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Hyperimmunoglobulinemia Abnormally high blood levels of immunoglobulins. Abnormally high blood levels of immunoglobulins. Polyclonal immunoglobulins Polyclonal

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  • Hyperimmunoglobulinemia Abnormally high blood levels of immunoglobulins. Abnormally high blood levels of immunoglobulins. Polyclonal immunoglobulins Polyclonal immunoglobulins Monoclonal immunoglobulins Monoclonal immunoglobulins
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  • Monoclonal immunoglobulins An uncontrolled proliferation of a single clone of plasma cells. An uncontrolled proliferation of a single clone of plasma cells. Electrophoresis of serum appears a narrow band or spike . Electrophoresis of serum appears a narrow band or spike . This protein is called paraprotein or This protein is called paraprotein or monoclonal protein M protein . monoclonal protein M protein . Paraproteinemia Paraproteinemia.
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  • Theory of Monoclonal Detection Immunoglobulins have a broad range of isoelectric points Immunoglobulins have a broad range of isoelectric points Results in a very diffuse band when subjected to electrophoresis Results in a very diffuse band when subjected to electrophoresis Each monoclonal antibody has a unique isoelectric point - all molecules of any given monoclonal antibody have the same isoelectric point Each monoclonal antibody has a unique isoelectric point - all molecules of any given monoclonal antibody have the same isoelectric point Results in a sharp distinct band when subjected to electrophoresis Results in a sharp distinct band when subjected to electrophoresis
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  • Monoclonal gammopathies A monoclonal serum (or urine) protein without clinical manifestations of plasma cell dyscrasia. A monoclonal serum (or urine) protein without clinical manifestations of plasma cell dyscrasia. Elevated levels of a single class and type of immunoglobulin. Elevated levels of a single class and type of immunoglobulin.
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  • Laboratory Methods and Testing Strategies for Detection and Monitoring Monoclonal gammopathies Serum viscosity Serum viscosity Radial immunodiffusion RID Radial immunodiffusion RID Nepherometry Nepherometry Serum electrophoresis SEP Serum electrophoresis SEP Immunoelectrophoreis IEP Immunoelectrophoreis IEP Immunofixation electrophoresis IFE Immunofixation electrophoresis IFE
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  • Monoclonal gammopathies Multiple myeloma Multiple myeloma Waldenstrom s macroglobulinemia Waldenstrom s macroglobulinemia Heavy chain diseases Heavy chain diseases Light chain diseases Light chain diseases Chronic lymphocytic leukemia ( CLL ) Chronic lymphocytic leukemia ( CLL )
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  • Multiple myeloma IgG is the most common form. IgG is the most common form. Between the ages of 40 and 70 years. Between the ages of 40 and 70 years. The male-to-female ratio is 1.6 1 The male-to-female ratio is 1.6 1 The causes of death are overwhelming infection (sepsis) and renal insufficiency. The causes of death are overwhelming infection (sepsis) and renal insufficiency.
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  • Multiple myeloma Signs and Symptoms Skeletal abnormalities Skeletal abnormalities --disseminated destruction of the skeleton. --disseminated destruction of the skeleton. --bone pain, consist of punched-out lytic --bone pain, consist of punched-out lytic areas, osteoporosis, and fractures. areas, osteoporosis, and fractures. Renal disorders Renal disorders --chronic renal failure and proteinuria are --chronic renal failure and proteinuria are common developments. common developments. --over half of all patients excreting abnormal --over half of all patients excreting abnormal amounts of Bence-Jones (BJ ) protein. amounts of Bence-Jones (BJ ) protein. Anemia or abnormal bleeding. Anemia or abnormal bleeding.
