is continued until tube 8 from which, after mixing, 0.1 ml of the serum dilution is discarded. 6. This process of decimal dilutions results in 0.1 ml of dilutions 1:10, 1:100, 1:1000, and so on, in each tube. 7. To each tube is then added 1 ml of antigen. The contents of the tube are thoroughly mixed. 8. Tube 9 is a positive control (positive serum plus antigen); tube 10 is a negative control, tube containing the diluent and the antigen only.  Serial decimal dilution (Tenfold dilution). Bibliography 1. MLAB 1235 Immunology/Serology and Lab. Panola college , USA 2. 2016 Advanced Instructional Systems, Inc. dba Web Assign, a Virginia Corporation and Cengage Company, and San Francisco State University Department of Chemistry and Biochemistry Faculty, Fall 2016 Update by Nancy Gerber 3. Ebtehal Nowfal Mahmmoud, BVMS, MSc. Lecturer, Department of Microbiology College of Veterinary Medicine, University of Mosul, Mosul, Iraq Republic of Iraq Ministry of Higher Education & Scientific Research Middle Technical University Baquba’h Technical institute Medical Laboratories Techniques Red blood cells suspension Practical serology By Msc. Saif A. Mohammed December, 2021 Msc Saif A. Mohammed Practical immunology 4. Red blood cells suspension Principle ; A red cell suspension is a common reagent used for many serologic procedures. Red cell suspensions provide the appropriate serum to cell ratio to allow for grading and interpretation of tests results. Specimens 1. EDTA anticoagulated whole blood 2. Cord blood collected in a plain tube Materials Reagents : - 1. Isotonic saline 2. Diluent 2 Supplies :- 1. Test tubes (10x75mm) 2. Transfer pipettes 3. Test tube rack Equipments : - 1. Serological centrifuge 2. Pipettor 3. Diluent Dispenser Scope and Related Policies Red cell suspension is used for the following tube examination procedures : - 1. ABO and Rh typing 2. Direct antiglobulin test and auto control 3. Donor unit compatibility (crossmatch) 4. Red cell phenotyping Msc Saif A. Mohammed Practical immunology Notes : - For best results red cell suspensions should be used for testing on the day of Preparation. Procedure of Red blood cells suspension 1. Add 1 drop of blood in the centrifuge tube 2. Adde saline in the tube until 1cm left from the tube mouth 3. centrifuge it at 2500-3000 rpm for about 1-2 minutes 4. After centrifuge the supernatant are removed and blood are mixed well with another saline 5. Repeat the step 2-3 times. 6. When all the blood are done centrifuge prepare 2% suspension (1 drop blood & 49 drop saline ) , 5% (1drop blood,19 saline), 10 % (1drop blood ,9 drop saline) Quality Control 1. Visually compare the 3-5% red cell suspension to commercially prepared red cell suspension to ensure the appropriate strength has been acheived 2. Mix the red cell suspension immediately before performing the serological procedure. 3. Diluent 2 must be visually inspected on each day of use for turbidity, discoloration or show any signs of bacterial contamination. Msc Saif A. Mohammed Practical immunology Blood Collection Tubes Most blood collection tubes contain an additive that either accelerates clotting of the blood (clot activator) or prevents the blood from clotting (anticoagulant). A tube that contains a clot activator will produce a serum sample when the blood is separated by centrifugation and a tube that contains an anticoagulant will produce a plasma sample after centrifugation. Some tests require the use of serum, some require plasma, and other tests require anticoagulated whole blood. The table below lists the most commonly used blood collection tubes. NO Tube cap color Additive Function of Additive Common laboratory tests Light-blue 3.2% Sodium citrate Prevents blood from 1 clotting by binding Coagulation calcium Red or gold Serum tube with or Clot activator Chemistry, without clot activator or promotes blood serology, 2 gel clotting with glass or immunology silica particles. Gel separates serum from cells. Green Sodium or lithium Prevents clotting by Stat and routine heparin with or without inhibiting thrombin chemistry 3 gel and thromboplastin Lavender or pink 4 Prevents clotting by Hematology and Potassium EDTA binding calcium blood bank Gray Glucose Fluoride inhibits (especially when Sodium fluoride, and 5 sodium or potassium glycolysis, and oxalate testing will be prevents clotting by delayed), blood oxalate precipitating calcium. alcohol, lactic acid Msc Saif A. Mohammed Practical immunology References 1. Manitoba Provincial Blood Coordinating Office. Manitoba transfusion quality manual for blood banks Version 2.0. Winnipeg (MB).Manitoba Provincial Blood Programs Coordinating Office; 2007. 2. Micro-Typing Systems, Inc. Preparing 0.8% cell suspensions. Markham, Ontario: Micro Typing Systems, Inc. 1996. 3. Micro-Typing Systems, Inc. Red blood cell diluent MTS™ diluent 2 plus instructions for use. Pompano Beach FL: Micro Typing Systems, Inc. 2010. 4. Roback, J., Grossman, B., Harris, T & Hillier, C. Technical manual 17th ed. Bethesda, Maryland: AABB; 2011. 5. Transfusion Ontario Programs Ottawa Office. Ontario regional blood coordinating network standard work instruction manual. Ottawa (ON): Transfusion Ontario Programs Ottawa Office; 2009 6. Western Memorial Hospital. MTS system (preparation of 0.8% cell suspension). Corner Brook NL: Western Memorial Hospital; Feb14/2011. Ministry of Higher Education & Scientific Research Middle Tech. University Baqubah Technical institute Medical laboratories Techniques. Practical Immunology By Complement Assist Lec. : Saifsystem A. Mohammed By Assist Lec. : Saif A. Mohammed November , 2022 M.sc Saif A. Mohammed Practical immunology complement system test? The complement system consists of almost 60 proteins, approximately 30 of which are circulating blood proteins that work together to promote immune and inflammatory responses. Complement tests measure the amount or activity of complement proteins in the blood. The complement system’s principal role is to help identify, destroy and remove foreign pathogens like bacteria and viruses, as well as damaged “self” materials (e.g., cells and proteins). Antigens Antigen is a substance that may be specifically bound by an antibody , A substance that induces specific immune response called antigen or immunogen or complete antigen ( Molecular weight > 100Kd ) M.sc Saif A. Mohammed Practical immunology Hapten / incomplete antigen The molecules that may bind to antibodies but can’t elicit immune response. These molecules are called hapten. Molecular weight