1. The document discusses the automated capillary zone electrophoresis (CZE) method for detecting monoclonal proteins using the Sebia CAPILLARYS instrument.
2. Key aspects of the CZE method include separating serum proteins into fractions in liquid phase without a gel, then using immunotyping to detect monoclonal bands through antigen-antibody reactions and observing changes in electrophoretic patterns.
3. Several case studies are presented to demonstrate interpretation of CZE scans and identify monoclonal bands, including one showing a restricted band superimposed on the C3 complement band.
The document summarizes a study comparing the automated serum protein electrophoresis system V8 from Sysmex to the Capillarys 2 system from Sebia. Over 1,600 patient samples were run on both systems using different buffers on the V8. The concordance between observers and systems for detecting monoclonal components was variable depending on the buffer used. While the V8 performance was similar to Capillarys 2 for monoclonal component detection, the V8 electropherograms had less defined outlines and lower run-to-run reproducibility compared to Capillarys 2.
Serum protein electrophoresis & their clinical importanceDr.M.Prasad Naidu
This document discusses serum proteins and electrophoresis techniques used to analyze them. It provides details on the major serum proteins - albumin and globulins - and their functions. Electrophoresis separates proteins based on their charge and size. Several electrophoresis methods are described, including agarose gel, SDS-PAGE, and capillary electrophoresis. Factors influencing electrophoresis results and common stains used are also outlined. The document concludes with descriptions of normal and abnormal serum protein electrophoresis patterns and their clinical significance.
The document discusses a study that measured the levels of prothrombin, apolipoprotein A-IV, and haptoglobin in the cerebrospinal fluid (CSF) and serum of Huntington's disease (HD) patients and control patients. The study found increased levels of all three proteins in the CSF of HD patients compared to controls. This suggests the proteins are produced in the central nervous system and may serve as biomarkers for HD. The results also indicate the blood-brain barrier remains intact in HD patients.
Structure and function of immunoglobulins(antibodies) Likhith KLIKHITHK1
Immunoglobulins (Ig) or antibodies are glycoproteins that are produced by plasma cells. B cells are instructed by specific immunogens.For, example, bacterial proteins, to differentiate into plasma cells, which are protein-making cells that participate in humoral immune responses against bacteria, viruses, fungi, parasites, cellular antigens, chemicals, and synthetic substances.
The immunogen or antigen reacts with a B-cell receptor (BCR) on the cell surface of B lymphocytes, and a signal is produced that directs the activation of transcription factors to stimulate the synthesis of antibodies, which are highly specific for the immunogen that stimulated the B cell. Furthermore, one clone of B cell makes an immunoglobulin (specificity). Besides, the immune system remembers the antigens that caused a previous reaction (memory) due to the development of memory B cells. These are intermediate, differentiated B cells with the capability to quickly become plasma cells. Circulating antibodies recognize antigen in tissue fluids and serum. This activity describes the physiology and pathophysiology of immunoglobulins
This study compared the effects of pre-storage vs post-storage pathogen reduction treatment on plasma constituents in fresh frozen plasma units. Biochemical, immune, and hemostatic parameters were analyzed in both groups. Results showed no clinically significant differences between the two groups in final plasma protein levels or activity of coagulation factors. Post-storage treated plasma remained high quality and could be effectively and safely inactivated prior to clinical use, providing an option for inactivating previously frozen units.
The complement fixation test detects antibodies in patient serum. It requires several components mixed at optimal concentrations, including sheep red blood cells bound to anti-sheep immunoglobulin G. The test consists of an indicator system using these red blood cells and an exogenous complement source. When the patient's serum contains a complement-fixing antibody, it will bind antigens and fix the complement, preventing lysis of the indicator red blood cells and yielding a positive result. While more demanding than other detection methods, complement fixation was commonly used but has been replaced by more sensitive techniques.
Immunologic tests play an important role in the diagnosis of autoimmune diseases. Autoantibodies are inappropriately produced against self-antigens and cause tissue damage. Identification of specific autoantibodies can help diagnose diseases as each is characterized by different autoantibody profiles. A variety of immunologic techniques exist to detect and quantify autoantibodies including gel diffusion, electrophoresis, enzyme immunoassays, and immunohistochemistry. Measurement of acute phase proteins, complement levels, cryoglobulins, and rheumatoid factors also provide clinically useful information for assessing disease activity and management in autoimmune conditions.
Serum protein electrophoresis is used to separate serum proteins into fractions based on their size and electrical charge. It has five major fractions: albumin, alpha1, alpha2, beta, and gamma globulins. Specific diseases and conditions cause abnormal patterns in the fractions. For example, liver disease decreases albumin levels while nephrotic syndrome increases alpha2 levels due to protein loss in the urine. The test is useful for identifying inflammatory states, immunodeficiencies, and monoclonal gammopathies.
The document summarizes a study comparing the automated serum protein electrophoresis system V8 from Sysmex to the Capillarys 2 system from Sebia. Over 1,600 patient samples were run on both systems using different buffers on the V8. The concordance between observers and systems for detecting monoclonal components was variable depending on the buffer used. While the V8 performance was similar to Capillarys 2 for monoclonal component detection, the V8 electropherograms had less defined outlines and lower run-to-run reproducibility compared to Capillarys 2.
Serum protein electrophoresis & their clinical importanceDr.M.Prasad Naidu
This document discusses serum proteins and electrophoresis techniques used to analyze them. It provides details on the major serum proteins - albumin and globulins - and their functions. Electrophoresis separates proteins based on their charge and size. Several electrophoresis methods are described, including agarose gel, SDS-PAGE, and capillary electrophoresis. Factors influencing electrophoresis results and common stains used are also outlined. The document concludes with descriptions of normal and abnormal serum protein electrophoresis patterns and their clinical significance.
The document discusses a study that measured the levels of prothrombin, apolipoprotein A-IV, and haptoglobin in the cerebrospinal fluid (CSF) and serum of Huntington's disease (HD) patients and control patients. The study found increased levels of all three proteins in the CSF of HD patients compared to controls. This suggests the proteins are produced in the central nervous system and may serve as biomarkers for HD. The results also indicate the blood-brain barrier remains intact in HD patients.
Structure and function of immunoglobulins(antibodies) Likhith KLIKHITHK1
Immunoglobulins (Ig) or antibodies are glycoproteins that are produced by plasma cells. B cells are instructed by specific immunogens.For, example, bacterial proteins, to differentiate into plasma cells, which are protein-making cells that participate in humoral immune responses against bacteria, viruses, fungi, parasites, cellular antigens, chemicals, and synthetic substances.
The immunogen or antigen reacts with a B-cell receptor (BCR) on the cell surface of B lymphocytes, and a signal is produced that directs the activation of transcription factors to stimulate the synthesis of antibodies, which are highly specific for the immunogen that stimulated the B cell. Furthermore, one clone of B cell makes an immunoglobulin (specificity). Besides, the immune system remembers the antigens that caused a previous reaction (memory) due to the development of memory B cells. These are intermediate, differentiated B cells with the capability to quickly become plasma cells. Circulating antibodies recognize antigen in tissue fluids and serum. This activity describes the physiology and pathophysiology of immunoglobulins
This study compared the effects of pre-storage vs post-storage pathogen reduction treatment on plasma constituents in fresh frozen plasma units. Biochemical, immune, and hemostatic parameters were analyzed in both groups. Results showed no clinically significant differences between the two groups in final plasma protein levels or activity of coagulation factors. Post-storage treated plasma remained high quality and could be effectively and safely inactivated prior to clinical use, providing an option for inactivating previously frozen units.
The complement fixation test detects antibodies in patient serum. It requires several components mixed at optimal concentrations, including sheep red blood cells bound to anti-sheep immunoglobulin G. The test consists of an indicator system using these red blood cells and an exogenous complement source. When the patient's serum contains a complement-fixing antibody, it will bind antigens and fix the complement, preventing lysis of the indicator red blood cells and yielding a positive result. While more demanding than other detection methods, complement fixation was commonly used but has been replaced by more sensitive techniques.
Immunologic tests play an important role in the diagnosis of autoimmune diseases. Autoantibodies are inappropriately produced against self-antigens and cause tissue damage. Identification of specific autoantibodies can help diagnose diseases as each is characterized by different autoantibody profiles. A variety of immunologic techniques exist to detect and quantify autoantibodies including gel diffusion, electrophoresis, enzyme immunoassays, and immunohistochemistry. Measurement of acute phase proteins, complement levels, cryoglobulins, and rheumatoid factors also provide clinically useful information for assessing disease activity and management in autoimmune conditions.
Serum protein electrophoresis is used to separate serum proteins into fractions based on their size and electrical charge. It has five major fractions: albumin, alpha1, alpha2, beta, and gamma globulins. Specific diseases and conditions cause abnormal patterns in the fractions. For example, liver disease decreases albumin levels while nephrotic syndrome increases alpha2 levels due to protein loss in the urine. The test is useful for identifying inflammatory states, immunodeficiencies, and monoclonal gammopathies.
This document provides an overview of electrophoresis. It discusses the history and development of the technique from moving boundary electrophoresis to zonal electrophoresis. It describes the principles behind electrophoresis, including the use of a solid support medium and buffer solutions. It also outlines various applications of electrophoresis like serum protein electrophoresis, CSF electrophoresis, and hemoglobin electrophoresis. The document provides examples of protein patterns seen in normal and diseased states for several conditions.
This document provides a lab manual covering various immunology and cell culture techniques. It includes 12 experiments on topics such as blood grouping, Rh factor testing, hemoglobin estimation, differential leukocyte counting, red blood cell counting using a Neubauer chamber, and total leukocyte counting with a Neubauer chamber. For each experiment, the document discusses the principle, requirements, procedure, results and interpretation. The experiments aim to teach fundamental immunology and cell culture methods relevant to research and clinical applications.