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  • Multiple myeloma Immunologic Manifestations IL-6 is the major growth factor. (also function as osteoclast-activating factors.) IL-6 is the major growth factor. (also function as osteoclast-activating factors.) Incessant synthesis of a dysfunctional single monoclonal protein or of Ig chains of fragments, with inhibits the synthesis of normal functional antibody. Incessant synthesis of a dysfunctional single monoclonal protein or of Ig chains of fragments, with inhibits the synthesis of normal functional antibody. M component is usually found in serum, urine or both. M component is usually found in serum, urine or both.
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  • Multiple myeloma
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  • Bence-Jones proteins Single-peptide chains with a molecular weight of 20000 or 22000, but dimerization occurs. Single-peptide chains with a molecular weight of 20000 or 22000, but dimerization occurs. Monoclonal kappa or lambda Ig light chains. Monoclonal kappa or lambda Ig light chains. Detected in serum, urine, or both. Detected in serum, urine, or both. Related to filtration, resorption, or catabolism of the protein by the kidney. Related to filtration, resorption, or catabolism of the protein by the kidney. Insoluble near 60-70 , then resoulubilize at 80 Insoluble near 60-70 , then resoulubilize at 80
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  • Waldenstrom s macroglobulinemia An indolent B-cell disorder characterized by excess secretion of IgM paraprotein. An indolent B-cell disorder characterized by excess secretion of IgM paraprotein. Involvement of the bone marrow by lymphoplasmacytic cells. Involvement of the bone marrow by lymphoplasmacytic cells. Age-specific incidence (60 to 64 years) Age-specific incidence (60 to 64 years)
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  • Waldenstrom s macroglobulinemia Signs and Symptoms High levels of IgM macroglobulin. High levels of IgM macroglobulin. IgM is precipitabled at cold temperatures. (when IgM levels exceed 2 to 3 g/dL) IgM is precipitabled at cold temperatures. (when IgM levels exceed 2 to 3 g/dL) Hyperviscosity Hyperviscosity Bleeding, weight loss, and infection. Bleeding, weight loss, and infection. Hepatomegaly, splenomegaly, and lymphadenopathy. (40%) Hepatomegaly, splenomegaly, and lymphadenopathy. (40%)
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  • Waldenstrom s macroglobulinemia Signs and Symptoms Hematologic abnormalities Hematologic abnormalities --forming bruising, purpura, bleeding --forming bruising, purpura, bleeding --quantities of platelets is normal, but --quantities of platelets is normal, but platelet function is abnormal. platelet function is abnormal. --IgM produces an increased erythrocyte --IgM produces an increased erythrocyte sedimentation rate. (rouleaux formation) sedimentation rate. (rouleaux formation) Blurred vision is abnormal Blurred vision is abnormal Glomerular lesions are the predominant form of real injury. Glomerular lesions are the predominant form of real injury. Congestive heart failure. Congestive heart failure.
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  • Waldenstrom s macroglobulinemia Immunologic Manifestations Accumulation of monoclonal IgM in the circulationg plasma and of plasmacytoid lymphocytes in the bone marrow. Accumulation of monoclonal IgM in the circulationg plasma and of plasmacytoid lymphocytes in the bone marrow. Cryoglobulin can be detected Cryoglobulin can be detected --precipitate or gel when cooled to 0c --precipitate or gel when cooled to 0c --fix complement and initiate an --fix complement and initiate an inflammatory reaction. inflammatory reaction. Occasionally, the macroglobulin is cryoprecipitable and capable of cold-induced, anti-i-mediated agglutination of red cells. Occasionally, the macroglobulin is cryoprecipitable and capable of cold-induced, anti-i-mediated agglutination of red cells.
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  • Heavy-Chain Disease The presence of monoclonal proteins composed of the heavy-chain portion of the Ig molecule. The presence of monoclonal proteins composed of the heavy-chain portion of the Ig molecule. Alpha heavy-chain disease is the most frequent. Alpha heavy-chain disease is the most frequent.
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  • Light-Chain Disease 10%~15% of monoclonal gammopathies. 10%~15% of monoclonal gammopathies. Only kappa or lambda monoclonal light chains or Bence Jones proteins are produced. Only kappa or lambda monoclonal light chains or Bence Jones proteins are produced.
