This document provides an overview of antibody identification in blood banking. It discusses:
1) The basics of antibody screens and the need to identify unexpected antibodies to ensure transfusion safety.
2) Key concepts like using patient serum as the unknown and reagent RBCs as knowns.
3) How antibody panels are performed using at least 10 RBCs and techniques like the immediate spin, 37C incubation, and antiglobulin phases.
4) Guidelines for interpreting panels by ruling out non-reactive antigens, circling reactive ones, considering antibody characteristics, and looking for a matching pattern to identify the antibody.
This document provides an overview of antibody identification in blood banking. It discusses the key steps in performing an antibody panel, including using a panel of known red blood cells to test against a patient's unknown serum. The goal is to identify any unexpected antibodies in the patient's serum. It also covers interpreting panel results, such as ruling out non-reactive antigens and looking for a matching antigen pattern. Techniques for identifying multiple antibodies like selected cells, neutralization, and chemical treatments are also outlined.
Pretransfusion testing final- ab screening - NAGLAA MAKRAM Naglaa Makram
1. Antibody screening tests patient serum against reagent red blood cells to detect unexpected antibodies that could destroy transfused donor cells.
2. Screening cells must contain many common antigens and include some cells with homozygous antigen expression to detect weakly reacting antibodies.
3. A positive antibody screen requires antibody identification testing to determine the antibody specificity so that antigen-negative blood can be transfused.
The document discusses ABO blood grouping methods and procedures. The two main methods are the slide method and spin tube method. The slide method uses glass slides while the spin tube method uses test tubes. Procedures include preparing red blood cell suspensions, adding blood and antisera to slides or tubes, incubating, and observing for agglutination. Quality control and potential sources of error are also outlined.
This document discusses various techniques used in blood banking and transfusion medicine, including:
1. Pretransfusion testing involves ABO/Rh typing, antibody screening, and crossmatching to select compatible blood and prevent hemolytic transfusion reactions.
2. Antibody identification uses a panel of red blood cells to identify the specific antibody in a patient's serum through various testing phases including immediate spin, LISS incubation, and antiglobulin.
3. Special techniques like elution, hemagglutination inhibition, and titration are used to further characterize antibodies or quantify their concentration.
Weak D testing is performed on all prenatal patients, Rh negative blood donors and transfusion candidates to identify those with the weak D phenotype. The procedure involves incubating patient red blood cells with anti-D, and if negative, adding anti-human globulin to look for weak agglutination indicating a weak D positive result. A true weak D will show at least a 2+ positive reaction; weaker results may be due to prior transfusions and require further investigation. All results are documented in the grouping register.
This document provides an overview of basic principles of immunohematology. It defines key terms like antigen and antibody. It describes the characteristics of antigens and factors that contribute to antigen immunogenicity. It also discusses the different types of immunoglobulins involved in blood group antibodies, and the differences between naturally occurring versus immune antibodies. Finally, it explains the stages of antigen-antibody reactions including sensitization and agglutination, and factors that can influence these reactions.
LABORATORY INVESTIGATION OF TRANSFUSION REACTION CASESSadd Alias
The document provides information about investigating transfusion reaction cases in the laboratory. It discusses the learning outcomes, defines transfusion reaction, and outlines the role of the laboratory. It describes the initial measures taken before testing and the preliminary tests conducted, including clerical checks, visual checks, and serology checks. If these tests produce suspicious results, additional tests are done, such as redoing ABO and Rh grouping, antibody screening, and repeating compatibility testing. The document also discusses tests used to investigate specific transfusion reactions like TRALI, TACO, and acute hemolytic transfusion reaction.
This document provides information about the Coombs test, which is used to detect antibody or complement coating of red blood cells. It describes the history and principles of the test, as well as the direct and indirect Coombs test procedures. The direct Coombs test detects in vivo coating of red blood cells and is used to diagnose conditions like hemolytic disease of the newborn. The indirect Coombs test detects in vitro coating of red cells and is used for compatibility testing and antibody screening. Factors affecting the tests and causes of false positive and negative results are also outlined.
This document provides an overview of antibody identification in blood banking. It discusses the key steps in performing an antibody panel, including using a panel of known red blood cells to test against a patient's unknown serum. The goal is to identify any unexpected antibodies in the patient's serum. It also covers interpreting panel results, such as ruling out non-reactive antigens and looking for a matching antigen pattern. Techniques for identifying multiple antibodies like selected cells, neutralization, and chemical treatments are also outlined.
Pretransfusion testing final- ab screening - NAGLAA MAKRAM Naglaa Makram
1. Antibody screening tests patient serum against reagent red blood cells to detect unexpected antibodies that could destroy transfused donor cells.
2. Screening cells must contain many common antigens and include some cells with homozygous antigen expression to detect weakly reacting antibodies.
3. A positive antibody screen requires antibody identification testing to determine the antibody specificity so that antigen-negative blood can be transfused.
The document discusses ABO blood grouping methods and procedures. The two main methods are the slide method and spin tube method. The slide method uses glass slides while the spin tube method uses test tubes. Procedures include preparing red blood cell suspensions, adding blood and antisera to slides or tubes, incubating, and observing for agglutination. Quality control and potential sources of error are also outlined.
This document discusses various techniques used in blood banking and transfusion medicine, including:
1. Pretransfusion testing involves ABO/Rh typing, antibody screening, and crossmatching to select compatible blood and prevent hemolytic transfusion reactions.
2. Antibody identification uses a panel of red blood cells to identify the specific antibody in a patient's serum through various testing phases including immediate spin, LISS incubation, and antiglobulin.
3. Special techniques like elution, hemagglutination inhibition, and titration are used to further characterize antibodies or quantify their concentration.
Weak D testing is performed on all prenatal patients, Rh negative blood donors and transfusion candidates to identify those with the weak D phenotype. The procedure involves incubating patient red blood cells with anti-D, and if negative, adding anti-human globulin to look for weak agglutination indicating a weak D positive result. A true weak D will show at least a 2+ positive reaction; weaker results may be due to prior transfusions and require further investigation. All results are documented in the grouping register.