May 2015 Antibodies Newsflash - BBI SolutionsBBISolutions
This month's Antibodies Newsflash features CRP recombinant FAB antibodies, D-Dimer and HbA1c monoclonal antibodies plus more.
Click through the slides to find out what makes these leading products in BBI's antibodies range.
This document summarizes key concepts in immunology. It defines antigens as substances recognized by B cells or T cells, and antibodies as antigen-binding proteins found on B cells and secreted by plasma cells. The immune system protects organisms from pathogens through innate immunity involving anatomical, physiological, phagocytic and inflammatory barriers, as well as adaptive immunity providing antigen specificity, diversity, immunological memory and self/non-self recognition via T and B lymphocytes. The complement system is a major effector of humoral immunity involving serum proteins that opsonize pathogens and induce inflammatory responses.
Electrophoresis is a technique used to separate charged biomolecules like proteins and nucleic acids. It works by applying an electric field to migrate these molecules through a buffer solution or gel based on their charge to mass ratio. The first major use of electrophoresis was in 1937. Key aspects include using a buffer to maintain pH and conductivity, as well as staining and destaining techniques to visualize separated biomolecules. Common types include gel electrophoresis using agarose or polyacrylamide gels as well as paper electrophoresis. Applications include clinical diagnosis and protein research.
The document discusses a study that measured concentrations of prothrombin, apolipoprotein A-IV, and haptoglobin in the cerebrospinal fluid (CSF) of patients with Huntington's disease (HD) compared to control patients. The study found increased levels of all three proteins in the CSF of HD patients. This suggests the proteins are produced directly in the central nervous system and their levels in CSF are not related to their serum concentrations. Elevated biomarkers in CSF could help track HD progression and indicate central nervous system involvement in the disease.
This document summarizes a comparison of two methods for antibody screening - the manual Polybrene (MP) method and the Column Agglutination Technology (CAT) method. The MP method is more commonly used in Taiwan but is less sensitive for detecting some antibodies that react at 37°C. The study found that the CAT method detected a higher rate of irregular alloantibodies (1% vs 0.86% for MP). While MP detected more Rh antibodies, CAT detected more antibodies like anti-M and anti-Lea. In conclusion, CAT screening had a higher detection rate and was better at detecting some clinically significant antibodies compared to the traditional MP method.
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.
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.
Cross-matching is a procedure performed prior to blood transfusion to check compatibility between donor blood and recipient blood. It involves testing donor red blood cells with recipient serum in a major cross-match, and testing recipient red blood cells with donor serum in a minor cross-match. The purposes of cross-matching are to detect any antibodies in the recipient that could react with antigens on donor red blood cells, as well as to check for errors in blood typing or sample collection. A positive result showing hemolysis or agglutination during the cross-match test would indicate blood incompatibility.
This document discusses transfusion reactions, including their classification, causes, and roles of technologists. It summarizes that transfusion reactions can be classified based on type (hemolytic vs non-hemolytic) or time of onset (immediate vs delayed). Hemolytic transfusion reactions involve red blood cell incompatibility and destruction, and can be fatal if not addressed. Causes include clerical errors, technical errors, or inadequate testing. Technologists must promptly stop transfusions if reactions are suspected and investigate the case.
Challenges in interpreting serum protein electrophoresis. Requires an approach to recognize pattern within the various protein fractions & differentiate systemic inflammatory response from abnormal antibody production due to neoplastic disorders.Presence of M-band does not always correlate with plasma cell disorders but can be seen some lymphomas, chronic leukaemias, systemic amyloidosis hence need further ancillary tests for diagnosis of aetiology for the M-band.
This document discusses principles of precipitation reactions and their applications in molecular immunogenetics testing. It describes how precipitation occurs when an antibody reacts with a soluble antigen, forming insoluble complexes. This reaction is used in tests like immunofixation electrophoresis and nephelometry. Nephelometry detects antigen-antibody complexes in solution by measuring light scattering. Immunofixation electrophoresis combines protein separation by electrophoresis with precipitation to identify monoclonal proteins. Precipitation reactions are useful for detecting interactions between antibodies and antigens.
This document discusses various types of antigen-antibody reactions including precipitation, agglutination, complement fixation, and immunodiffusion. It then focuses on the complement fixation test (CFT), providing details on its principle, components, procedure, advantages, and disadvantages. CFT detects antibodies that fix complement by inhibiting lysis of red blood cells. The document also covers immunodiffusion techniques like single and double diffusion involving the movement of antigens and antibodies in gel mediums, forming visible precipitation lines. Related techniques involving an electric field like immunoelectrophoresis and rocket electrophoresis are also summarized.
The document discusses serum protein electrophoresis and immunoelectrophoresis, which are used to detect monoclonal gammopathies like multiple myeloma. These tests separate serum proteins and identify abnormal immunoglobulins using electrophoresis and immunofixation. The results are used in the diagnosis and management of conditions involving monoclonal gammopathies.
Immunoelectrophoresis is a technique used in clinical laboratories to detect the presence or absence of proteins in serum. It works by electrophoresing the serum sample to separate its components, then exposing it to antisera specific to different proteins. Lines will form where antigens and antibodies interact, identifying the proteins present. It is useful for determining if a patient produces abnormal amounts of certain proteins, as seen in immunodeficiency diseases or multiple myeloma. While qualitative, it can detect large departures from normal protein levels. A related quantitative technique, rocket electrophoresis, measures antigen levels based on the height of precipitate "rockets" formed during electrophoresis.
Biochemistry Immunochemical techniques part 1.pdfRinaDas9
Immunochemical techniques can be used to determine hormone and protein levels in serum to diagnose endocrine and infectious diseases. These techniques rely on the highly specific binding between antigens and antibodies. Depending on the assay format, immunoassays can be qualitative or quantitative. Common immunochemical techniques include precipitation methods like single radial immunodiffusion where antigen diffuses from a well into an agarose gel containing antibody, forming a visible precipitate ring. Double immunodiffusion allows both antigen and antibody to diffuse and form precipitation lines where they interact. Immunoelectrophoresis separates antigens by charge before allowing diffusion with antibody. Immunofixation identifies monoclonal immunoglobulins by separating serum proteins by electrophoresis then using specific antibodies. Precip
Characterization of intact antibodies by pre-fractionation using gel electrop...Expedeon
Antibodies represent an important class of proteins due to their central role in the immune response. Moreover, there is an increasing interest in the use of recombinant antibodies as novel drug therapies.
The document describes the complement fixation test (CFT), a serological test used to detect antibodies. It involves two steps - in the first step, complement is fixed to an antigen-antibody complex. In the second step, free complement is detected using a hemolytic system containing sheep red blood cells, which will cause lysis in the absence of fixed complement, indicating a negative test result. The CFT requires five reagents - antigen, antibody, complement, red blood cells, and amboceptor antibody against red blood cells. A positive test shows no lysis, while a negative test shows lysis.
1. The authors investigated whether antibodies in intravenous immunoglobulin (IVIg) can alter the aggregation states of β-amyloid (Aβ) and alpha-synuclein (AS).
2. Using techniques like size exclusion chromatography, transmission electron microscopy, and atomic force microscopy, they found that IVIg binds to oligomers of Aβ and AS and promotes their assembly into higher-order oligomeric and fibrillar structures.
3. Preliminary evidence suggests IVIg may form heteropolymers of IgG and Aβ, and that naturally occurring complexes of IgG and Aβ have been found in Alzheimer's patients, potentially part of an immune-mediated clearance mechanism for amyloid aggregates.
This document discusses advanced immuno molecular diagnostic techniques for infectious diseases. It provides an overview of principles of antigen-antibody reactions, factors that influence antigen-antibody binding, and various methods for detecting antigens or antibodies including agglutination, precipitation, complement fixation, immunoassays like ELISA, immunofluorescence, radioimmunoassay, and immunoblotting. It also describes methods for detecting immune cell function such as isolation of immune cells from blood, lymphocyte proliferation assays, and cytotoxicity assays. The document emphasizes the importance of immunoassays in disease diagnosis, immune surveillance, and their applications.
This document describes various immunological techniques used for diagnosing diseases and conditions. It discusses methods like radioallergosorbent testing (RAST) for diagnosing allergies, indirect immunofluorescence assays (IFA) for detecting autoantibodies, bacterial agglutination tests like Widal for typhoid, latex agglutination tests like CRP, enzyme-linked immunosorbent assays (ELISA) for detecting antibodies, indirect hemagglutination assays (IHA) for diagnosing infections, nephelometry for measuring protein and antibody levels, chemiluminescent microparticle immunoassays (CMIA) like syphilis screening, immunoblot techniques for diagnosing autoimmune diseases, micro
This document provides an overview of electrophoresis. It discusses the history and development of the technique from moving boundary electrophoresis to zonal electrophoresis. It describes the principles behind electrophoresis, including the use of a solid support medium and buffer solutions. It also outlines various applications of electrophoresis like serum protein electrophoresis, CSF electrophoresis, and hemoglobin electrophoresis. The document provides examples of protein patterns seen in normal and diseased states for several conditions.
This document provides a lab manual covering various immunology and cell culture techniques. It includes 12 experiments on topics such as blood grouping, Rh factor testing, hemoglobin estimation, differential leukocyte counting, red blood cell counting using a Neubauer chamber, and total leukocyte counting with a Neubauer chamber. For each experiment, the document discusses the principle, requirements, procedure, results and interpretation. The experiments aim to teach fundamental immunology and cell culture methods relevant to research and clinical applications.
May 2015 Antibodies Newsflash - BBI SolutionsBBISolutions
This month's Antibodies Newsflash features CRP recombinant FAB antibodies, D-Dimer and HbA1c monoclonal antibodies plus more.
Click through the slides to find out what makes these leading products in BBI's antibodies range.