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  • Laboratory Methods and Testing Strategies for Detection and Monitoring Serum viscosity Serum viscosity Radial immunodiffusion RID Radial immunodiffusion RID Nepherometry Nepherometry Serum electrophoresis SEP Serum electrophoresis SEP Immunoelectrophoreis IEP Immunoelectrophoreis IEP Immunofixation electrophoresis IFE Immunofixation electrophoresis IFE
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  • Serum viscosity Elevated serum viscosity (SV) levels commonly occur in patients with Waldenstrom s macroglobulinemia (WM )as a result of excess serum IgM production. Elevated serum viscosity (SV) levels commonly occur in patients with Waldenstrom s macroglobulinemia (WM )as a result of excess serum IgM production. The normal SV ranges from 1.4-1.8 units. The normal SV ranges from 1.4-1.8 units. hyperviscosity : viscosity greater than 5 units. hyperviscosity : viscosity greater than 5 units.
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  • Nepherometry: Quantitative Immunoglobulins (G,A,M) ( 0) ( 0) PEG6000 PEG6000
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  • Serum Electrophoresis (SEP) excellent low cost screen for initial discovery of gammopathies excellent low cost screen for initial discovery of gammopathies If Negative, go no further If Negative, go no further If positive, order Immunoelectrophoresis (IEP) or Immunofixation (IFE) If positive, order Immunoelectrophoresis (IEP) or Immunofixation (IFE) Under SEP test instructions, can state Do IEP if abnormal gamma region detected Under SEP test instructions, can state Do IEP if abnormal gamma region detected Good monitoring tool - Can quantitate grams in monoclonal peak Good monitoring tool - Can quantitate grams in monoclonal peak
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  • Serum electrophoresis (SEP) Thin layer agarose - High resolution and Cellulose acetate - 5 zone resolution Thin layer agarose - High resolution and Cellulose acetate - 5 zone resolution To trained eye 5 Zone sensitivity equal to IFE To trained eye 5 Zone sensitivity equal to IFE Scaning by densitometry yields quantitative measure of monoclonal peak relative to total protein Scaning by densitometry yields quantitative measure of monoclonal peak relative to total protein
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  • Immunoelectrophoresis 1. Identifies Ig Class: Qualitative not quantitative 2.Not good monitoring tool 3. Presently, if IEP is questionable or inconclusive, IFE is automatically performed 4. Being replaced by IFE
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  • Immunofixation Identifies Ig Class Identifies Ig Class Qualitative not quantitative Qualitative not quantitative Not good monitoring tool Not good monitoring tool More sensitive than IEP at identifying monoclonal gammopathie More sensitive than IEP at identifying monoclonal gammopathie
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  • Immunodeficiency Immunodeficiency
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  • Classification Primary vs. secondary immunodeficiency Primary vs. secondary immunodeficiency Antibody (B cell) immunodeficiency diseases Antibody (B cell) immunodeficiency diseases T cell immunodeficiency diseases T cell immunodeficiency diseases Combined B cell and T cell immunodeficiency diseases Combined B cell and T cell immunodeficiency diseases Phagocyte dysfunction diseases Phagocyte dysfunction diseases Complement abnormalities Complement abnormalities
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  • B cell immunodeficiency diseases
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  • X-linked agammaglobulinemia Definition: An inherited disorder characterized by very low levels of protective immunoglobulins; affected people develop repeated bacterial infections. An inherited disorder characterized by very low levels of protective immunoglobulins; affected people develop repeated bacterial infections. IgG
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  • XLA involves an absence of mature,peripheral B cells.