This document provides an overview of basic principles of immunohematology. It defines key terms like antigen and antibody. It describes the characteristics of antigens and factors that contribute to antigen immunogenicity. It also discusses the different types of immunoglobulins involved in blood group antibodies, and the differences between naturally occurring versus immune antibodies. Finally, it explains the stages of antigen-antibody reactions including sensitization and agglutination, and factors that can influence these reactions.
LABORATORY INVESTIGATION OF TRANSFUSION REACTION CASESSadd Alias
The document provides information about investigating transfusion reaction cases in the laboratory. It discusses the learning outcomes, defines transfusion reaction, and outlines the role of the laboratory. It describes the initial measures taken before testing and the preliminary tests conducted, including clerical checks, visual checks, and serology checks. If these tests produce suspicious results, additional tests are done, such as redoing ABO and Rh grouping, antibody screening, and repeating compatibility testing. The document also discusses tests used to investigate specific transfusion reactions like TRALI, TACO, and acute hemolytic transfusion reaction.
This document provides information about the Coombs test, which is used to detect antibody or complement coating of red blood cells. It describes the history and principles of the test, as well as the direct and indirect Coombs test procedures. The direct Coombs test detects in vivo coating of red blood cells and is used to diagnose conditions like hemolytic disease of the newborn. The indirect Coombs test detects in vitro coating of red cells and is used for compatibility testing and antibody screening. Factors affecting the tests and causes of false positive and negative results are also outlined.
The document provides an overview of crossmatching procedures in blood banking. It discusses the types of crossmatches, including major and minor crossmatches. It describes the steps involved in immediate spin, tube method, and gel card crossmatches. It also covers causes and approaches to dealing with incompatible crossmatches. The roles of antibody screening, direct antiglobulin test, and autocontrol are explained. The document discusses common alloantibodies and autoantibodies. It provides details on Coombs testing and antibody titration.
Pretransfusion testing involves several important steps to ensure blood compatibility and prevent transfusion reactions:
1) Blood typing to determine the patient's ABO and Rh blood group is performed along with antibody screening to detect any unexpected antibodies.
2) Crossmatching tests the patient's serum against donor red blood cells to identify any antibodies that could cause a transfusion reaction.
3) Computerized crossmatching can detect ABO incompatibility but requires strict data entry and confirmation of patient and donor blood types to ensure accuracy.
Check cell, Preparation and Importance.pptxUVAS,Lahore
This document discusses the preparation and importance of check cells, which are red blood cells that have IgG antibodies attached to their surface. It describes how check cells are made by pooling O+ red blood cells from multiple donors and incubating them with IgG antibodies to allow the antibodies to attach. Check cells are used as controls in tests like the Coombs test to validate the test results. They help determine if a negative Coombs test is a true negative or a false result. Check cells can be stored for 7 days at 2-6°C or 24 hours at 25°C and are important for validating reagents, tests, and detecting antibodies in other tests.
Pre-transfusion tests are performed to ensure blood compatibility and safety. These include determining the recipient's blood group and Rh type, screening the recipient's serum for antibodies, and performing a cross-match test between the recipient's serum and donor red blood cells to detect any agglutination. Additional safety tests are done on donor blood to identify infections like HIV, hepatitis B and C, and syphilis. Together these tests help select immunologically compatible blood to minimize adverse transfusion reactions in recipients.
Blood can be separated into components like red blood cells, platelets, cryoprecipitate, and frozen plasma which are useful for different medical purposes. Whole blood is rarely used now due to the risk of volume overload. The Coombs test, also known as the antiglobulin test, detects the presence of antibodies and can be performed directly on a patient's red blood cells or indirectly by incubating their serum with donor red blood cells. A positive result in either test indicates the presence of antibodies.
coombs test, introduction with principle and whole laboratory procedure, also u will read about ,how to perform direct and indirect coombs test? and how to report them?
Weak D testing is performed on all prenatal patients, Rh negative blood donors and transfusion candidates to identify those with the weak D phenotype. The procedure involves incubating patient red blood cells with anti-D, and if negative, adding anti-human globulin to look for weak agglutination indicating a weak D positive result. A true weak D will show at least 2+ agglutination; weaker results may be due to prior transfusions and require checking the transfusion history. All results are documented in the grouping register.
The document discusses the steps for compatibility testing prior to blood transfusion, which includes correctly identifying the donor and recipient, testing their blood samples, and performing a cross-match. It aims to detect any errors in grouping, labeling, or identification and to find any antibodies in the recipient's serum that could react with donor antigens to prevent adverse reactions.
This document discusses pre-transfusion testing procedures, including patient identification, blood sample collection and handling, compatibility testing, and crossmatching. The key steps are:
1) Performing ABO and Rh typing on the recipient's sample to determine blood type.
2) Screening for unexpected antibodies and identifying any present to guide compatible blood unit selection.
3) Crossmatching a recipient's plasma with donor red blood cells to confirm compatibility and detect antibodies.
4) Labeling and releasing crossmatched blood units for transfusion only after resolving any discrepancies.
The document discusses compatibility testing protocols for blood transfusions. It describes how compatibility testing includes ABO and Rh grouping of donor and recipient samples, screening for unexpected antibodies, and a cross-match. Proper identification of donor and recipient samples is critical to avoid errors. The purpose is to select appropriately compatible blood and ensure the best results for the transfusion by preventing hemolysis or antibody-mediated destruction of transfused red blood cells.
This document discusses the Direct Antiglobulin (Coombs) Test and Indirect Antiglobulin (Coombs) Test. The Direct Antiglobulin Test detects in vivo sensitization of a patient's red blood cells by antibodies and is used to investigate hemolytic disease of the fetus and newborn, transfusion reactions, and autoimmune hemolytic anemia. The Indirect Antiglobulin Test detects in vitro sensitization of reagent red blood cells by antibodies in a patient's serum and is used for full blood typing, antibody screening, and antibody identification. Both tests use antihuman globulin to detect antibody-coated red blood cells, but the Direct Test requires no incubation while the Indirect Test
This document discusses the ABO blood group system. It notes that there are over 20 known blood group systems that are genetically determined. The ABO and Rh systems are most important for blood transfusions. The ABO system involves antigens on red blood cells and corresponding antibodies in plasma. People are categorized into one of the main blood groups - A, B, AB, or O - depending on which antigens are present on their red blood cells and which antibodies are present in their plasma. The exact genetic basis and inheritance of the ABO system is also described.