This document summarizes key concepts in immunology. It defines antigens as substances recognized by B cells or T cells, and antibodies as antigen-binding proteins found on B cells and secreted by plasma cells. The immune system protects organisms from pathogens through innate immunity involving anatomical, physiological, phagocytic and inflammatory barriers, as well as adaptive immunity providing antigen specificity, diversity, immunological memory and self/non-self recognition via T and B lymphocytes. The complement system is a major effector of humoral immunity involving serum proteins that opsonize pathogens and induce inflammatory responses.
Electrophoresis is a technique used to separate charged biomolecules like proteins and nucleic acids. It works by applying an electric field to migrate these molecules through a buffer solution or gel based on their charge to mass ratio. The first major use of electrophoresis was in 1937. Key aspects include using a buffer to maintain pH and conductivity, as well as staining and destaining techniques to visualize separated biomolecules. Common types include gel electrophoresis using agarose or polyacrylamide gels as well as paper electrophoresis. Applications include clinical diagnosis and protein research.
The document discusses a study that measured concentrations of prothrombin, apolipoprotein A-IV, and haptoglobin in the cerebrospinal fluid (CSF) of patients with Huntington's disease (HD) compared to control patients. The study found increased levels of all three proteins in the CSF of HD patients. This suggests the proteins are produced directly in the central nervous system and their levels in CSF are not related to their serum concentrations. Elevated biomarkers in CSF could help track HD progression and indicate central nervous system involvement in the disease.
This document summarizes a comparison of two methods for antibody screening - the manual Polybrene (MP) method and the Column Agglutination Technology (CAT) method. The MP method is more commonly used in Taiwan but is less sensitive for detecting some antibodies that react at 37°C. The study found that the CAT method detected a higher rate of irregular alloantibodies (1% vs 0.86% for MP). While MP detected more Rh antibodies, CAT detected more antibodies like anti-M and anti-Lea. In conclusion, CAT screening had a higher detection rate and was better at detecting some clinically significant antibodies compared to the traditional MP method.
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.
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.
Cross-matching is a procedure performed prior to blood transfusion to check compatibility between donor blood and recipient blood. It involves testing donor red blood cells with recipient serum in a major cross-match, and testing recipient red blood cells with donor serum in a minor cross-match. The purposes of cross-matching are to detect any antibodies in the recipient that could react with antigens on donor red blood cells, as well as to check for errors in blood typing or sample collection. A positive result showing hemolysis or agglutination during the cross-match test would indicate blood incompatibility.
This document discusses transfusion reactions, including their classification, causes, and roles of technologists. It summarizes that transfusion reactions can be classified based on type (hemolytic vs non-hemolytic) or time of onset (immediate vs delayed). Hemolytic transfusion reactions involve red blood cell incompatibility and destruction, and can be fatal if not addressed. Causes include clerical errors, technical errors, or inadequate testing. Technologists must promptly stop transfusions if reactions are suspected and investigate the case.
Challenges in interpreting serum protein electrophoresis. Requires an approach to recognize pattern within the various protein fractions & differentiate systemic inflammatory response from abnormal antibody production due to neoplastic disorders.Presence of M-band does not always correlate with plasma cell disorders but can be seen some lymphomas, chronic leukaemias, systemic amyloidosis hence need further ancillary tests for diagnosis of aetiology for the M-band.
This document discusses principles of precipitation reactions and their applications in molecular immunogenetics testing. It describes how precipitation occurs when an antibody reacts with a soluble antigen, forming insoluble complexes. This reaction is used in tests like immunofixation electrophoresis and nephelometry. Nephelometry detects antigen-antibody complexes in solution by measuring light scattering. Immunofixation electrophoresis combines protein separation by electrophoresis with precipitation to identify monoclonal proteins. Precipitation reactions are useful for detecting interactions between antibodies and antigens.
This document discusses various types of antigen-antibody reactions including precipitation, agglutination, complement fixation, and immunodiffusion. It then focuses on the complement fixation test (CFT), providing details on its principle, components, procedure, advantages, and disadvantages. CFT detects antibodies that fix complement by inhibiting lysis of red blood cells. The document also covers immunodiffusion techniques like single and double diffusion involving the movement of antigens and antibodies in gel mediums, forming visible precipitation lines. Related techniques involving an electric field like immunoelectrophoresis and rocket electrophoresis are also summarized.
The document discusses serum protein electrophoresis and immunoelectrophoresis, which are used to detect monoclonal gammopathies like multiple myeloma. These tests separate serum proteins and identify abnormal immunoglobulins using electrophoresis and immunofixation. The results are used in the diagnosis and management of conditions involving monoclonal gammopathies.
Immunoelectrophoresis is a technique used in clinical laboratories to detect the presence or absence of proteins in serum. It works by electrophoresing the serum sample to separate its components, then exposing it to antisera specific to different proteins. Lines will form where antigens and antibodies interact, identifying the proteins present. It is useful for determining if a patient produces abnormal amounts of certain proteins, as seen in immunodeficiency diseases or multiple myeloma. While qualitative, it can detect large departures from normal protein levels. A related quantitative technique, rocket electrophoresis, measures antigen levels based on the height of precipitate "rockets" formed during electrophoresis.
Biochemistry Immunochemical techniques part 1.pdfRinaDas9
Immunochemical techniques can be used to determine hormone and protein levels in serum to diagnose endocrine and infectious diseases. These techniques rely on the highly specific binding between antigens and antibodies. Depending on the assay format, immunoassays can be qualitative or quantitative. Common immunochemical techniques include precipitation methods like single radial immunodiffusion where antigen diffuses from a well into an agarose gel containing antibody, forming a visible precipitate ring. Double immunodiffusion allows both antigen and antibody to diffuse and form precipitation lines where they interact. Immunoelectrophoresis separates antigens by charge before allowing diffusion with antibody. Immunofixation identifies monoclonal immunoglobulins by separating serum proteins by electrophoresis then using specific antibodies. Precip
Characterization of intact antibodies by pre-fractionation using gel electrop...Expedeon
Antibodies represent an important class of proteins due to their central role in the immune response. Moreover, there is an increasing interest in the use of recombinant antibodies as novel drug therapies.
The document describes the complement fixation test (CFT), a serological test used to detect antibodies. It involves two steps - in the first step, complement is fixed to an antigen-antibody complex. In the second step, free complement is detected using a hemolytic system containing sheep red blood cells, which will cause lysis in the absence of fixed complement, indicating a negative test result. The CFT requires five reagents - antigen, antibody, complement, red blood cells, and amboceptor antibody against red blood cells. A positive test shows no lysis, while a negative test shows lysis.
1. The authors investigated whether antibodies in intravenous immunoglobulin (IVIg) can alter the aggregation states of β-amyloid (Aβ) and alpha-synuclein (AS).
2. Using techniques like size exclusion chromatography, transmission electron microscopy, and atomic force microscopy, they found that IVIg binds to oligomers of Aβ and AS and promotes their assembly into higher-order oligomeric and fibrillar structures.
3. Preliminary evidence suggests IVIg may form heteropolymers of IgG and Aβ, and that naturally occurring complexes of IgG and Aβ have been found in Alzheimer's patients, potentially part of an immune-mediated clearance mechanism for amyloid aggregates.
This document discusses advanced immuno molecular diagnostic techniques for infectious diseases. It provides an overview of principles of antigen-antibody reactions, factors that influence antigen-antibody binding, and various methods for detecting antigens or antibodies including agglutination, precipitation, complement fixation, immunoassays like ELISA, immunofluorescence, radioimmunoassay, and immunoblotting. It also describes methods for detecting immune cell function such as isolation of immune cells from blood, lymphocyte proliferation assays, and cytotoxicity assays. The document emphasizes the importance of immunoassays in disease diagnosis, immune surveillance, and their applications.
This document describes various immunological techniques used for diagnosing diseases and conditions. It discusses methods like radioallergosorbent testing (RAST) for diagnosing allergies, indirect immunofluorescence assays (IFA) for detecting autoantibodies, bacterial agglutination tests like Widal for typhoid, latex agglutination tests like CRP, enzyme-linked immunosorbent assays (ELISA) for detecting antibodies, indirect hemagglutination assays (IHA) for diagnosing infections, nephelometry for measuring protein and antibody levels, chemiluminescent microparticle immunoassays (CMIA) like syphilis screening, immunoblot techniques for diagnosing autoimmune diseases, micro
Immunoblotting techniques like ELISA, Western blotting, and Southern blotting utilize the binding specificity between antigens and antibodies. Western blotting involves separating protein mixtures by gel electrophoresis, transferring the proteins to a membrane, and detecting specific proteins using labeled antibodies. It is used to detect the presence of target proteins in complex samples. The key steps are tissue lysis and preparation, gel electrophoresis, protein transfer, membrane blocking, primary/secondary antibody probing, and colorimetric detection. This allows visualization and quantification of proteins separated by size on the membrane.
Antibodies, also known as immunoglobulins, are glycoproteins produced by plasma cells that recognize and bind to specific antigens. There are five classes of antibodies - IgG, IgM, IgA, IgD, and IgE - which are distinguished by their structure and functions. Antibodies are made up of two light polypeptide chains and two heavy polypeptide chains that form antigen binding sites. The different classes of antibodies play important but distinct roles in the immune response.
This document summarizes the development of an ELISA method to detect osteoprotegerin (OPG) using OPG ligand (OPGL) for analyte capture. The researchers found that an OPGL capture with monoclonal antibody detection provided the best results. They tested various OPG analogs and found dimeric OPG had greater affinity for OPGL than monomeric OPG. Additionally, they developed a human serum substitute using human serum albumin and fetal bovine serum that could be used as an assay diluent. This ELISA method using OPGL capture and defined diluent will enable analysis of OPG in clinical trial samples.
