Professional Documents
Culture Documents
Laboratory
Tests
Immunologic
Laboratory Tests
❖ Direct Coombs test (DAT)
❖ Immunofluorescence
❖ Western blot
❖ Flow Cytometry
❖ ELISA
❖ Agglutination reactions
Questions to always
ask
• What does it measure ?
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
https://www.slideshare.net/dream10f/immunologic-laboratorytests
❑ 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://www.slideshare.net/dream10f/immunologic-laboratorytests
❑ What does it measure?
❑ Where does Ag-Ab
Immunofluoresence 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
https://www.slideshare.net/dream10f/immunologic-laboratorytests
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
• 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
https ://www.slideshare.net/dream10f/immunologic-laboratorytests
❑ What does it measure?
❑ Where does Ag-Ab
Flow Cytometry 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
https://www.slideshare.net/dream10f/immunologic-laboratorytests
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
https://www.slideshare.net/dream10f/immunologic-laboratorytests
❑ 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/figure/Positive-number-5-and-negative-number-6-reactions-of-qualitative-latex-agglutination_fig1_273063322
https://www.slideshare.net/dream10f/immunologic-laboratorytests