Immunologic
Laboratory
Tests
Immunologic
Laboratory Tests
❖ Direct Coombs test (DAT)
❖ Indirect Coombs test (IAT)
❖ Immunofluorescence
❖ Western blot
❖ Flow Cytometry
❖ ELISA
❖ Agglutination reactions
Questions to always
ask
• What does it measure ?
• Where does the Ag-Ab
interaction occur?
• How is the Ag-Ab complex
detected?
❑ What does it measure?
❑ Where does Ag-Ab
Direct Coombs interaction occur?
❑ How is the Ag-Ab
detected
Detection of Ab or (Complement) on patient’s RBCs
Examples
aka DAT – Direct Antiglobulin Test • Primarily used for testing in hemolytic
anemia
❑ What does it measure?
❑ Where does Ag-Ab
Direct Coombs interaction occur?
❑ How is the Ag-Ab
detected
• Method
• Patients’ RBCs are retrieved
• Anti-human globulin (AHG/Coombs reagent) is added
• Presence of agglutination is looked for
❑ What does it measure?
Direct Coombs ❑ Where does Ag-Ab
interaction occur?
❑ How is the Ag-Ab
detected
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❑ What does it measure?
❑ Where does Ag-Ab
Indirect Coombs interaction occur?
❑ How is the Ag-Ab
detected
• Detection of Ab to RBCs’ antigens
• aka IAT – Indirect Antiglobulin Test
• Performed as part of pre-transfusion testing
• Ab screening
• Cross match
❑ What does it measure?
❑ Where does Ag-Ab
Indirect Coombs interaction occur?
❑ How is the Ag-Ab
detected
• Method
• Patient’s serum is retrieved
• Donor’s RBCs are added
• Anti-human globulin (AHG/Coombs reagent) is added
• Presence of agglutination is looked for
❑ What does it measure?
❑ Where does Ag-Ab
Indirect Coombs interaction occur?
❑ How is the Ag-Ab
detected
https ://laboratoryi nfo.com/wp-content/uploads/2015/05/Indirect-Coombs-Test.jpg
❑ What does it measure?
Indirect Coombs ❑ Where does Ag-Ab
interaction occur?
❑ How is the Ag-Ab
detected
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❑ What does it measure?
❑ Where does Ag-Ab
Immunofluoresence interaction occur?
❑ How is the Ag-Ab
detected
Detection of a specific Ag in a specimen
Detection of bacterial organisms
Examples include Detection of Ag-Ab complexes
❑ What does it measure?
❑ Where does Ag-Ab
Immunofluoresence - Methods interaction occur?
❑ How is the Ag-Ab
detected
• Direct • Indirect
• Fix specimen on slide • Fix specimen on slide
• Add Ab specific for the • Add Ab specific for the
desired Ag attached to a desired Ag
fluorochrome • Add second Ab attached
• Look for Fluorescence to a fluorochrome
• Look for Fluorescence
Immunofluorescence
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Immunofluorescence
https://bmcmicrobiol.biomedcentral.com/articles/10.1186/1471-2180-11-73#Sec7
❑ What does it measure?
❑ Where does Ag-Ab
Western Blot interaction occur?
❑ How is the Ag-Ab
detected
• Detection of Abs in a patient’s sample
• Examples include
• HIV testing
❑ What does it measure?
❑ Where does Ag-Ab
Western Blot interaction occur?
❑ How is the Ag-Ab
detected
• Methods
• Make a protein suspension of the targeted Ag (e.g. HIV)
• Electrophorese the suspension onto a gel strip
• Apply the patient’s serum to the strip
• Add AHG with an attached enzyme
• Add substrate and look for bands
❑ What does it measure?
❑ Where does Ag-Ab
Western Blot interaction occur?
❑ How is the Ag-Ab
detected
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❑ What does it measure?
❑ Where does Ag-Ab
Flow Cytometry interaction occur?
❑ How is the Ag-Ab
detected
• Characterisation of cell size complexity and Ags
• Examples include
• Diagnosis of leukemia and lymphoma
• Determination of Immune cell counts
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❑ What does it measure?
❑ Where does Ag-Ab
ELISA interaction occur?
❑ How is the Ag-Ab
detected
• Detection of Abs in patients’ sample
• Examples include
• Home pregnancy tests
• HIV
• Cytokines
• Autoantibodies
• Coagulation factors
❑ What does it measure?
❑ Where does Ag-Ab
ELISA interaction occur?
❑ How is the Ag-Ab
detected
• Methods
• Add patient specimen to wells coated with ligand
• Add AHG with attached enzyme
• Add substrate
• Measure colour change
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ELISA - Variations
• Sandwich immunoassay • Radioimmunoassay
• Detects Ag • Detects Ab or Ag
• Coat wells with Ab • Detector is a radioactive substance
• Steps following are same as in • Similar to ELISA or sandwich
ELISA immunoassay
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❑ What does it measure?
❑ Where does Ag-Ab
Agglutination Reactions interaction occur?
❑ How is the Ag-Ab
detected
• Detection of Ag or Ab in patient specimen
• Examples
• Blood typing
• Testing for antibodies to infectious antigens
❑ What does it measure?
❑ Where does Ag-Ab
Agglutination Reactions interaction occur?
❑ How is the Ag-Ab
detected
• Method
• Particles are coated with Ag or Ab
• Patient’s serum is added – containing either Ag or Ab
• Observe if agglutination (clumping) occurs
Agglutination Reactions ❑ What does it measure?
❑ Where does Ag-Ab
interaction occur?
❑ How is the Ag-Ab
detected
https://www.researchgate.net /figu re/Diagram-of-lat ex-agglutin ation-A-Reaction-between-gp43-sensitized-latex-p articl es_fi g6_272184562
Sensitised latex particles
Agglutination Reactions
https://www.researchgate.net/figure/Positive-number-5-and-negative-number-6-reactions-of-qualitative-latex-agglutination_fig1_273063322
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