Historical aspect of transfusion medicinetashagarwal
Transfusion medicine has evolved greatly over centuries from early attempts at blood transfusions in the 15th century that proved fatal, to modern safe practices. Some key developments include the first successful animal-animal transfusion in 1665, first human-human transfusion in 1818, discovery of blood groups in 1901 which aided compatibility testing, development of anticoagulants and storage techniques in the early 20th century, establishment of the first blood bank in 1936, and advances in screening and testing that have made transfusions much safer procedures over the past few decades.
The Rh blood group system is the second most important after ABO in transfusion medicine. The principal antigen is D, with Rh+ and Rh- referring to presence or absence of D. Rh antigens, especially D, are highly immunogenic and can cause hemolytic disease of the newborn or transfusion reactions. The Rh system is controlled by two closely linked genes, RHD encoding D and RHCE encoding CE antigens. The Fisher-Race and Wiener theories proposed different models to explain Rh inheritance and antigen combinations. Precautions must be taken to prevent anti-D formation in Rh- individuals through blood transfusions or pregnancies.
Group I discrepancies involve weak or missing antibodies in reverse grouping due to conditions like newborn status, immunosuppression, or subgroups. Group II discrepancies involve weak or missing antigens in forward grouping, which can be caused by subgroups or diseases weakening antigens. Group III discrepancies are due to rouleaux formation or pseudoagglutination from elevated proteins. Group IV involves miscellaneous issues like cold antibodies, mixed field RBCs, or unexpected antibodies. Resolving discrepancies involves investigating patient history, enhancing weak reactions, addressing rouleaux or cold antibodies, determining if multiple RBC populations exist, and following an algorithmic approach.
1. The document describes procedures for titrating anti-D antibodies, performing Du testing, cross matching donor and patient blood, and direct and indirect Coombs testing.
2. Key steps for titration include serially diluting a test serum, adding diluted serum to test tubes containing red blood cells, and determining the titer by the highest dilution showing agglutination.
3. Du testing, cross matching, and Coombs testing involve incubating patient and donor blood components together, then checking for agglutination with or without the addition of anti-globulin reagent to indicate blood compatibility.
This document summarizes ABO and Rh(D) blood grouping systems. It discusses the key points of:
- The ABO system including the antigens, antibodies produced, and inheritance patterns. Group O is the universal donor.
- The Rh system focuses on the D antigen. About 85% of people are Rh positive. Sensitization can be prevented with anti-D immunoglobulin.
- Testing methods for ABO and Rh(D) typing including cell typing with monoclonal antibodies and serum typing. Weak D phenotypes require additional testing to determine Rh status.
- Clinical significance of blood group matching for transfusions to prevent hemolytic transfusion reactions. Group AB is the universal recipient.
This example shows mixed field agglutination in the forward grouping. The causes could be a recent blood transfusion of group O cells or a bone marrow transplant. To resolve it, the tests should be repeated on a new sample or the cells washed and retested.
Compatibility testing involves mixing a patient's serum with donor red blood cells to check for compatibility and detect any antibodies present in the patient's serum that could react with antigens on the donor's cells. The major cross-match test incubates this mixture to detect clinically significant agglutinating or hemolyzing antibodies, while the minor cross-match tests the donor's plasma with the patient's cells. Factors like specimen collection, storage time, temperature during testing, and contamination can affect compatibility testing results if not properly controlled. The goal of compatibility testing is to safely select blood for transfusion that will not cause harm to the recipient.
1. ELISA (Enzyme-linked immunosorbent assay) is an immunoassay technique used to detect antibodies, proteins, peptides, and other molecules. It relies on an antigen-antibody reaction to detect the presence of a substance.
2. The document provides detailed information on the basic principles and steps of ELISA, including coating a plate with antibodies, adding samples and reagents, washing steps, and detecting reactions using enzymes and substrates.
3. Key aspects of performing ELISA are discussed such as sample treatment and storage, controlling humidity and air flow during incubations, and troubleshooting poor results. Direct, indirect, sandwich, and competitive ELISA techniques are also summarized.
CQ blood cell and anti-serums reagents.pdfSalamSawadogo1
This document discusses quality control procedures for reagents used in immunohematology testing. It describes evaluating antisera used for blood grouping, including checking for appearance, specificity, avidity, potency, and titers. It also discusses quality control of anti-D reagents, antiglobulin reagents, lectins, and reagent red blood cells to ensure accurate serological testing in blood banks. Maintaining the quality of these reagents through regular testing is important for the reliability of immunohematology test results.
The document provides an overview of crossmatching procedures in blood banking. It discusses the types of crossmatches, including major and minor crossmatches. It describes the steps involved in immediate spin, tube method, and gel card crossmatches. It also covers causes and approaches to dealing with incompatible crossmatches. The roles of antibody screening, direct antiglobulin test, and autocontrol are explained. The document discusses common alloantibodies and autoantibodies. It provides details on Coombs testing and antibody titration.
Pretransfusion testing involves several important steps to ensure blood compatibility and prevent transfusion reactions:
1) Blood typing to determine the patient's ABO and Rh blood group is performed along with antibody screening to detect any unexpected antibodies.
2) Crossmatching tests the patient's serum against donor red blood cells to identify any antibodies that could cause a transfusion reaction.
3) Computerized crossmatching can detect ABO incompatibility but requires strict data entry and confirmation of patient and donor blood types to ensure accuracy.
Check cell, Preparation and Importance.pptxUVAS,Lahore
This document discusses the preparation and importance of check cells, which are red blood cells that have IgG antibodies attached to their surface. It describes how check cells are made by pooling O+ red blood cells from multiple donors and incubating them with IgG antibodies to allow the antibodies to attach. Check cells are used as controls in tests like the Coombs test to validate the test results. They help determine if a negative Coombs test is a true negative or a false result. Check cells can be stored for 7 days at 2-6°C or 24 hours at 25°C and are important for validating reagents, tests, and detecting antibodies in other tests.
