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Manual of Immunopathological Techniques
Usha, Gyanendra Kumar Sonkar, Sangeeta Singh
CHAPTER 1:
Introduction
INTRODUCTION
COMMON INVESTIGATIONS FOR IMMUNOLOGICAL DISORDERS
Investgations For Natural Immunity
Investigations For Acquired Immunity
Tests For Cell Mediated Immunity
Tests For Humoral Immunity
LABORATORY TESTS FOR ANTIGEN AND ANTIBODY DETECTION
Tests Based on immunoprecipitation
Qualitative AGAR Diffusion Tests
Quantitative AGAR Diffusion Tests
Test Based on Agglutination Reactions
Agglutination Reactions
RADIO IMMUNOASSAY (RIA)
Application
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
Heterogenous ELISA
Indirect ELISA
Sandwich ELISA Test
Homogenous ELISA Tests
Multiplex ELISA
Enzyme-Linked Immunosorbent Spot (ELISpot) Assay
Application of ELISpot
FLOW CYTOMETRY
Main Uses
Immunohistochemistry
Immunofluorescent Method
Indirect Fluorescent
Direct Fluorescent
Fluorescent Microscope
IMMUNOPEROXIDASE TEST
Advantages of Immunoperoxidase Test Over Immunofluorescence Test
MOLECULAR GENETIC TECHNIQUE IN IMMUNOPATHOLOGY
Hybridization Based Test
Dot Blot Hybridization
Reverse Dot or Line Blot Assay
Southern Blot Hybridization
Northern Blot
In Situ Hybridization (ISH)
Fluorescence in Situ Hybridization (FISH) Technique
Chromogenic in Situ Hybridization (CISH) Technique
Comparison of FISH and CISH
Advantage of CISH over FISH
POLYMERASE CHAIN REACTION (PCR)
Precaution
Application of PCR
Variations in PCR
Touchdown PCR
DNA CHIP TECHNOLOGY
CHAPTER 2:
Tests for Cell Mediated Immunity (CMI)
TEST FOR LYMPHOCYTE ACTIVATION AND PROLIFERATION
CYTOKINES
Assessment of Cytotoxic T Cells
T-CELL Rosette Count1
Principle
Material
Reagents
Sheep Red Blood Cells (SRBC)
Alsever's Solution
Cutaneous Hypersensitivity Test
Delayed Type of Hypersensitivity Response (DTH) to Recall Antigens
DTH Response to Purified Protein Derivative (PPD) and Candida Albicans Antigen2
DIAGNOSIS OF TUBERCULOSIS IN HIGH RISK AREA BY DTH TEST3
Mantoux Test
Modified Tuberculin Test
Tine Test
Heaf Test
Advantages and disadvantages of these tests
DELAYED TYPE HYPERSENSITIVITY (DTH) RESPONSE IN LEPROSY (LEPROMIN TEST)
Lepromin Test
Two Types of Reaction is Same
APPLICATION OF DTH RESPONSE IN LEISHMANIA INFECTION
Leishmanin (Montenegro) Test
CONTACT SENSITIZATION WITH 2:4 DINITROCHLOROBENZENE (DNCB) TO TEST CMI4
Principle
CHAPTER 3:
Lymphocytes Typing by Flow Cytometry
OBJECTIVES
METHOD
Isolation of PBMC
Acquisition to Flow Cytometry
MATERIALS REQUIRED
Collection of Blood up to Laboratory
Extraction of PBMC
Staining of Cell
STANDARD OPERATING PROCEDURE
Separation of Cells
Staining of the Cells
Acquiring the Stained Cells
ANTIBODY PANELS
Precautions
INTERPRETATIONS OF CMI TESTS
CHAPTER 4:
Diagnosis of Kala-Azar by Polymerase Chain Reaction (PCR) Technique
OBJECTIVE
METHOD
MATERIALS REQUIRED
Isolation of Genomic DNA from Peripheral Blood
POLYMERASE CHAIN REACTION (PCR)
Evaluation of the PCR Product by Agarose Gel Electrophoresis
STANDARD OPERATING PROCEDURE
Polymerase Chain Reaction
Evaluation of the PCR Product by Agarose Gel Electrophoresis
Precautions
CHAPTER 5:
Test for Myeloma Profile and Qualitative Assessment of Serum Proteins