The document discusses immunodiffusion techniques for detecting antigens and antibodies, specifically the Ouchterlony double immunodiffusion assay. It provides background on immunodiffusion basics, including antigen-antibody reactions, precipitation, and single and double immunodiffusion. It also defines key terms like antigen, antibody, monoclonal and polyclonal antibodies, and discusses their characteristics and functions.
Since antigen and antibody reactions are specific, they can be used to identify each other.
These diagnostic tests are particularly useful in diagnosing for examples: infectious diseases, autoimmune diseases, and in typing of blood and tissues prior to transplantation.
Immunoelectrophoresis is a technique that combines electrophoresis and immunodiffusion to separate and characterize proteins based on their charge and reaction with antibodies. It involves electrophoresing an antigen mixture to separate components by charge, cutting troughs in the gel for antiserum, and detecting lines of precipitation where antibodies and antigens meet. Immunoelectrophoresis is used qualitatively in clinical laboratories to detect the presence or absence of proteins in serum and identify normal and abnormal proteins. It can detect immunodeficiencies or overproduction of proteins but is limited for quantitative analysis.
This document provides an overview of the ELISA (Enzyme Linked Immuno Sorbent Assay) technique. It was developed in 1971 as a method to detect antigens or antibodies. The principle involves forming an antigen-antibody complex that is detected using an enzyme-conjugated secondary antibody. There are four main types of ELISA: direct, indirect, sandwich, and competitive. ELISA has various applications in diagnostics, food testing, and more due to its sensitivity, availability of equipment, and low cost of reagents.
DECLARATION OF HELSINKI - History and principlesanaghabharat01
This SlideShare presentation provides a comprehensive overview of the Declaration of Helsinki, a foundational document outlining ethical guidelines for conducting medical research involving human subjects.
NAVIGATING THE HORIZONS OF TIME LAPSE EMBRYO MONITORING.pdfRahul Sen
Time-lapse embryo monitoring is an advanced imaging technique used in IVF to continuously observe embryo development. It captures high-resolution images at regular intervals, allowing embryologists to select the most viable embryos for transfer based on detailed growth patterns. This technology enhances embryo selection, potentially increasing pregnancy success rates.
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptxHolistified Wellness
We’re talking about Vedic Meditation, a form of meditation that has been around for at least 5,000 years. Back then, the people who lived in the Indus Valley, now known as India and Pakistan, practised meditation as a fundamental part of daily life. This knowledge that has given us yoga and Ayurveda, was known as Veda, hence the name Vedic. And though there are some written records, the practice has been passed down verbally from generation to generation.
Lecture 6 -- Memory 2015.pptlearning occurs when a stimulus (unconditioned st...AyushGadhvi1
learning occurs when a stimulus (unconditioned stimulus) eliciting a response (unconditioned response) • is paired with another stimulus (conditioned stimulus)
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
low birth weight presentation. Low birth weight (LBW) infant is defined as the one whose birth weight is less than 2500g irrespective of their gestational age. Premature birth and low birth weight(LBW) is still a serious problem in newborn. Causing high morbidity and mortality rate worldwide. The nursing care provide to low birth weight babies is crucial in promoting their overall health and development. Through careful assessment, diagnosis,, planning, and evaluation plays a vital role in ensuring these vulnerable infants receive the specialize care they need. In India every third of the infant weight less than 2500g.
Birth period, socioeconomical status, nutritional and intrauterine environment are the factors influencing low birth weight
Test bank for karp s cell and molecular biology 9th edition by gerald karp.pdfrightmanforbloodline
Test bank for karp s cell and molecular biology 9th edition by gerald karp.pdf
Test bank for karp s cell and molecular biology 9th edition by gerald karp.pdf
Test bank for karp s cell and molecular biology 9th edition by gerald karp.pdf
Cell Therapy Expansion and Challenges in Autoimmune DiseaseHealth Advances
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.
Despite these headwinds, industry leaders and investors remain confident that cell therapies are poised to address significant unmet need in patients suffering from autoimmune disorders. However, the extent of this impact on the treatment landscape remains to be seen, as the industry rapidly approaches an inflection point.
These lecture slides, by Dr Sidra Arshad, offer a simplified look into the mechanisms involved in the regulation of respiration:
Learning objectives:
1. Describe the organisation of respiratory center
2. Describe the nervous control of inspiration and respiratory rhythm
3. Describe the functions of the dorsal and respiratory groups of neurons
4. Describe the influences of the Pneumotaxic and Apneustic centers
5. Explain the role of Hering-Breur inflation reflex in regulation of inspiration
6. Explain the role of central chemoreceptors in regulation of respiration
7. Explain the role of peripheral chemoreceptors in regulation of respiration
8. Explain the regulation of respiration during exercise
9. Integrate the respiratory regulatory mechanisms
10. Describe the Cheyne-Stokes breathing
Study Resources:
1. Chapter 42, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 36, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 13, Human Physiology by Lauralee Sherwood, 9th edition
How to Control Your Asthma Tips by gokuldas hospital.Gokuldas Hospital
Respiratory issues like asthma are the most sensitive issue that is affecting millions worldwide. It hampers the daily activities leaving the body tired and breathless.
The key to a good grip on asthma is proper knowledge and management strategies. Understanding the patient-specific symptoms and carving out an effective treatment likewise is the best way to keep asthma under control.
How to Control Your Asthma Tips by gokuldas hospital.
compendium-first-edition-april-2008-Dr-Zia
1. 1
(04-30-2008 11:19A.M.)
INTRODUCTION
The detection and identification of monoclonal proteins in serum/urine, for the
diagnosis of multiple myeloma and other diseases by solid phase zone
electrophoresis and immunofixation has been traditionally a slow and labor intensive
laboratory procedure. In view of the recent availability of the automated capillary
zone electrophoresis (CZE) instruments for the identification of monoclonal proteins
(immunotying) in serum, the laboratory results are provided to the physician more
rapidly than before. There are two automated capillary zone electrophoresis
instruments that are currently available, i.e. Paragon CZE 2000 (Beckman Coulter,
USA), and CAPILLARYS (Sebia, France). We have assembled this compendium of
selected cases for the educational purposes of the medical technologists, residents
in pathology, pathologists, oncologists, and clinical chemists. The objective is to
familiarize the reader with the salient features of the results of the automated method
(Sebia CAPILLARYS) so that an accurate interpretation is made for diagnostic
purposes, especially when the laboratory is switching from the manual/semi-
automated solid phase electrophoretic to the liquid phase automated procedure.
We have briefly described the semi-automated electrophoretic and
immunofixation methods, and the automated methods used for the detection and
identification of the monoclonal bands in serum. Serum immunoglobulins (IgG, IgA,
IgM) were quantified using Beckman Coulter IMMAGE nephelometer.
Serum Protein Electrophoresis: Sebia Hydragel Beta1-Beta2 kit was used
to separate serum proteins on alkaline buffered agarose gels (pH 8.5) into six
fractions, i.e., albumin, alpha-1 globulin, alpha-2 globulin, beta-1 globulin, beta-2
globulin, and gamma globulin. The kits were used in conjunction with the semi-
automated HYDRASYS instrument (Sebia). Amidoblack was used to stain the gels,
and the percent fractions were quantified using the scanner.
Serum Protein Immunofixation: Sebia HYDRAGEL IF kit was used first to
separate the proteins into six fractions (see above) in six different lanes or tracks.
The reference track was overlaid with fixative reagents and each of the five
additional tracks were overlaid with different antisera (IgG, IgA, IgM, kappa and
lambda). The immunoglobulin-antiserum complex was trapped in the agarose, and
then stained with amidoblack for visual examination of the protein electrophoresis
pattern and only bound specific polyclonal or monoclonal immunoglobulins in each
track. Any protein restriction (monoclonal band) was thus determined.
2. 2
Track # 1 Serum Protein Electrophoresis-six fractions
Track # 2 Immunoglobulin G
Track # 3 Immunoglobulin A
Track # 4 Immunoglobulin M
Track # 5 Kappa Chains
Track # 6 Lambda Chains
Capillary Zone Electrophoresis: The proteins in serum were separated into
six fractions using a liquid-based system (Sebia CAPILLARYS), without the use of a
solid support medium i.e., agarose gel. In this method a fused silica capillary (into
which a small volume of serum is aspirated) with strong negative charge on the
interior of the capillary provided a large negative surface area. Under the conditions
of electrophoresis (movement of protein molecules under charge) and endosmotic
flow of cations toward the cathode, the proteins were separated into six fractions,
and quantified by an ultraviolet detector using the absorbance of the amide band of
the proteins at 200-215 nm. All the serum CZE scans wherever illustrated in this
compendium are in green color.
Immunotyping of Serum Proteins: The monoclonal protein present in
serum was detected and identified by an antigen-antibody reaction in liquid phase
followed by CZE using the Sebia CAPILLARYS. In this automated methodology the
specific immunoglobulin and the antisera react to provide an antigen-antibody
complex (pretreatment step). The pretreated samples (reference, IgG, IgA, IgM,
kappa and lambda) were electrophoresed simultaneously in six capillaries. The
antigen-antibody complexes migrated slowly than an unbound immunoglobulin, i.e.,
migrated anodically to albumin, or migrated as an increased baseline co-migrating
with albumin, alpha-1 and/or alpha-2 globulin. This procedure is obverse of
immunofixation (see above), as the monoclonal proteins were removed and thus not
detected on the electropherogram after CZE. The interpretation (identification of the
monoclonal band) was made by the “overlay” of the untreated reference
electrophoretic curve on each of the five electrophoretic curves of the pretreated
samples. All the serum CZE scans after the each pretreatment step
(imunosubtraction process with anti-IgG, anti-IgA, anti-IgM, anti-kappa, and
anti-lambda) wherever illustrated in this compendium are in red color.