Pre-transfusion tests are performed to ensure blood compatibility and safety. These include determining the recipient's blood group and Rh type, screening the recipient's serum for antibodies, and performing a cross-match test between the recipient's serum and donor red blood cells to detect any agglutination. Additional safety tests are done on donor blood to identify infections like HIV, hepatitis B and C, and syphilis. Together these tests help select immunologically compatible blood to minimize adverse transfusion reactions in recipients.
Blood can be separated into components like red blood cells, platelets, cryoprecipitate, and frozen plasma which are useful for different medical purposes. Whole blood is rarely used now due to the risk of volume overload. The Coombs test, also known as the antiglobulin test, detects the presence of antibodies and can be performed directly on a patient's red blood cells or indirectly by incubating their serum with donor red blood cells. A positive result in either test indicates the presence of antibodies.
coombs test, introduction with principle and whole laboratory procedure, also u will read about ,how to perform direct and indirect coombs test? and how to report them?
Weak D testing is performed on all prenatal patients, Rh negative blood donors and transfusion candidates to identify those with the weak D phenotype. The procedure involves incubating patient red blood cells with anti-D, and if negative, adding anti-human globulin to look for weak agglutination indicating a weak D positive result. A true weak D will show at least 2+ agglutination; weaker results may be due to prior transfusions and require checking the transfusion history. All results are documented in the grouping register.
The document discusses the steps for compatibility testing prior to blood transfusion, which includes correctly identifying the donor and recipient, testing their blood samples, and performing a cross-match. It aims to detect any errors in grouping, labeling, or identification and to find any antibodies in the recipient's serum that could react with donor antigens to prevent adverse reactions.
This document discusses pre-transfusion testing procedures, including patient identification, blood sample collection and handling, compatibility testing, and crossmatching. The key steps are:
1) Performing ABO and Rh typing on the recipient's sample to determine blood type.
2) Screening for unexpected antibodies and identifying any present to guide compatible blood unit selection.
3) Crossmatching a recipient's plasma with donor red blood cells to confirm compatibility and detect antibodies.
4) Labeling and releasing crossmatched blood units for transfusion only after resolving any discrepancies.
The document discusses compatibility testing protocols for blood transfusions. It describes how compatibility testing includes ABO and Rh grouping of donor and recipient samples, screening for unexpected antibodies, and a cross-match. Proper identification of donor and recipient samples is critical to avoid errors. The purpose is to select appropriately compatible blood and ensure the best results for the transfusion by preventing hemolysis or antibody-mediated destruction of transfused red blood cells.
This document discusses the Direct Antiglobulin (Coombs) Test and Indirect Antiglobulin (Coombs) Test. The Direct Antiglobulin Test detects in vivo sensitization of a patient's red blood cells by antibodies and is used to investigate hemolytic disease of the fetus and newborn, transfusion reactions, and autoimmune hemolytic anemia. The Indirect Antiglobulin Test detects in vitro sensitization of reagent red blood cells by antibodies in a patient's serum and is used for full blood typing, antibody screening, and antibody identification. Both tests use antihuman globulin to detect antibody-coated red blood cells, but the Direct Test requires no incubation while the Indirect Test
This document discusses the ABO blood group system. It notes that there are over 20 known blood group systems that are genetically determined. The ABO and Rh systems are most important for blood transfusions. The ABO system involves antigens on red blood cells and corresponding antibodies in plasma. People are categorized into one of the main blood groups - A, B, AB, or O - depending on which antigens are present on their red blood cells and which antibodies are present in their plasma. The exact genetic basis and inheritance of the ABO system is also described.
Historical aspect of transfusion medicinetashagarwal
Transfusion medicine has evolved greatly over centuries from early attempts at blood transfusions in the 15th century that proved fatal, to modern safe practices. Some key developments include the first successful animal-animal transfusion in 1665, first human-human transfusion in 1818, discovery of blood groups in 1901 which aided compatibility testing, development of anticoagulants and storage techniques in the early 20th century, establishment of the first blood bank in 1936, and advances in screening and testing that have made transfusions much safer procedures over the past few decades.
The Rh blood group system is the second most important after ABO in transfusion medicine. The principal antigen is D, with Rh+ and Rh- referring to presence or absence of D. Rh antigens, especially D, are highly immunogenic and can cause hemolytic disease of the newborn or transfusion reactions. The Rh system is controlled by two closely linked genes, RHD encoding D and RHCE encoding CE antigens. The Fisher-Race and Wiener theories proposed different models to explain Rh inheritance and antigen combinations. Precautions must be taken to prevent anti-D formation in Rh- individuals through blood transfusions or pregnancies.
Group I discrepancies involve weak or missing antibodies in reverse grouping due to conditions like newborn status, immunosuppression, or subgroups. Group II discrepancies involve weak or missing antigens in forward grouping, which can be caused by subgroups or diseases weakening antigens. Group III discrepancies are due to rouleaux formation or pseudoagglutination from elevated proteins. Group IV involves miscellaneous issues like cold antibodies, mixed field RBCs, or unexpected antibodies. Resolving discrepancies involves investigating patient history, enhancing weak reactions, addressing rouleaux or cold antibodies, determining if multiple RBC populations exist, and following an algorithmic approach.
1. The document describes procedures for titrating anti-D antibodies, performing Du testing, cross matching donor and patient blood, and direct and indirect Coombs testing.
2. Key steps for titration include serially diluting a test serum, adding diluted serum to test tubes containing red blood cells, and determining the titer by the highest dilution showing agglutination.
3. Du testing, cross matching, and Coombs testing involve incubating patient and donor blood components together, then checking for agglutination with or without the addition of anti-globulin reagent to indicate blood compatibility.
This document summarizes ABO and Rh(D) blood grouping systems. It discusses the key points of:
- The ABO system including the antigens, antibodies produced, and inheritance patterns. Group O is the universal donor.
- The Rh system focuses on the D antigen. About 85% of people are Rh positive. Sensitization can be prevented with anti-D immunoglobulin.
- Testing methods for ABO and Rh(D) typing including cell typing with monoclonal antibodies and serum typing. Weak D phenotypes require additional testing to determine Rh status.