INTRODUCTION
PAPER ELECTROPHORESIS OF SERUM AND URINE
Principle
Material
Method
Interpretation
IN PLASMA CELL DYSCRASIA
APPENDIX
Light Green Stain Preparation
Other Alternative Buffer for Paper Electrophoresis
Points to be Noted
Disadvantage
Other Uses
TESTS FOR BENCE JONES PROTEINS IN URINE
Principle
Heat Test1
Chemical Test by Cohen and Raducha Technique2
Osgood and Haskins Test3
CHAPTER 6:
Agarose Gel Electrophoresis for Qualitative Assessment of Serum Proteins
INTRODUCTION
PRINCIPLE
MATERIAL
METHOD
Points to be Noted
Adavantages
APPENDIX
Staining Solution
Destaining Solution
CHAPTER 7:
Cellulose Acetate Electrophoresis
PRINCIPLE
METHOD
SOLUTIONS
APPLICATIONS
POINTS TO BE NOTED
ADVANTAGES
CHAPTER 8:
Agar Gel Immunoelectrophoresis for Typing of Myeloma
PRINCIPLE
EQUIPMENT
REAGENTS
MICROSLIDES
PROCEDURE
PREPARATION OF AGAR GEL SLIDES
Electrophoresis
Application of antisera
Washing of Slides
Drying of Slides
Staining of the Slides
Interpretation
CHAPTER 9:
Micro Agar Gel Double Diffusion Technique
PRINCIPLE
REAGENTS
PREPARATION OF AGAR PLATE
Filling of Wells
Washing of the Slides
Drying of Slides
Staining of Slides
Uses
CHAPTER 10:
Counter Current Immunoelectrophoresis
PRINCIPLE
LABORATORY PROCEDURE
CHAPTER 11:
Single Radial Immunodiffusion and Immunoturbidometric Methods for the Estimation of Immunoglobulins
INTRODUCTION
SINGLE RADIAL IMMUNODIFFUSION (SRID)
Principle
Standard Serum for Serum Immunoglogulins
Preparation of Phosphate Buffer Solution pH 7.4
Preparation of Agar Solution
Preparation of Antisera
Preparation of Immnodiffusion Plates
Placement of Antigen in Immunodiffusion Plate
Incubation of Immunodiffusion Plates
Reading of the Antigen-Antibody Diffusion Rings
Plotting of the Standard Time and Calculate the Immunoglobulin Concentration of the Sample
Points to be Noted
IMMUNOTURBIDOMETRIC METHOD FOR ESTIMATION OF IMMUNOGLOBULINS
Principle
Estimation of Serum Immunoglobulin A (IgA)
Estimation of Serum Immunoglobulin G (IgG)
Estimation of Serum Immunoglobulin M (IgM)
Precautions in Turbidometric Test
COMPARISON OF SRID AND TURBIDOMETRY TEST FOR ESTIMATION OF IMMUNOGLOBULINS AND COMPLEMENTS
Interpretation (Table 11.5 and 11.6)
Decrease in Immunoglobulin
Increase in Immunoglobulin
Polyclonal increase in Gammaglobulin
CHAPTER 12:
Estimation of Complement C3, C4 and Hemolytic Assay for Serum Complement
ESTIMATION OF COMPLEMENT C3 AND C4
Estimation of Complement Components
Collection of Blood
Single Radial Immunodiffusion Method for Estimation of Complement
IMMUNOTURBIDOMETRIC ASSAY FOR DETERMINATION OF COMPLEMENTS C3 AND C4
Principle of the Test
Estimation of Complement Component C3
Estimation of Complement Component C4
HEMOLYTIC ASSAYS FOR COMPLEMENT ESTIMATION
CH 50 Hemolytic Assay
Preparation of Rabbit Antibody Coated Sheep Erythrocytes (EA) for CH50 Assay
AH 50 Hemolytic Assay
APPENDIX
Alsever's Solution
Preparation of Buffers for Estimation of Complements by Classical Pathway
Stock Solutions
Preparation of Buffers for Estimation of Complements by Alternate Pathway
Stock Solution
Working Solution
Interpretation
CHAPTER 13:
Demonstration of Immune Complexes and Complements by Indirect Immunoperoxidase Method