3. 3
Selection of Patients for Case Studies: We performed serum protein
electrophoresis on over 5000 patients over a period of nine months. All the sera
having either abnormal band or atypical pattern were sequestered. The patient’s
medical record was reviewed to obtain clinical information. For each case we have
presented in this compendium, a brief medical history, prior diagnosis and the
diagnosis after hospitalization (if applicable), and any other information that may be
helpful in the understanding of the electrophoretic patterns. First the serum protein
electrophoretic (agrose gel) scan, and serum immunofixation (agarose gel) picture
were presented. We also presented a picture of the electrophoretic separation on the
agarose gel. The five fractions of serum (albumin, alpha-1, alpha-2, beta, and
gamma globulin) in gram/dL along with the quantified immunoglobulins were also
provided. An enlarged version of the serum protein CZE scan was also presented
separately for interpretative purposes. Finally, the serum protein electrophoresis
pattern obtained from the automated CZE method (Sebia CAPILLARYS) was
presented six different times on a separate page. One of these scan was used as a
reference. On the remaining five scans the electrophoretic pattern after the antigen-
antibody pretreatment reaction of the serum with each of the five immunoglobulins
(IgG, IgA, IgM, kappa and lambda chains) was electronically superimposed for the
indentification of monolclonal bands. In some cases the identification of the mini-
monoclonal band was facilitated by the electronic magnification of the area under
study of the electrophoretic pattern. Comments were made in some cases in order to
explain the laboratory results otherwise the scans of protein electrophoresis,
immunofixation and immunotyping were obvious to interpret.
Detection and Identification of Monoclonal Bands: The majority of the
readers of this compendium are familiar with the interpretation of the serum protein
electrophoresis and immunofixation patterns. The interpretation of the serum protein
electrophoresis by CZE is virtually identical to the agarose gel serum protein
electrophoresis, and thus needs no further comments.
The detection of protein bands with restricted mobility by immunotyping needs
careful evaluation of the five individual CZE scans of the pretreated samples ( anti-
IgG, anti-IgA, anti-IgM, anti-kappa, and anti-lambda) in conjunction with CZE scan of
the neat serum.
4. 4
In order to familiarize with the salient features of electrophoresis, immunofixation
electrophoresis, immunotyping based on CZE either after immunosubtraction using
antibodies coated to Sepharose beads (Beckman Coulter Paragon CZE 2000), or
after immunosubtraction using liquid phase antigen-antibody reaction (Sebia
CAPILLARYS), the reader is advised to review the first three chapters of the “Protein
Electrophoresis in Clinical Diagnosis” by David F. Keren, MD , 2003 Edition (Arnold,
a member of the Holder Headline Group, Great Britain), ISBN 0340 812133.
The interpretation of the immunotyping patterns requires the visualization of the
subtle changes in the shape of the electrophoretic scans after the antigen-antibody
reaction. A large monoclonal band (M-protein greater than 100 mg/dL) can be
accurately identified without much difficulty. It is the minimonoclonal bands (M-
protein less than 25 mg/dL) that could not be identified easily due to not so obvious
large changes in the shape of the electrophoretic curve, especially if the
minimonoclonal band is either embedded with a polyclonal gamma globulin,
transferrin, complement C3, or associated with an artifact (fibrinogen, contrast
media, etc.).
First, let us examine the symmetrical changes in the shape of the electrophoretic
curve after the immunosubtraction step in a normal person. Please see the
hypothetical figure No A.
5. 5
The difference between the two curves (green minus red) is plotted in violet color.
This violet colored curve is plotted only to exhibit symmetrical polyclonal
immunoreduction process in a normal person, and one will never notice it on the
immunotyping scans of any of the five tracks (anti-IgG, anti-IgA, anti-IgM, anti-kappa,
and anti-lambda). A smooth and symmetrical shaped curve after the
immunoreduction step (red curve) indicates a polyclonal reduction of the
immunoglobulins from the serum. This kind of smooth and symmetrical shaped curve
(red color) is most commonly seen in a normal person after the liquid phase reaction
with anti-IgG. Uniform immunoreduction in the green curve, Fig No. A, cannot be
construed as a monoclonal band, as polyclonal immunosubtraction results in the
reduction of the gamma globulin fraction in a symmetrical manner.
In a normal person the serum IgA is lower than the serum IgG, therefore a very small
difference is observed between the serum CZE scan and the serum CZE scan after
the anti-IgA reaction. This immunoreduction after reaction with anti-IgA is primarily
witnessed in the beta-globulin region. Similarly in a normal person the serum IgM is
even lower than IgA, therefore again a very little reduction without change of the
symmetry of the fraction is observed (virtually mirror image of each other).
6. 6
The situation in case of kappa and lambda chains is slightly different. Kappa chains
are normally present in an approximate 2:1 ratio to lambda chains. Therefore in a
normal person after liquid phase reaction with anti-kappa, one should expect 2/3
reduction in the gamma region. Obversely, in a normal person after liquid phase
reaction with anti-lambda, one should expect 1/3 reduction in the gamma region. The
magnitude of the reduction in the gamma globulin region for both the kappa and
lambda chains is very low as compared to the IgG, and this is obviously due to the
high concentration of serum IgG as compared to kappa and lambda chains in serum.
Let us consider another hypothetical case with a protein restriction (monoclonal
band) in the gamma globulin region (Fig No. B).
It is pointed out that in this hypothetical case of monoclonal band detected in the
gamma globulin region from CZE (green color, Fig No. B), there is also a
symmetrical shaped immunoreduction of the gamma globulin region after the
reaction with anti-IgG (red curve, Fig No. B). The difference between the green and
the red curve is illustrated by violet colored curve, thus indicating a sharp monoclonal
7. 7
peak. It is again pointed out that in actual practice the violet colored curve will
never be depicted on the CZE scan after the immunosubtraction step.
Assignment of monoclonal bands: The first step is the visual examination
of serum CZE scan to note the presence of, a) sharp peak, b) unsymmetrical band
(from the alpha-2 to the farthest cathode end of gamma globulin region), c) distortion
(similar to electrical noise of a signal) of the shape of any of the alpha-2, beta-1,
beta-2, and gamma globulin bands, and d) decreased gamma globulin. A decreased
gamma globulin band also suggests the electrophoretic migration of the possible
restricted immunoglobulin towards the anode, therefore one might observe
unsymmetrical bands for any of the alpha-2, beta-1, and beta-2 globulin bands. The
location of the abnormal / unsymmetrical band on the CZE scan must be kept in
mind while comparing it with the five pretreated (anti-IgG, anti-IgA, anti-IgM, anti-
kappa, and anti-lambda) CZE scans.
Generally speaking (except in a very rare case of heavy chain disease associated
with gamma, alpha, or mu chains), any abnormal / unsymmetrical disappearance of
the immunoglobulin fraction (IgG, IgA, IgM) in the liquid phase immunosubtraction
step followed by CZE must be associated with a concomitant abnormal /
unsymmetrical disappearance of either kappa or lambda chains or both.
Also the position (electrophoretic mobility) of the abnormal / unsymmetrical heavy
chain and the light chain must be the same on the CZE scan (after
immunosubtraction step) in order to correctly assign the monoclonal band. There are
rare cases in which abnormal / unsymmetrical disappearance of the light chain is
observed after the immunosubtraction step without any concomitant subtraction of
the IgG, IgA, and IgM. In these cases the laboratory must rule out the possibility of
either IgD or IgE monoclonal gammopathy, and also the possibility of light chain
gammopathy.
8. 8
Selected Case Studies:
Section A: Case # 1 Normal Person
Case # 2 Restricted band superimposed on
C3 complement band
Case # 3 Atypical shape of the Beta1-Beta2
region in the capillary zone electrophoresis,
and heavy staining of the beta1 region
(transferrin) in agarose gel electrophoresis
Hint: The presence of fibrinogen in serum due to any reason, e.g. patient on
heparin therapy, improper processing of specimen, etc., can mimic the
pattern of monoclonal gammopathy.
Case # 1 Normal Person
Serum of a 28 years old male employee, presented for annual physical
examination. No abnormality observed in the laboratory tests and
examination by the physician.
Total Protein: 7.5 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 4.5 g/dL (3.8-5.8 g/dL) IgG: 902 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 208 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 81 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.2 g/dL (0.5-1.3g/dL)
11. 11
Case # 2 Restricted band superimposed on C3 complement band
51 years male admitted with hypotension, acute renal failure,
protein malnutrition, and was diagnosed with staphylococcus aureus
septicemia during hospitalization.
Total Protein: 2.9 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 1.3 g/dL (3.8-5.8 g/dL) IgG: 786 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 208 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.4 g/dL (0.4-0.9 g/dL) IgM: 32 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.5 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 0.5 g/dL (0.5-1.3g/dL)
Comments: The intense band detected in both the agarose gel and capillary zone
electrophoresis (superimposed on the C3 complement band position) is an artifact,
perhaps due to fibrinogen, as no monoclonal band was detected either by
immunofixation or immunotyping. We did not confirm it by reacting the patient’s
specimen with anti-fibrinogen. In view of the high intensity of this band, we believe
that the patient sample was most likely plasma and not serum. A follow up inquiry
confirmed that indeed it was plasma specimen.
13. 13
NOTE: The apparent increase in -2 globulin was persistent as indicated by an
arrow, perhaps due to fibrinogen. Similar bands in the -2 globulin region as an
artifact due to fibrinogen were reported in the CZE (Xavier Bossuyt, et at, Automated
serum protein electrophoresis by Capillarys, Clin Chem Lab Med 2003; 41:704-710).
14. 14
Case # 3 Atypical shape of the Beta1-Beta2 region in the capillary zone
electrophoresis, and heavy staining of the beta1 region (transferrin) in
agarose gel electrophoresis.