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  • XLA is now known to be largely due to a defect in a gene encoding a tyrosine kinase - btk (Brutons tyrosine kinase). 90-95% of patients with XLA have a mutation in btk. Btk is involved in activation of PLC during B cell activation (cooperatively with Syk and BLNK)
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  • X-linked hyper IgM syndrome(CD40L deficiency) Definition: Definition: Normal to elevated serum levels of IgM Normal to elevated serum levels of IgM 150~1000mg/dl, reduced IgG and IgA 150~1000mg/dl, reduced IgG and IgA Defective CD154 (CD40L) on T cells, defective isotype switching to IgG and IgA isotypes Defective CD154 (CD40L) on T cells, defective isotype switching to IgG and IgA isotypes Limited IgM responses to antigens Limited IgM responses to antigens
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  • Selective deficiency of IgA Definition: Low levels of or complete absence of IgA (
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  • Selective IgG subclasses deficiency Definition: These patients are susceptible to bacterial infections. These patients are susceptible to bacterial infections. deficiency of one ore more IgG subclass deficiency of one ore more IgG subclass IgG1 Response to protein antigen IgG2 Response to polysaccharide IgG3 Response to protein antigen IgG4 High in allergic dz;block IgE response
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  • T cell immunodeficiency diseases
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  • Digeorges Syndrome Definition: Absence of T cell function in peripheral blood due to defective development of 3 rd and 4 th pharyngeal pouches in Absence of T cell function in peripheral blood due to defective development of 3 rd and 4 th pharyngeal pouches in hypoplasia of the thymus hypoplasia of the thymus Athymic mouse or rat model Athymic mouse or rat model Alternative Names: catches 22 catches 22
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  • Combined B cell and T cell immunodeficiency diseases
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  • Severe combined immunodeficiency disease Definition: Over half of all SCID cases are due to a defect in a gene on the X chromosome. Over half of all SCID cases are due to a defect in a gene on the X chromosome. The gene involved encodes the gamma subunit of the IL-2 receptor. The gene involved encodes the gamma subunit of the IL-2 receptor. This is also the gamma subunit of the receptors for (at least): This is also the gamma subunit of the receptors for (at least): - IL-4 - IL-7 - IL-9 - IL-15
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  • Wiskott-Aldrich syndrome (WAS) Definition: 1 in 250,000 births, 1 in 250,000 births, normal T cell numbers with reduced functions, normal T cell numbers with reduced functions, IgM are reduced but IgG levels are normal. IgM are reduced but IgG levels are normal. Both IgA and IgE levels are elevated. Both IgA and IgE levels are elevated. Syndrome develop severe eczema, petechia (due to platelet defect and thrombocytopenia). Syndrome develop severe eczema, petechia (due to platelet defect and thrombocytopenia). They respond poorly to polysaccharide antigens and are prone to pyogenic infection. They respond poorly to polysaccharide antigens and are prone to pyogenic infection. defect in WAS protein (WASP) which binds Cdc42, a GTP binding protein known to regulate cytoskeletal reorganization and important for collaboration of T and B cells defect in WAS protein (WASP) which binds Cdc42, a GTP binding protein known to regulate cytoskeletal reorganization and important for collaboration of T and B cells
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  • Initial Screening evaluation
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  • Antibody-mediated immunity IgG IgM IgA level IgG IgM IgA level Anti-A anti-B antibody titer Anti-A anti-B antibody titer Specific antibody titer Specific antibody titer
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  • Cell-mediated immunity Complete blood count (CBC) Complete blood count (CBC) Different count Different count Total T cells and T cell subsets Total T cells and T cell subsets Skin test Skin test
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  • Therapy Intravenous immunoglobulin (IVIG) Intravenous immunoglobulin (IVIG) Bone marrow transplantation Bone marrow transplantation Gene therapy Gene therapy