INTRODUCTION
PRINCIPLE OF THE TEST
PROCEDURE
INTERPRETATION
DISADVANTAGE
ADDITIONAL NOTES
CHAPTER 14:
Test for Autoimmune Diseases
DEMONSTRATION OF LUPUS ERYTHEMATOSUS (LE) CELLS IN PERIPHERAL BLOOD1
Principle
Procedures
Method of Leishman Staining2
Preparation of Leishman Stain
Sorensen's Buffer
Working Buffer
ANTINUCLEAR ANTIBODY (ANA) BY INDIRECT IMMUNOFLUORESCENT TEST
Introduction
Principle
Substrate
Preparation of Frozen Section of Composite Block of Rat Liver, Kidney and Stomach
Procedure for Staining
Interpretation
Speckled
Nucleolar Pattern
Points to be Noted
INDIRECT FLUORESCENT TEST FOR AUTOIMMUNE CHRONIC HEPATITIS, PRIMARY BILIARY CIRRHOSIS, ATROPHIC GASTRITIS AND GOOD PASTEUR'S SYNDROME
Autoimmune Hepatitis
Primary Billiary Cirrhosis
INDIRECT IMMUNOFLUORESCENT TEST FOR AUTOIMMUNE HEPATIC DISEASE
Substrate
Principle
Dilution of Patient Serum
Staining
Antismooth Muscle Antibody (ASMA) Test
Interpretation
ASMA Positive Conditions
Antiliver/Kidney Microsomal Antibody (Anti-LKM Ab)
Interpretation
Points to be Noted
Antimitochondrial Antibody
Substrate
Procedure and Principle
Dilution Sample
Interpretation
Utility of Antimitochondrial Antibody
Points to be Noted
Antiparietal Cell Antibody (APCA)
Substrate
Interpretation
Utility of APA
Antiglomerular Basement Membrane Antibody (AGBMA)
Substrate
Method
Interpretation
Utility of this Test
ANTINEUTROPHIL CYTOPLASMIC ANTIBODY (ANCA) TEST
Indirect Fluorescence Test (IFT) for ANCA
Principle of the Test
Interpretation
Points to be noted
DETECTION OF ANTIKERATIN ANTIBODY, ANTIENDOMYSIAL ANTIBODY AND ANTIDESMOSOMAL (DESMOGLEIN) ANTIBODY BY INDIRECT FLUORESCENT METHOD
Technique
Method of Staining
Interpretation
Advantage of Indirect Immunofluorescent Test on Monkey Esophagus
CHAPTER 15:
Test for Autoantibodies by Enzyme-Linked Immunosorbent Assay Technique
ENZYME-LINKED IMMUNOSORBENT ASSAY FOR IGM RHEUMATOID FACTOR (RF)1
Material Required
Procedure
CHAPTER 16:
Acid Fuchsin Orange G in Kidney Biopsy for Demonstration of Immune Complexes
ACID FUCHSIN ORANGE G (AFOG) STAINING
Technique
Result
Advantage
APPENDIX
Preparation of Stains
CHAPTER 17:
Detection of Tumor Markers In Paraffin-Embedded Sections By Immunohistochemistry
INDIRECT IMMUNOPEROXIDASE STAINING FOR C-ERB B2 ANTIGEN IN TISSUE SECTION
Principle
Indirect Immunoperoxidase Method
Interpretation
Points to be Noted
P53 ANTIGEN DETECTION IN PARAFFIN-EMBEDDED SECTIONS
Immunohistochemistry for P53 Demonstration
Additional step of pretreatment is necessary for P53 antigen retrieval
Interpretation
APPENDIX FOR IMMUNOHISTOCHEMISTRY
Buffers for Antigen Retrieval
Washing Buffer
CHAPTER 18:
Detection of Circulating Immune Complexes in the Serum
INTRODUCTION
PRINCIPLE
METHOD
PEG INDEX: MULTIPLY READING OF ABSORBANCE WITH 100
THRESHOLD VALUE
APPENDIX
CHAPTER 19:
Demonstration of Immune Complexes, Antibodies and Complements in Renal Biopsy by Immunofluorescence
DIRECT IMMUNOFLUORESCENT TEST
Material
Method
INDIRECT IMMUNOFLUORESCENT TEST
Interpretation
Other Utility of Indirect Immunofluorescent Test in Renal Biopsy
CHAPTER 20:
Human Leukocyte Antigen Typing and Crossmatch Test in Solid Organ Transplantation