76 years old female with severe symptomatic anemia (hemoglobin of
5.3%) due to gastrointestinal bleeding, bleeding gastric ulcer,
obstructive pulmonary disease, coronary artery disease, atrial
fibrillation, congestive heart failure, and diabetes mellitus Type II
complicated with peripheral neuropathy. Cord compression secondary
to malignant lymphoma involving the spine.
Total Protein: 5.8 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 3.1 g/dL (3.8-5.8 g/dL) IgG: 701 mg/dL(751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 668 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 170 mg/dL ( 46-304 mg/dL)
Beta Globulin: 1.2 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 0.6 g/dL (0.5-1.3g/dL)
Comments: The serum protein electrophoresis by agarose gel indicated increased
concentration of the beta1 globulin region (suggesting increased transferrin due to
serum iron deficiency as the patient is severely anemic). Another possibility of
increased transferrin may be double transferrin bands due to genetic reasons in
some Afro-Americans. The third possibility is of embedded monoclonal band under
the transferrin band. The serum protein electrophoresis by capillary zone
electrophoresis indicated asymmetrical beta1-beta2 globulin region with a minor
restriction on the cathode side of the C3 complement band. It is obvious from the
study of Cases # 2- 4 that fibrinogen can be the possible cause of the atypical
electrophoretic pattern. A monoclonal IgA band is known to migrate between alpha-2
globulin to any place in the gamma globulin region. Indeed immunofixation and
immunotyping indicated the presence of monoclonal IgA-kappa in serum of this
patient. Therefore it is imperative to perform immunotyping or immunofixation of
serum, whenever a minor restriction band or atypical shape of the fractions are
observed on electrophoresis in the area of beta 1-beta 2 globulin region.
17. 17
Section B: Acute-Phase Reaction Pattern
The serum protein electrophoresis is a useful tool to detect acute
tissue damage ( infection, tissue injury, tumor necrosis), with or
without inflammation. In general a group of hepatocyte-derived
proteins are increased, and these proteins are labeled as acute
phase reactants. In a classical acute-phase serum protein
electrophoresis pattern, one detects, a) decreased albumin,
b) increased alpha-1 globulin, c) increased alpha-2 globulin,
d) decreased transferrin if there is no concomitant decrease in
serum iron, e) and small band in the gamma globulin region due
to C-reactive protein (which can be confused with a mini-monoclonal
band). It must be pointed out that C-reactive protein band is never
detected with capillary zone electrophoresis. Fibrinogin is increased
within 24 hour of the injury, and C3 complement elevations without
consistency are noticed within a week after acute tissue damage. In
clinical practice the acute–phase reaction pattern of serum protein
electrophoresis is not used for making any differential diagnosis,
however if detected it can be useful in a variety of clinical conditions,
e.g. predicting infections in patients, etc.
Case # 1 Acute-phase reaction pattern with monoclonal gammopathy
63 years old male with a known history of anemia, nephrotic
syndrome, hypomagnesemia, edema, hypertension, neuropathy,
deep venous thrombosis was admitted to the hospital and a colon
cancer diagnosis with metastasis to the liver was made.
Total Protein: 4.3 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 0.9 g/dL (3.8-5.8 g/dL) IgG: 924 mg/dL(751-1560 mg/dL)
Alpha-1 Globulin: 0.3 g/dL (0.1-0.2 g/dL) IgA: 280 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 1.0 g/dL (0.4-0.9 g/dL) IgM: 401 mg/dL ( 46-304 mg/dL)
Beta Globulin: 1.0 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.1 g/dL (0.5-1.3g/dL)
Comments: The serum electrophoretic pattern by both the agarose gel and the
capillary zone electrophoresis depicts features of acute-phase reaction pattern. In
view of the fact that gamma globulin concentration though not increased but greater
than albumin in serum, this pattern suggested active, chronic inflammation.
19. 19
NOTE: The difference between the red and green curve for the IgM and Kappa
chain is not symmetrical (indicated by an arrow), thus suggesting monoclonal band.
20. 20
Section C: Double Gammopathy and Biclonal Gammopathy
The presence of two distinct bands of restricted mobility on the
serum electrophoretic pattern are rarely observed in the laboratory.
Biclonal gammopathy: If the light chains identified by either
immunofixation or immunotyping differ, i.e. both kappa and lambda
chains are detected then it is called “biclonal gammopathy” as these
light chains originate from two separate clones.
Double gammopathy: If the light chains identified by either
immunofixation or immunotyping are the same, i.e. both are either
“kappa” or “Lambda” then it is called “double gammopathy” as these
light chains originate from a same clone.
The presence of the heavy chain can be the same or different in either
the double or biclonal gammopathy.
It is pointed out that the clinical course and treatment is the same for
biclonal and double gammopathies.
Case # 1 Double gammopathy (IgG-Lambda and IgM-Lambda)
63 years old female under chemotherapy for a previously known case
of multiple myeloma, residing at the nursing home was routinely
checked by the oncologist for several laboratory tests including serum
protein electrophoresis and quantitative immunoglobulins.
Total Protein: 6.4 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 3.2 g/dL (3.8-5.8 g/dL) IgG: 1940 mg/dL(751-1560 mg/dL)
Alpha-1 Globulin: 0.1 g/dL (0.1-0.2 g/dL) IgA: 165 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.6 g/dL (0.4-0.9 g/dL) IgM: 904 mg/dL ( 46-304 mg/dL)
Beta Globulin: 1.6 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 0.9 g/dL (0.5-1.3g/dL)
29. 29
Case # 4 Double Gammopathy (IgA1 – Kappa and IgA2 – Kappa)
55 years old unconscious female admitted to the hospital for liver
cirrhosis, asthma, hypertension, etc. Her kidney function was normal.
Serum creatinine was 0.6 mg/dL (0.7 – 1.5 mg/dL), and the calculated
glomerular filtration rate (GFR) was 105 (> 60 ml/min/1.73 m2 ).
Total Protein: 6.3 g/dL Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 2.9 g/dL (3.8 – 5.8 g/dL) IgG: 1300 mg/dL (751 –1560 mg/dL)
Alpha-1 Globulin: 0.1 g/dL (0.1 – 02 g/dL) IgA: 1640 mg/dL (82 – 453 mg/dL)
Alpha-2 Globulin: 0.6 g/dL (0.4 – 0.9 g/dL) IgM: 105 mg/dL (46 - 304 mg/dL)
Beta Globulin: 1.4 g/dL (0.5 – 1.1 g/dL) Free 37.6 mg//L (3.3 – 19.4 mg/L)
Gamma Globulin: 1.3 g/dL (0.5 – 1.3 g/dL) Free 20.7 mg/L (5.7 – 26.3 mg/L)
Free / 1.82 (0.26 – 1.65)
Comments: Serum protein electrophoresis (Sebia, HYDRASIS) showed a dark
staining for C3 band, and another monoclonal band adjacent to the C3 band
(cathode side). According to Feinstein et al (Nature 212: 1496-1498, 1966), IgA in
human beings is composed of two antigenically different subclasses IgA1 and IgA2,
and approximately 80-90% is IgA1 in serum (Gray et al., Exp. Med. 128: 1233-1236,
1969).
32. 32
Section D: IgA Monoclonal Gammopathies
The monoclonal bands due to IgA migrate in the agarose gel
anywhere from alpha-2 region to the gamma globulin region, however
in the capillary zone electrophoresis (Sebia) the IgA restrictions were
mostly observed in the beta globulin region. The identification of these
monoclonal bands is complicated by the superimposed bands of C3
complement and lipoproteins. In normal population the twin bands of
C3 complement and transferrin (beta globulin region) are very
symmetrical in shape. The shape and the concentration of transferrin
in serum deviates in several clinical conditions, notably serum iron
deficiency (increased transferrin), acute phase reactions (decreased
transferrin), etc. Similarly the shape and the concentration of C3
complement band are altered in some clinical conditions. Serum
samples accidentally exposed to elevated temperatures (above
refrigeration) for a long period of time commonly exhibit lower
concentration (decreased height of the band) of C3 complement.
Upon visual examination of the electrophoretic pattern, if any distortion
of the beta globulin region is detected, it is advised to perform
immunotyping or immunofixation.
33. 33
Case # 1 IgA monoclonal band masking the beta globulin region
67 years old male admitted to hospital and was diagnosed with
anemia, acute renal failure, urinary tract infection, congestive heart
failure, coronary artery disease, and gastrointestinal hemorrhage.
Positive for Bence Jones protein in urine.
Total Protein: 6.8 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 3.1 g/dL (3.8-5.8 g/dL) IgG: 441 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 2410 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 43 mg/dL (46-304 mg/dL)
Beta Globulin: 2.4 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 0.4 g/dL (0.5-1.3g/dL)
39. 39
Section E: IgM Monoclonal Gammopathies:
IgM monoclonal gammopathies when detected are generally
associated with either plasma cell proliferations (IgM myeloma) or a
low-grade B-cell lymphoplasmacytic disorder (Waldenstrom’s
macroglobulinemia). In both the diseases the bone marrow is involved,
however one major characteristic of the Waldenstrom’s
macrglobulinemia is the absence of lytic skeletal lesions.
Case # 1 IgM monoclonal gammopathy in multiple myeloma
92 years old male with several clinical disorders was on
chemotherapy, and the treatment was monitored by serum protein
electrophoresis and quantitative IgM. In view of the hyperviscosity
usually associated with increased concentration of the large molecule
IgM in serum (1770 mg/dL; Normals: 46-304 mg/dL), other clinical
problems may be present, e.g. cryoglobulinemia either with or without
hepatitis C.
Total Protein: 7.1 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 3.9 g/dL (3.8-5.8 g/dL) IgG: 695 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 128 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.6 g/dL (0.4-0.9 g/dL) IgM: 1770 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.5 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.9 g/dL (0.5-1.3g/dL)
42. 42
Case # 2 IgM monoclonal gammopathy in malignant lymphoma (IgM-Kappa)
60 years old male with a past medical history of malignant lymphoma
was examined after first round of chemotherapy and radiation for bone
marrow and flow cytometric tests.
