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
• Detects Ag
• Coat wells with Ab
• Steps following are same as in
ELISA
• Radioimmunoassay
• Detects Ab or Ag
• Detector is a radioactive substance
• Similar to ELISA or sandwich
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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