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  • Phagocytic dysfunction diseases susceptible to bacterial but not viral infection susceptible to bacterial but not viral infection extrinsic defects: deficiency of antibody and complement, or immunosuppression extrinsic defects: deficiency of antibody and complement, or immunosuppression intrinsic defects: enzyme or adhesion molecules defect. intrinsic defects: enzyme or adhesion molecules defect. Acquired vs. congenital phagocytic defect Acquired vs. congenital phagocytic defect
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  • Phagocytic dysfunction diseases Neutropenia Neutropenia Disorders of adhesion (Leukocyte adhesion deficiency, LAD) Disorders of adhesion (Leukocyte adhesion deficiency, LAD) inherited deficiency of CR3 receptor and LFA-1 (integrin) inherited deficiency of CR3 receptor and LFA-1 (integrin) Disordrs of mobility Disordrs of mobility Lazy leukocyte syndrome Lazy leukocyte syndrome Disorders of chemotaxis Disorders of chemotaxis Chediak-Higashi syndrome (CHS) Chediak-Higashi syndrome (CHS) Pelger-Huet anomaly Pelger-Huet anomaly Hyper IgE (Jobs) syndrome Hyper IgE (Jobs) syndrome
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  • Disorder of adhesion : CR3 deficiency Complement receptor 3 deficiency Complement receptor 3 deficiency Autosomal recessive trait Autosomal recessive trait Present as a leukocyte adhesion deficiency Present as a leukocyte adhesion deficiency Marked abnormalities of adherence related functions Marked abnormalities of adherence related functions Lack an intravascular marginating pool Lack an intravascular marginating pool
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  • Disorder of adhesion: Lazy leukocyte syndrome Defective leukocyte locomotion Defective leukocyte locomotion
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  • Granular disorders Chediak Higashi Syndrome Chediak Higashi Syndrome Myeloperoxidase Deficiency Myeloperoxidase Deficiency Specific Granule Deficiency Specific Granule Deficiency
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  • Chediak Higashi Syndrome Autosomal recessive Autosomal recessive Have giant granules display delay killing of ingested bacteria Have giant granules display delay killing of ingested bacteria Mutation in a protein involved in the regulation of intracellular trafficking Mutation in a protein involved in the regulation of intracellular trafficking
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  • Chediak Higashi Syndrome
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  • Myeloperoxidase Deficiency Autosomal recessive trait on chromosome 17 Autosomal recessive trait on chromosome 17 MPO negative MPO negative Not usually associated with increased infection Not usually associated with increased infection Mild to marked defect in bacteria-killing ability of neutrophils Mild to marked defect in bacteria-killing ability of neutrophils
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  • Disorders of oxidative metabolism Chronic granulomatosus disease (CGD) Chronic granulomatosus disease (CGD) Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency)
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  • Chronic granulomatosus disease (CGD) X-linked or autosomal recessive X-linked or autosomal recessive NADPH oxidase defect NADPH oxidase defect NBT negative NBT negative
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  • G6PD deficiency Glucose-6-phosphate dehydrogenase deficiency Glucose-6-phosphate dehydrogenase deficiency X-linked X-linked Unable to generate enough NADPH Unable to generate enough NADPH
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  • Nitro blue tetrazolium test (NBT) Principle: Fresh heparinized blood incubated with NBT dye, allowing phagocytosis to occur. Following ingestion, NBT reacts with superoxide and is reduced to a black substance called formazan. Principle: Fresh heparinized blood incubated with NBT dye, allowing phagocytosis to occur. Following ingestion, NBT reacts with superoxide and is reduced to a black substance called formazan. Interpretation: normal > 14%. Stimulated (add latex particles) results are usually 2 to 3 times the normal range. Interpretation: normal > 14%. Stimulated (add latex particles) results are usually 2 to 3 times the normal range. Chemiluminescence, and Flow cytometry have been used to replace NBT Chemiluminescence, and Flow cytometry have been used to replace NBT
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  • Positive Reduced NBT is seen as dark-blue crystals Negative Unstimulated neutrophils, or stimulated neutrophils from patients who have CGD fail to reduce NBT http://www.immunologyclinic.com/figure.asp?chap=03&fig=6-12a