MAJOR HISTOCOMPATIBILITY COMPLEX
HLA Class I Antigen
Functions of HLA Class IA (A, B, C) and Class IB (E, F, G) Antigens
HLA Class II Antigens
Functions of HLA class II Antigens
Other Genes in MHC Class II Region
MHC Class III Region
INHERITANCE OF HLA GENES
TYPING OF HLA CLASS I (A, B, C) ANTIGENS
Principle
Materials Required
Preparation of Typing Trays
Procedure for Class I Typing
TYPING OF HLA CLASS II (DR, DQ) ANTIGENS
Principle
Materials Required
Procedure for Class II Typing
Interpretation of Result
CROSS MATCH TEST FOR RENAL TRANSPLANTATION
Principle
Procedure
Interpretation
DETERMINATION OF HLA CLASS II BY IMMUNOBEAD METHOD
Principle
Materials
Isolation of Cells
Tissue Typing Procedure
Interpretation of Result
APPENDIX
Staining Solution
Working Solution
Blocking Solution
Advantages of Immunobead Method
Disadvantages of Immunobead Method
CHAPTER 21:
Human Leukocyte Antigen B27 Typing by Polymerase Chain Reaction Method
PRINCIPLE
METHOD OF DNA EXTRACTION
Setting up Reaction
GEL ELECTROPHORESIS OF THE PCR PRODUCTS
Ineterpretation
Precautions
APPENDIX
SIGNIFICANCE OF HUMAN LEUKOCYTE ANTIGEN (HLA) TYPING
HUMAN LEUKOCYTE ANTIGEN IN NORMAL POPULATION
CHAPTER 22:
Fluorescence In Situ Hybridization: Methods and Applications
INTRODUCTION
What is FISH?
PROBES
Probe Types
Locus Specific Probes
Centromeric Repeat Probes
Telomere Specific Probes
Whole Chromosome Painting Probes
Single, Double and Multiple Color FISH Probes
Single Color Probes
Dual Color Probes
Multiple Color Probes
FISH ON PARAFFIN-EMBEDDED TISSUE SECTIONS
Preparing Specimen Slides
Deparaffinizing Slides
Pretreating Slides
Treating Slides with Protease
Chromosome Denaturation and Probe Hybridization
Posthybridization Steps
FISH ON BLOOD SMEAR
INTERPRETING FISH SIGNALS
TIPS AND TROUBLESHOOTING
APPLICATIONS
CHAPTER 23:
Detection of Cytokines—Interleukin (IL)-6, Interleukin (IL)-8 and Tumor Necrosis Factor (TNF)-α
DETECTION OF IL-6 BY ELISA TEST
Principal
Procedure
APPENDIX FOR ELISA TEST
CHAPTER 24:
Western Blot Tests for Detection of Antibody to Extractable Nuclear Antigen
SEPARATION OF EXTRACTABLE NUCLEAR ANTIGENS FROM RABBIT THYMUS
Rabbit Thymus Extract (RTE)
PREPARATION OF SDS-PAGE ELECTROPHORESIS
PREPARE REAGENTS IN DOUBLE DISTILLED WATER
STAINING PROCEDURES
Points to be Noted
APPENDIX
CHAPTER 25:
Detection of Antithyroglobulin Antibodies by Hemagglutination Method
HYPOTHYROIDISM
DETECTION OF ANTITHYROGLOBULIN ANTIBODIES (ATG Ab)
Principle
Material
Preparation of Human Thyroglobulin
Results
INDIRECT FLUORESCENT TEST FOR ANTITHYROGLOBULIN AND ANTITHYROID PEROXIDASE ANTIBODY
Material and Method
Interpretation
Points to be Noted
GELATIN AGGLUTINATION METHOD FOR ANTITHYROID MICROSOMAL ANTIBODIES
CHAPTER 26:
Distillation and Demineralization of Water
WATER DISTILLATION APPARATUS
Principle
Components
Working
Applications
Precautions
Additional Information
Glass Distilled Water
DEIONIZER (DEMINERALIZER)
Specific Features
General Features
Quality Control Devises
CHAPTER 27:
Raising of Polyclonal Anti-Human Anti-IgG in Rabbit
METHOD
BLEEDING OF RABBITS
POINTS TO BE NOTED
INDEX
TOC
Index
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