- Clinical significance of blood group matching for transfusions to prevent hemolytic transfusion reactions. Group AB is the universal recipient.
This example shows mixed field agglutination in the forward grouping. The causes could be a recent blood transfusion of group O cells or a bone marrow transplant. To resolve it, the tests should be repeated on a new sample or the cells washed and retested.
Compatibility testing involves mixing a patient's serum with donor red blood cells to check for compatibility and detect any antibodies present in the patient's serum that could react with antigens on the donor's cells. The major cross-match test incubates this mixture to detect clinically significant agglutinating or hemolyzing antibodies, while the minor cross-match tests the donor's plasma with the patient's cells. Factors like specimen collection, storage time, temperature during testing, and contamination can affect compatibility testing results if not properly controlled. The goal of compatibility testing is to safely select blood for transfusion that will not cause harm to the recipient.
1. ELISA (Enzyme-linked immunosorbent assay) is an immunoassay technique used to detect antibodies, proteins, peptides, and other molecules. It relies on an antigen-antibody reaction to detect the presence of a substance.
2. The document provides detailed information on the basic principles and steps of ELISA, including coating a plate with antibodies, adding samples and reagents, washing steps, and detecting reactions using enzymes and substrates.
3. Key aspects of performing ELISA are discussed such as sample treatment and storage, controlling humidity and air flow during incubations, and troubleshooting poor results. Direct, indirect, sandwich, and competitive ELISA techniques are also summarized.
CQ blood cell and anti-serums reagents.pdfSalamSawadogo1
This document discusses quality control procedures for reagents used in immunohematology testing. It describes evaluating antisera used for blood grouping, including checking for appearance, specificity, avidity, potency, and titers. It also discusses quality control of anti-D reagents, antiglobulin reagents, lectins, and reagent red blood cells to ensure accurate serological testing in blood banks. Maintaining the quality of these reagents through regular testing is important for the reliability of immunohematology test results.
This document provides information on the preparation and separation of plasma and serum from whole blood. It explains that plasma can be separated from whole blood by transferring blood to a tube containing anticoagulant and centrifuging, while serum is separated from clotted whole blood. The document discusses proper storage methods for plasma and serum, such as refrigeration or freezing. It also outlines some common errors in plasma/serum preparation like hemolysis and provides tips to prevent hemolysis.
This document provides guidance on using an antibody identification (ABID) panel to rule out possible alloantibodies in a patient's serum.
The summary is:
1. Reactions on the ABID panel indicate different reaction strengths, suggesting multiple antibodies or different antibody dosages. The patient's auto control is negative.
2. To rule out antigens, only use panel cells with no reaction. Mark out antigens on each cell until only antigens shared by the patient are left.
3. For the example panel, antigens K and s could not be ruled out using panel cells alone. Phenotyping the patient showed they were positive for K but negative for s, indicating anti-s is the likely antibody
This document provides guidance on using an antibody identification (ABID) panel to rule out potential alloantibodies in a patient's serum.
The summary is:
1. Reactions on the ABID panel indicate different reaction strengths, suggesting multiple antibodies or different antibody dosages. The patient's auto control is negative.
2. To rule out antigens, only use panel cells with no reaction. Mark out antigens on each cell until one antigen remains.
3. After ruling out on all cells, if one antigen meets the "Rule of Three" and matches the reaction pattern, it cannot be ruled out yet. Phenotype the patient's cells for that antigen to determine the antibody.
4
Agglutination is the clumping of particulate antigens caused by antibody binding. It was first observed in 1896 using bacterial cells and serum antibody. Antibodies that cause agglutination are called agglutinins. Agglutination involves an initial antigen-antibody binding step followed by lattice formation into large aggregates. Erythrocytes, bacteria, and latex particles can all participate. The reaction is influenced by factors like ionic strength, pH, temperature, and viscosity. It can be directly observed on cell surfaces or indirectly using antigen-coated carriers like latex beads. Agglutination tests are used to diagnose various infectious diseases.
This document provides an overview of serological diagnostic techniques, including concepts of antigens, antibodies, and their interactions. It discusses various types of agglutination assays like direct agglutination tests, indirect latex agglutination, and indirect hemagglutination. It also covers immunochromatographic strip tests, qualitative and semi-quantitative test results, and precautions for accurate serology work. The key techniques covered include detection of antigens or antibodies using agglutination of particles, flocculation, or hemagglutination on test strips or plates.
The document provides guidelines for performing ELISA (Enzyme-Linked Immunosorbent Assay), including:
1. ELISA is a popular immunological technique that uses antibodies and enzymes to detect antigens or antibodies. It is very sensitive and can be used both qualitatively and quantitatively.
2. The basic steps of ELISA involve coating a plate with an antigen, adding a primary antibody, adding a secondary antibody linked to an enzyme, adding a substrate that reacts with the enzyme to produce a colored product.
3. Factors that can cause troubleshooting issues include contamination, improper washing, expired reagents, incorrect procedures, temperature issues, poor pipetting technique, and more. Proper controls and validation
Antigen and immunogens, types and mitogens .pptVetico
An antigen is a molecule that stimulates an immune response, while an immunogen is a foreign substance that can induce antibody formation or sensitized lymphocytes. All immunogens are antigens, but not all antigens are immunogens. Epitopes are small parts of antigens that interact specifically with antibodies or T cell receptors. Haptens are low molecular weight molecules that gain immunogenicity when combined with carrier proteins. Factors like antigen dose, route of administration, and adjuvants can influence a substance's immunogenicity. The ABO blood group system classifies human blood based on the presence of A and B antigens, while the Rh system determines if the D antigen is present. Cross-reacting antigens share similar epitopes, allowing
This document discusses blood group systems, specifically ABO and Rh blood groups. It provides details on:
- The antigens found on red blood cell membranes that determine blood type
- Landsteiner's discovery of the ABO blood group system in 1900 and the four main blood types (A, B, AB, and O)
- The antigens and antibodies present in each blood type
- Rh blood group system including the Rho(D) antigen and typing only for Rho(D) to determine Rh status
- Techniques for blood typing including tube, slide, microplate, and newer gel/cassette methods
- Interpreting and resolving discrepancies in blood typing results
ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of antibodies or antigens in a sample. It involves affixing an unknown antigen to a surface, applying a specific antibody linked to an enzyme, and adding a substrate that the enzyme converts to a detectable signal, most commonly a color change. ELISA has been used as a diagnostic tool in medicine, plant pathology, and the food industry for quality control.