Total Protein: 6.7 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 4.2 g/dL (3.8-5.8 g/dL) IgG: 922 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.1 g/dL (0.1-0.2 g/dL) IgA: 195 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 373 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.8 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 0.9 g/dL (0.5-1.3g/dL)
45. 45
Case # 3 IgM monoclonal gammopathy (MGUS) without any clinical symptoms
(IgM-Kappa)
72 years old female with a history of hypothyroid and ovarian cancer
without any prior cardiac history was admitted for chest pain. Stress
test and heart catheterization was performed, but no evidence of
coronary artery disease was found. The left ventricular ejection volume
was normal.
Total Protein: 7.1 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 4.1 g/dL (3.8-5.8 g/dL) IgG: 1240 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 253 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.8 g/dL (0.4-0.9 g/dL) IgM: 355 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.1 g/dL (0.5-1.3g/dL)
48. 48
Section F: IgG Monoclonal Gammopathies
The gamma globulin region of the electrophoretic pattern consists of
five immunoglobulins (IgG, IgA, IgM, IgD, and IgE), and in normal
person the shape of this is symmetrical akin to half moon. IgG is the
major component of these five immunoglobulins in a normal person.
Several diseases are associated with either the increase or decrease
in serum IgG. It is also known that more than 50% of the monoclonal
bands in serum are linked to IgG. The IgG monoclonal bands can
migrate any place from beta globulin to the most cathodal area of
gamma globulin region. Therefore, anytime a deviation of the
symmetrical shape of the band (beta/gamma globulin) is observed, a
possibility of monoclonal band must be ruled by immunofixation or
immunotyping. The presence of large monoclonal band (IgG
concentration greater than1.0 gram/dL) can be easily detected,
however it is the mini-monoclonal bands that requires follow up, e.g.
free kappa and lambda chain studies in serum.
The following cases are selected to show the various shapes of the
electrophoretic pattern that are linked to monoclonal gammopathies.
Case # 1 IgG-Kappa multiple myeloma
73 years old female with a past medical history of multiple myeloma
was examined during her treatment and laboratory tests were
performed.
Total Protein: 7.2 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 4.1 g/dL (3.8-5.8 g/dL) IgG: 1440 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 202 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.9 g/dL (0.4-0.9 g/dL) IgM: 22 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.8 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.2 g/dL (0.5-1.3g/dL)
57. 57
Case # 4 IgG-Lambda monoclonal gammopathy of undetermined significance
(MSUG)
48 years old male was admitted to the hospital for a viral illness, and
was placed on acylclovir but with no relief of his hyperpyrexia. After the
antiviral therapy the patient was administered antibiotics, and the
pulmonary symptoms were resolved. Several serological tests were
performed during hospitalization, but all of them were negative.
Total Protein: 5.9 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 2.6 g/dL (3.8-5.8 g/dL) IgG: 1150 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.5 g/dL (0.1-0.2 g/dL) IgA: 202 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 1.0 g/dL (0.4-0.9 g/dL) IgM: 77 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.0 g/dL (0.5-1.3g/dL)
60. 60
Case # 5 IgG-Kappa monoclonal gammopathy with persistent anemia
and elevated serum ferritin
61 years old female with persistent anemia, weakness, multiple falling
episodes, refractory lumbosacral pain, hypothyroidism, hypertension,
pelvic ramus fracture in 2003 and sacral fracture in 2003 was
examined by an oncologist, but no morphologic cause for the patient’s
anemia was demonstrated. The mini-monoclonal band detected
(indicated by the shaded area) at the most cathode part of the
electrophoretic pattern was very faint in both the agarose and the
capillary zone electrophoresis (approximately 0.08 gram/dL).
Total Protein: 5.0 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 2.4 g/dL (3.8-5.8 g/dL) IgG: 794 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.3 g/dL (0.1-0.2 g/dL) IgA: 146 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 1.1 g/dL (0.4-0.9 g/dL) IgM: 53 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.7 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 0.6 g/dL (0.5-1.3g/dL)
63. 63
Case # 6 IgG-Kappa monoclonal gammopathy without manifestation of
myeloma from the bone marrow and clinical symptoms
56 years old male with IgG-Kappa monoclonal gammopathy was
examined for bone marrow aspirate, however the bone marrow
features and other hematopathology assays did not meet the criteria of
multiple myeloma.
Total Protein: 7.9 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 4.8 g/dL (3.8-5.8 g/dL) IgG: 1890 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.1 g/dL (0.1-0.2 g/dL) IgA: 149 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.6 g/dL (0.4-0.9 g/dL) IgM: 26 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.8 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.6 g/dL (0.5-1.3g/dL)
66. 66
Case # 7 IgG-Lambda monoclonal gammopathy (MGUS)
74 years old female was presented at the emergency department with
complaints of dizziness, weakness, and inability to walk. She had lost
weight of over thirty pounds during the last six months. Preliminary
work-up diagnosed cerebrovascular accident. Final diagnoses
included neuropathy secondary to diabetes mellitus Type II, renal
tubular acidosis with chronic kidney disease, chronic obstructive
pulmonary disease, anemia, and hypertension.
Total Protein: 6.4 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 3.5 g/dL (3.8-5.8 g/dL) IgG: 1270 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 307 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.8 g/dL (0.4-0.9 g/dL) IgM: 77 mg/dL ( 46-304 mg/dL)
Beta Globulin: 1.0 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.0 g/dL (0.5-1.3g/dL)
69. 69
Section G Hypogammaglobulinemia and Hypergammaglobulinemia
A decrease or increase in the gamma globulins noticed from serum
protein electrophoresis requires a careful evaluation of the over all
electrophoretic pattern. Both of these situations may be associated
with some clinical condition. The hypogamma globulinemia pattern in a
person over 50 years old and with no aberration in other bands of the
protein electrophoresis pattern may suggest Bence Jones proteinuria.
This requires urine protein electrophoresis and immunofixation to
detect free light chains in urine or alternatively assay of free kappa and
free lambda chains in serum. In certain cases it is possible that there is
decrease in the gamma globulin fraction, however some other fraction,
e.g., the beta1-beta2 globulin fraction is increased due to the migration
of the monoclonal immunoglobulin in this region. This situation also
dictates a follow-up of the patient with immunotyping, etc. The
hypergammaglobulinemia pattern of the serum electrophoresis must
be examined for a superimposed (embedded) monoclonal band, and
thus immunotyping is required to rule out mini-monoclonal
gammopathy.
Case # 1 Polyclonalgammopathy
68 years old female admitted for rectal bleeding, anemia
and infection
Total Protein: 6.5 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 3.2 g/dL (3.8-5.8 g/dL) IgG: 2090 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.1 g/dL (0.1-0.2 g/dL) IgA: 446 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.5 g/dL (0.4-0.9 g/dL) IgM: 119 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.6 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 2.1 g/dL (0.5-1.3g/dL)
75. 75
Case # 3 Apparent Hypogammaglobulinemia with monoclonalgammopathy
65 years old female previously diagnosed for multiple myeloma was
checked for clinical response for chemotherapy, and serum protein
electrophoresis along with quantitative immunoglobulins assay was
performed on out-patient basis.
Total Protein: 7.1 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 4.4 g/dL (3.8-5.8 g/dL) IgG: 1170 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 34 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.7 g/dL (0.4-0.9 g/dL) IgM: 45 mg/dL ( 46-304 mg/dL)
Beta Globulin: 1.5 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 0.2 g/dL (0.5-1.3g/dL)
Comment: In this patient though the gammaglobulin region appears to be low, it is an
aberration as the monoclonal gammaglobulin band (IgG-Kappa) migrates with C3
complement. In this manner the concentration of C3 complement and the total
beta1-beta2 region appears to be increased, which in fact is not.
78. 78
Section H: Oligoclonal Bands in Serum Gammaglobulin Region
The term “oligo” means few (more than one). The gammaglobulin
region of the serum protein electrophoresis very rarely exhibits
oligoclonal bands of different intensities spaced at equal distance or
crowded very close to each other. Sometimes one band is very
prominent as compared to the other bands. In general there is
polyclonal increase in the serum gammaglobulins, and the
concomitant presence of oligoclonal bands are observed in few clinical
conditions, e.g. chronic infection, autoimmune diseases and less
frequently in lymphoproliferative processes. In this situation
immunotyping and/or immunofixation is helpful in the interpretation of
these bands. In cases the clinical condition of the patient requires the
diagnosis of light chain multiple myeloma, it is suggested to perform
urine protein electrophoresis and immunofixation or alternatively the
assay of free kappa and lambda chains in serum.
Case # 1 Oligoclonal bands without any clinical significance
79 years old female with known peripheral neuropathy was evaluated
to rule out lumbar radiculopathy (EMG was negative for radiculopathy).
She was having some gait dysfunction and parathesia in her feet. The
bone X-ray and CT scan were negative. A few laboratory tests were
slightly abnormal (BUN, creatinine, PTT, WBC, and Vitamin B12), but
were not diagnostic of any settled clinical condition.
Total Protein: 6.3 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 3.5 g/dL (3.8-5.8 g/dL) IgG: 763 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 107 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 1.1 g/dL (0.4-0.9 g/dL) IgM: 59 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 0.6 g/dL (0.5-1.3g/dL)
Comments: The oligoclonal bands (indicated by dashed lines) were detected in
both the agarose gel and capillary zone electrophoresis, however, they were not
present on the immunofixation plate and the immunotyping scan. The laboratory
report was signed off (without any prior access to the clinical and other laboratory
data), a) oligoclonal bands present perhaps due to immune complex formation, and
b) Suggest urine protein electrophoresis to rule out Bence Jones proteinuria.