Serology is the study of blood serum and the detection of antibodies and antigens. Key events in the history of serology include Karl Landsteiner's 1901 discovery of the A, B, and O blood groups. Serological tests can be classified as primary, secondary, or tertiary based on their level of sensitivity and directness of measurement. Common serological techniques include ELISA, immunofluorescence, agglutination tests, precipitation reactions, and complement fixation tests. These methods are used to detect infections and other medical conditions.
Gel technology provides an innovative approach to performing various tests in immunohaematology with improved sensitivity and specificity compared to conventional tube techniques. It involves centrifuging red blood cells through a gel column where agglutination reactions occur. The distribution of red blood cells throughout the column allows for easy grading of reaction strength. Gel technology is used for blood grouping, antibody screening and identification, compatibility testing, and other immunohaematology applications. It provides standardized, efficient and reliable results compared to conventional methods.
ELISA (Enzyme-Linked Immunosorbent Assay) is a biochemical technique used to detect antibodies or antigens in a sample. There are three main types of ELISA: competitive ELISA where a labeled antigen competes for antibody binding sites; sandwich ELISA where antibodies coat a surface to detect a specific antigen; and indirect ELISA where a test antigen is coated on a surface and secondary antibodies are used to detect primary antibodies in a serum sample. The amount of color change produced corresponds to the concentration of antigen or antibody in the original sample.
ELISA (Enzyme-Linked Immunosorbent Assay) is a biochemical technique used to detect antibodies or antigens in a sample. There are three main types of ELISA: competitive ELISA, sandwich ELISA, and indirect ELISA. Sandwich ELISA coats a plate with capture antibodies and detects antigens bound between the capture and detection antibodies. Indirect ELISA coats antigens on a plate and detects antibodies in samples using enzyme-linked secondary antibodies. ELISA is used to test samples like blood, urine, and tissue extracts for proteins, hormones, antibodies, and other molecules.
The document discusses the Coombs test, also known as the antiglobulin test. It is used to detect antibodies attached to red blood cells, such as the Rh antibody. There are two types of Coombs tests - direct and indirect. The direct test detects antibodies directly attached to red blood cells, while the indirect test detects antibodies in the patient's serum that could cause red blood cell agglutination. The document provides detailed procedures for performing both the direct and indirect Coombs test in a laboratory setting. It also lists conditions where a positive Coombs test could indicate, such as hemolytic anemia.
principle instrumentation and application of capillary electrophoresisAnimikh Ray
Immunochemical assays are based on antibody-antigen interactions in vitro. An immunoglobulin molecule contains two heavy chains and two light chains, each composed of variable and constant domains. Antigen is any foreign molecule that provokes an antibody response. Radioimmunoassay uses radioactive isotopes to detect antigens or antibodies with high sensitivity down to picogram levels. Enzyme-linked immunosorbent assay uses an enzyme label for detection and can be used for both qualitative and quantitative analysis.
1. ELISA (Enzyme-linked Immunosorbent Assay) is an immunoassay technique used to detect the presence of antibodies and antigens in a solution. It relies on antibodies and antigens binding specifically together.
2. There are several types of ELISA including direct, indirect, sandwich, competitive, and reverse ELISA. Each uses a different approach but all rely on an enzyme-labeled antibody or antigen binding to produce a detectable color change.
3. ELISA is a very sensitive technique that can detect ng/mL to pg/mL concentrations and is widely used for applications like detecting hormones, infectious agents, drugs and more.
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There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
In addition to infrastructure and capacity constraints, CAR-Ts face a very different risk-benefit dynamic in autoimmune compared to oncology, highlighting the need for tolerable therapies with low adverse event risk. CAR-NK and Treg-based therapies are also being developed in certain autoimmune disorders and may demonstrate favorable safety profiles. Several novel non-cell therapies such as bispecific antibodies, nanobodies, and RNAi drugs, may also offer future alternative competitive solutions with variable value propositions.
Widespread adoption of cell therapies will not only require strong efficacy and safety data, but also adapted pricing and access strategies. At oncology-based price points, CAR-Ts are unlikely to achieve broad market access in autoimmune disorders, with eligible patient populations that are potentially orders of magnitude greater than the number of currently addressable cancer patients. Developers have made strides towards reducing cell therapy COGS while improving manufacturing efficiency, but payors will inevitably restrict access until more sustainable pricing is achieved.
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2. The Basics…..
Screens….
Antibody Screens use 2 or 3 Screening
Cells to “detect” if antibodies are
present in the serum
If antibodies are detected, they must
be identified…
present
Not present
3. Why do we need to identify?
Antibody identification is needed for
Transfusion purposes
Important component of compatibility
testing
It will identify any unexpected
antibodies in the patient’s serum
If a person with an antibody is
exposed to donor cells with the
corresponding antigen, serious side
effects may occur
4. Key Concepts
In blood banking, we test “knowns” with
“unknowns”
When detecting and/or identifying
antibodies, we test patient serum
(unknown) with reagent RBCs (known)
Known: Unknown:
Reagent RBCs + patient serum
Reagent antisera + patient RBCs
5. Reagent RBCs
Screening Cells and Panel Cells are
the same with minor differences:
both are group O cells.
Screening cells
Antibody detection
Sets of 2 or 3 vials
Panel cells
Antibody identification
At least 10 vials per set
6. Antibody Panel vs. Screen
An antibody panel is just an
extended version of an antibody
screen
The screen only uses 2-3 group O
cells:
7. Antibody Panel
An antibody panel usually includes
at least 10 group O panel cells:
9. Panel
Each of the panel cells has been
antigen typed (shown on antigram)
+ refers to the presence of the antigen
0 refers to the absence of the antigen
Example: Panel Cell #10 has 9 antigens present: c, e, f, M, s, Leb, k, Fya, and Jka
10. Panel
An autocontrol should also be run
with ALL panels
Autocontrol
Patient RBCs
+
Patient serum
11. Panel
The same phases used in an
antibody screen are used in a panel
• IS
• 37°
• AHG
12. Antibody ID Testing
A tube is labeled for each of the
panel cells plus one tube for AC:
AC
1 2 3 4 5 6 7 8 9 10 11
1 drop of each panel cell
+
2 drops of the patients serum
13. IS Phase
Perform immediate spin (IS) and
grade agglutination; inspect for
hemolysis
Record the results in the
appropriate space as shown:
2+
0
0
Last
tube
14. (LISS) 37°C Phase
2 drops of LISS are added, mixed
and incubated for 10-15 minutes
Centrifuge and check for
agglutination
Record results
18. IAT Phase (or AHG)
Indirect Antiglobulin Test (IAT) –
we’re testing whether or not
possible antibodies in patient’s
serum will react with RBCs in vitro
To do this we use the Anti-Human
Globulin reagent (AHG)
Polyspecific
Anti-IgG
Anti-complement
19. AHG Phase
Wash cells 3 times with saline
(manual or automated)
Add 2 drops of AHG and gently mix
Centrifuge
Read
Record reactions
24. Interpreting Antibody Panels
There are a few basic steps to
follow when interpreting panels
1. “Ruling out” means crossing out
antigens that did not react
2. Circle the antigens that are not
crossed out
3. Consider antibody’s usual reactivity
4. Look for a matching pattern
25. An antibody will only react
with cells that have the
corresponding antigen;
antibodies will not react with
cells that do not have the
antigen
Always remember:
29. 3. Consider antibody’s usual reactivity
2+
0
0
2+
0
0
2+
0
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
Lea is normally a Cold-Reacting antibody (IgM), so it makes
sense that we see the reaction in the IS phase of testing;
The E antigen will usually react at warmer temperatures
30. 4. Look for a matching pattern
2+
0
0
2+
0
0
2+
0
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
…Yes, there is a matching pattern!
E doesn’t match and
it’s a warmer Rx Ab
32. Guidelines
Again, it’s important to look at:
Autocontrol
Negative - alloantibody
Positive – autoantibody or DTR (i.e.,alloantibodies)
Phases
IS – cold (IgM)
37° - cold (some have higher thermal range) or
warm reacting
AHG – warm (IgG)…significant!!
Reaction strength
One consistent strength – one antibody
Different strengths – multiple antibodies or dosage
33. About reaction strengths……
Strength of reaction may be due to
“dosage”
If panel cells are homozygous, a strong
reaction may be seen
If panel cells are heterozygous,
reaction may be weak or even non-
reactive
Panel cells that are heterozygous
should not be crossed out because
antibody may be too weak to react
(see first example)
34. Guidelines (continued)
Matching the pattern
Single antibodies usually shows a pattern
that matches one of the antigens (see
previous panel example)
Multiple antibodies are more difficult to match
because they often show mixed reaction
strengths
35. Rule of three
The rule of three must be met to
confirm the presence of the antibody
A p-value ≤ 0.05 must be observed
This gives a 95% confidence interval
How is it demonstrated?
Patient serum MUST be:
Positive with 3 cells with the antigen
Negative with 3 cells without the antigen
36. Our previous example fulfills the
“rule of three”
2+
0
0
2+
0
0
2+
0
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
3 Negative
cells
3 Positive
cells
Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin
Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at immediate spin
37. What if the “rule of three” is not fulfilled?
If there are not enough cells in the
panel to fulfill the rule, then
additional cells from another panel
could be used
Most labs carry different lot
numbers of panel cells
38. Phenotyping
In addition to the rule of three,
antigen typing the patient red cells
can also confirm an antibody
How is this done?
Only perform this if the patient has NOT
been recently transfused (donor cells
could react)
If reagent antisera (of the suspected
antibody) is added to the patient RBCs, a
negative reaction should result…Why?
40. Multiple antibodies
Multiple antibodies may be more of
a challenge than a single antibody
Why?
Reaction strengths can vary
Matching the pattern is difficult
41. So what is a tech to do?
Several procedures can be
performed to identify multiple
antibodies
Selected Cells
Neutralization
Chemical treatment
Proteolytic enzymes
Sulfhydryl reagents
ZZAP
42. Selected Cells
Selected cells are chosen from other
panel or screening cells to confirm
or eliminate the antibody
The cells are “selected” from other
panels because of their
characteristics
The number of selected cells
needed depends on how may
antibodies are identified
43. Selected Cells
Every cell should be positive for
each of the antibodies and negative
for the remaining antibodies
For example:
Let’s say you ran a panel and identified
3 different antibodies: anti-S, anti-Jka,
and anti-P1
Selected cells could help…
44. Selected Cells
Selected
cells
S Jka P1 IS LISS
37°
AHG
#1 + 0 0 0 0 2+
#5 0 + 0 0 0 3+
#8 0 0 + 0 0 0
These results show that instead of 3 antibodies, there
are actually 2: anti-S and anti-Jka
45. Neutralization
Some antibodies may be neutralized
as a way of confirmation
Commercial “substances” bind to
the antibodies in the patient serum,
causing them to show no reaction
when tested with the corresponding
antigen (in panel)
46. Neutralization
Manufacturer’s directions should be
followed and a dilutional control
should always be used
The control contains saline and serum
(no substance) and should remain
positive
A control shows that a loss of reactivity
is due to the neutralization and not to
the dilution of the antibody strength
when the substance is added
47. Neutralization
Common substances
P1 substance (sometimes derived from hydatid cyst
fluid)
Lea and Leb substance (soluble antigen found in
plasma and saliva)
I substance can be found in breast milk
Sda substance derived from human or guinea pig
urine
**you should be aware that many of these substances
neutralize COLD antibodies; Cold antibodies can
sometimes mask more clinically significant antibodies
(IgG), an important reason to use neutralization
techniques
48. Enzymes (proteolytic)
Can be used to enhance or destroy
certain blood group antigens
Several enzymes exist:
Ficin (figs)
Bromelin (pineapple)
Papain (papaya)
In addition, enzyme procedures
may be
One-step
Two-step
49. Enzymes
Enzymes remove the sialic acid
from the RBC membrane, thus
“destroying” it and allowing other
antigens to be “enhanced”
Antigens destroyed: M, N, S, s,
Duffy
Antigens enhanced: Rh, Kidd,
Lewis, I, and P
50. Enzyme techniques
One-stage
Enzyme is added directly to the
serum/cell mixture
Two-stage
Panel cells are pre-treated with
enzyme, incubated and washed
Patient serum is added to panel cells
and tested
51. Enzyme techniques
If there is no agglutination after
treatment, then it is assumed the
enzymes destroyed the antigen
53. Sulfhydryl Reagents
Cleave the disulfide bonds of IgM
molecules and help differentiate
between IgM and IgG antibodies
Good to use when you have both
IgG and IgM antibodies (warm/cold)
Dithiothreitol (DTT) is a thiol and will
denature Kell antigens
2-mercaptoethanol (2-ME)
54. ZZAP
A combination of proteolytic
enzymes and DTT
Denatures Kell, M, N, S, Duffy and
other less frequent blood group
antigens
Does not denature the Kx antigen
Good for adsorption techniques
“frees” autoantibody off patient’s cell, so that
autoantibody can then be adsorbed onto another
RBC
56. Autoantibodies
Autoantibodies can be cold or
warm reacting
A positive autocontrol or DAT may
indicate that an auto-antibody is
present
Sometimes the autocontrol may be
positive, but the antibody screening
may be negative, meaning
something is coating the RBC
57. Getting a positive DAT
We have focused a lot on the IAT
used in antibody screening and ID,
but what about the DAT?
The direct antiglobulin test
(DAT) tests for the in vivo coating
of RBCs with antibody (in the body)
AHG is added to washed patient red
cells to determine this
58. What can the DAT tell us?
Although not always performed in
routine pretransfusion testing, a
positive DAT can offer valuable
information
If the patient has been transfused, the
patient may have an alloantibody
coating the transfused cells
If the patient has NOT been transfused,
the patient may have an
autoantibody coating their own cells
60. Cold autoantibodies
React at room temperature with
most (if not all) of the panel cells
and give a positive autocontrol
The DAT is usually positive with
anti-C3 AHG (detects complement)
Could be due to Mycoplasma
pneumoniae, infectious mono, or
cold agglutinin disease
61. Cold autoantibodies
Mini-cold panels can be used to help
identify cold autoantibodies
Since anti-I is a common
autoantibody, cord blood cells (no I
antigen) are usually included
Group O
individual with
cold autoanti-I
Group A
individual with
cold autoanti-IH
Anti-IH is reacting weakly with the cord
cells (some H antigen present)
62. Avoiding reactivity
Cold autoantibodies can be a nuisance
at times. Here are a few ways to avoid
a reaction:
Use anti-IgG AHG instead of polyspecific.
Most cold antibodies react with polyspecific
AHG and anti-C AHG because they fix
complement
Skipping the IS phase avoids the
attachment of cold autoantibodies to the red
cells
Use 22% BSA instead of LISS
63. Other techniques
If the antibodies remain, then
prewarmed techniques can be
performed:
Red cells, serum, and saline are incubated
at 37° before being combined
Autoadsorption is another technique
in which the autoantibody is removed
from the patients serum using their
own red cells
The serum can be used to identify any
underlying alloantibodies
64. Warm autoantibodies
More common that cold
autoantibodies
Positive DAT due to IgG antibodies
coating the red cell
Again, the majority of panel or
screening cells will be positive
The Rh system (e antigen) seems to
be the main target although others
occur
65. Warm autoantibodies
Cause warm autoimmune hemolytic
anemia (WAIHA)…H&H
How do you get a warm autoantibody?
Idiopathic
Known disorder (SLE, RA, leukemias, UC,
pregnancy, infectious diseases, etc)
Medications
Several techniques are used when
warm autoantibodies are suspected…
66. Elution (whenever DAT is positive)
Elution techniques “free”
antibodies from the sensitized
red cells so that the antibodies
can be identified
Y
Y
Y
Y
Sensitized
RBC
Positive DAT
Elution
Y
Frees antibody Antibody ID
67. Elution
The eluate is a term used for the
removed antibodies
Testing the eluate is useful in
investigations of positive DATs
HDN
Transfusion reactions
Autoimmune disease
The red cells can also be used after
elution for RBC phenotyping if needed
When tested with panel cells, the eluate
usually remains reactive with all cells if a
warm autoantibody is present
68. Elution Methods
Acid elutions (glycine acid)
Most common
Lowers pH, causing antibody to
dissociate
Organic solvents (ether, chloroform)
Dissolve bilipid layer of RBC
Heat (conformational change)
Freeze-Thaw (lyses cells)
ABO
antibodies
69. Adsorption
Adsorption procedures can be used
to investigate underlying
alloantibodies
ZZAP or chloroquine
diphosphate can be used to
dissociate IgG antibodies from the
RBC (may take several repeats)
After the patient RBCs are
incubated, the adsorbed serum is
tested with panel cells to ID the
alloantibody (if present)
70. Adsorption
Two types:
Autoadsorption
No recent transfusion
Autoantibodies are removed using patient
RBCs, so alloantibodies can be identified
Allogenic (Differential) adsorption
If recently transfused
Uses other cells with the patients serum
71. Wash x3 after
incubation
Centrifuge after
incubating; and
transfer serum to 2nd
tube of treated cells;
incubate and
centrifuge again
2
tubes
Remove
serum and
test for
alloantibody
72. More reagents….
Many of elution tests can damage
the antigens on the RBC
Choroquine diphosphate (CDP)
and glycine acid EDTA reagents can
dissociate IgG from the RBC without
damaging the antigens
Very useful if the RBC needs to be
antigen typed
73. Chloroquine diphosphate
Quinilone derivative often used as
an antimalarial
May not remove autoantibody
completely from DAT positive cells
Partial removal may be enough to
antigen type the cells or to be used
for autoadsorption of warm
autoantibodies