81. 81
Section I: Light Chain Multiple Myeloma
Case #1 Lambda Chains Myeloma
72 years old male (previously known case of light chain
multiple myeloma for more than three years), was admitted
to the hospital due to renal failure, fever, and urinary
tract infection. In the most recent serum specimen, there
was no evidence of a monoclonal band from agarose gel
electrophoresis. The shape of the C3 complement band
was not symmetrical, but diffuse and slanted, and this triggered
serum immunofixation studies. The initial laboratory interpretation
provided to the physician was: " Acute inflammation pattern,
possibility of monoclonal band." "Final interpretation to
follow after immunofixation studies."
Serum immunofixation studies indicated a monoclonal band
due to free lmbda chains, and no precipitation band was
detected for any of the heavy chains (IgG, IgA, IgM, IgD,
and IgE). The immunofixation results using the anti-IgD and
anti-IgE are not displayed on next page.
Total Protein: 5.8 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 2.9 g/dL (3.8-5.8 g/dL) IgG: 522 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.4 g/dL (0.1-0.2 g/dL) IgA: 29 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 1.2 g/dL (0.4-0.9 g/dL) IgM: 6 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.9 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 0.4 g/dL (0.5-1.3g/dL)
87. 87
Section J: Cryoglobulins
Agarose gel serum protein electrophoresis is not the procedure for the detection of
the cryoglobulins, however in some cases due to the employment of cooler
temperatures, one might observe a precipitate at the application point / abnormal
electrophoretic pattern. This formation of the precipitate or abnormal pattern triggers
further investigation, e.g., serum immunofixation. It is the observation of several
lanes with precipitate upon imuunofixation (and repeat immunofixation upon dilution
of the serum and also replacement of one of the antisera with buffer or saline), that
alerts the laboratorian about the possibility of cryoglobulins. It is emphasized that the
confirmation of cryoglobulins in serum requires establishment of the cryocrit, and
immunofixation of the washed cryoglobulin. The assay for Hepatitis C virus antibody,
rheumatoid factor and complement C4 for other diagnostic reasons are also
recommended. Conversely the CZE and the immunotyping by the Sebia System is
performed at 35.5o C, thus the cryoglobulins remain dissolved and do not precipitate,
thus the presence of cryoglobulins are eluded. Quantitative analysis for IgG, IgA,
igM, Free , Free were performed at 37o C, therefore accurate results were obtained.
Case # 1 Brouet Type I
68 year old male, with a known history of several clinical disorders,
e.g., hypentension, anemia, weakness in the legs, deep venous
thrombosis, etc. was admitted to the hospital. Negative for Hepatitis C.
Total Protein: 7.7 g/dL Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 4.2 g/dL (3.8 – 5.8 g/dL) IgG: 1480 mg/dL (751 –1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1 – 02 g/dL) IgA: 160 mg/dL (82 – 453 mg/dL)
Alpha-2 Globulin: 0.8 g/dL (0.4 – 0.9 g/dL) IgM: 943 mg/dL (46 - 304 mg/dL)
Beta Globulin: 1.2 g/dL (0.5 – 1.1 g/dL) Free 56.9 mg//L (3.3 – 19.4 mg/L)
Gamma Globulin: 1.4 g/dL (0.5 – 1.3 g/dL) Free 31.3 mg/L (5.7 – 26.3 mg/L)
Free / 1.82 (0.26 – 1.65)
88. 88
Comments: The serum protein electrophoresis by agarose gel electrophoresis
(HYDRASYS) indicated three restrictions. Serum immunofixation indicated
precipitate lanes in all five sectors (serum protein electrophoresis, IgG, IgA, IgM,
kappa, and lambda). Repeat serum immunofixation after 1:5 dilution of the serum
again indicated the precipitate lanes in all the five areas. The CZE at 35.5o C
indicated no such phenomenon and the elctrophoretic pattern indicated a
monoclonal band in the gamma globulin region. Immunotyping indicated a distinct
IgM-Kappa monoclonal band.
92. 92
Section K : Seven Cases for Interpretation
In this section we have presented seven cases and the reader is
requested to provide the interpretation of the laboratory results.
Case # 1 74 years old male was admitted from the emergency department with
complaints of dizziness and lightheadedness, difficulty in walking and
near syncopal episode. He had a history of atrial fibrillation. The
patient had a history of seizure disorder and was on dilantin. The
patient had a history of coronary artery disease and had angioplasty in
the past. Chest x-ray showed cardiomegaly. Echocardiogram showed
left ventricular ejection fraction of 40%. The patient was seen by a
neurologist and a psychiatrist. The dilantin dose was adjusted in the
therapeutic range, and the patient was sent to the nursing home, and
as a follow-up advised to see the primary physician in two weeks.
Total Protein: 6.9 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 4.3 g/dL (3.8-5.8 g/dL) IgG: 1020 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 317 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.6 g/dL (0.4-0.9 g/dL) IgM: 414 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.7 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.1 g/dL (0.5-1.3g/dL)
Hint: A band of restricted mobility is present between alpha-1 globulin and alpha-2
globulin in both the agarose gel electrophoresis and the capillary zone
electrophoresis. Neither the immunofixation and nor the immunotyping by capillary
zone electrophoresis indicated any monoclonal band. Sometimes in the agarose gel
electrophoresis system (Sebia, HYDRASYS), the beta-lipoprotein band migrates
between the alpha-1 and alpha-2 globulin.
Interpretation please:
95. 95
Case # 2 68 years old female, resident of a nursing home complained of severe
abdominal pain, and was brought to the emergency department. Acute
pancreatitis was the admitting diagnosis. The discharge diagnoses
were pleural effusion, chronic obstructive pulmonary disease,
pneumonia, congestive heart failure, anemia and arteriosclerotic heart
disease, etc.
Test Result Normal Value
Amylase 290 50 – 130 U/L
Lipase 913 23 – 300 U/L
Iron 14 37 – 170 ug/dL
Ferritin 381 8 – 120 ng/mL
WBC 25.6 4.0 – 11.0 k/cumm
C-Reactive Protein 10.9 Less than 1.0 mg/dL
Haptoglobin 379 16 – 200 mg/dL
(component of alpha-2 globulin)
Total Protein: 3.9 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 1.4 g/dL (3.8-5.8 g/dL) IgG: 658 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.3 g/dL (0.1-0.2 g/dL) IgA: 175 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.9 g/dL (0.4-0.9 g/dL) IgM: 27 mg/dL ( 46-304 mg/dL)
Beta Globulin: 0.7 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 0.6 g/dL (0.5-1.3g/dL)
Hints: Apparent increase in transferrin is due to low serum iron in a patient that also
has high acute phase protein (C-Reactive Protein). Another acute phase protein
(haptoglobin) was elevated, however there was no evidence of intracellular
hemolysis or elevation of LD-1 isoenzyme. Patient had lower lobe pneumonia and
was treated with antibiotics.
Interpretation please:
98. 98
Case # 3 86 years old female came to the emergency department from the
nursing home in view of ataxia and slurred speech. She had been
diagnosed for atherosclerotic heart disease and a permanent
pacemaker was inserted past coronary artery bypass graft. She suffers
from degenerative arthritis and chronic joint pain. A final diagnosis of
cerebrovascular accident was made.
Bone marrow examination in 1998 and 2000 were essentially similar
(normocellular with normoblastic erythropoiesis, maturing
granulopoieses and megakaryopoiesis).
Total Protein: 6.6 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 3.5 g/dL (3.8-5.8 g/dL) IgG: 1430 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 396 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.9 g/dL (0.4-0.9 g/dL) IgM: 94 mg/dL ( 46-304 mg/dL)
Beta Globulin: 1.0 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.0 g/dL (0.5-1.3g/dL)
Interpretation please:
101. 101
Case # 4 59 years old male with a past medical history of thrombocytopenia was
admitted for bone marrow examination to rule out myelodysplasia and
assess the megakarocyte population. Intravenous gamma globulin
was administered during hospitalization along with the higher dose of
prednisone.
Total Protein: 6.9 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 4.0 g/dL (3.8-5.8 g/dL) IgG: 876 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 260 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.8 g/dL (0.4-0.9 g/dL) IgM: 314 mg/dL ( 46-304 mg/dL)
Beta Globulin: 1.0 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.0 g/dL (0.5-1.3g/dL)
Interpretation please:
104. 104
Case # 5 80 years old female during routine check-up at her primary physician’s
office presented pale, with hemoglobin of 6.9 gram/dL. She was
admitted on the basis of profound anemia with probable GI blood loss
versus bone marrow hypoplasia-neoplasia. She had a history of more
than ten other diseases. Bone marrow did not demonstrate any
evidence of monoclonality, acute leukemia or non-Hodgkin’s
lymphoma. No diagnostic morphologic evidence of myelodysplastic
syndrome.
Total Protein: 6.7 g/dL (6.4-8.3 g/dL) Immunoglobulins:
Agarose gel electrophoresis: Beckman Coulter IMMAGE:
Albumin: 3.7 g/dL (3.8-5.8 g/dL) IgG: 1050 mg/dL (751-1560 mg/dL)
Alpha-1 Globulin: 0.2 g/dL (0.1-0.2 g/dL) IgA: 472 mg/dL (82-453 mg/dL)
Alpha-2 Globulin: 0.8 g/dL (0.4-0.9 g/dL) IgM: 108 mg/dL ( 46-304 mg/dL)
Beta Globulin: 1.1 g/dL (0.5-1.1 g/dL)
Gamma Globulin: 1.0 g/dL (0.5-1.3g/dL)
Interpretation please:
107. 107
Case # 6A and 6B We have only presented the electrophoretic, immunofixation,
and immunotyping data for these two patients.
Interpretation please: