Immunologic Laboratory Tests
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Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot • Flow cytometry
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Things to remember about each test
• What does it measure? • Where does the Ag-Ab interaction occur? • How is the Ag-Ab complex detected?
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Immunologic Lab Tests Outline • Agglutination reactions
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Agglutination Reactions: Purpose
• Detection of Ag or Ab in patient specimen • Examples: •
blood typing
•
testing for antibodies to infectious agents
•
testing for Hemophilus influenzae type B capsular antigen in CSF
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Agglutination Reactions: Method
• Use particles coated with Ag or Ab • Add patient’s serum (containing Ab or Ag) • See if particles clump
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Agglutination Reactions: Interpretation
Clumping = patient has the antibody (or antigen)
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negative
positive
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Immunologic Lab Tests Outline • Agglutination reactions • DAT
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Direct Antiglobulin Test: Purpose
• Detection of Ab (or C’) on patient’s red cells • Also called the direct Coombs Test • Performed in patients with hemolytic anemia
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DAT: Method
• Use patient’s red cells (coated with Ab) • Add anti-human globulin (AHG) (Coombs reagent) • Look for agglutination
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patient red cells
+
AHG
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=
agglutination
DAT: Interpretation
Clumping = patient red cells are coated with antibody and/or complement
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Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT
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Indirect Antiglobulin Test: Purpose • Detection of antibodies to red cell antigens • Also called the indirect Coombs Test • Performed as part of pre-transfusion testing •
antibody screen
•
cross-match
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IAT: Method
• Use patient serum (containing Ab) • Add donor RBCs (coated with Ag) • Add anti-human globulin (Coombs reagent) • Look for agglutination
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patient Ab
+
donor RBC
=
Ab-coated + donor RBC
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AHG = agglutination
ANTIBODY SCREENING patient serum (without red cell Ab)
patient serum (with red cell Ab)
reagent RBC (with red cell Ag)
reagent RBC (with red cell Ag)
AHG
AHG
no agglutination (negative test)
agglutination (positive test) www.indiandentalacademy.com
IAT: Interpretation
Clumping = patient has an antibody to the donor (or reagent) red cells
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Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence
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Immunofluorescence: Purpose • Detection of a specific antigen in a specimen • Examples: •
detection of bacterial organisms
•
detection of antigen-antibody complexes
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Immunofluorescence: Methods Direct
Indirect
• Fix specimen on slide
•
Fix specimen on slide
• Add antibody specific for
•
Add antibody specific for the desired antigen
•
Add second antibody
•
Look for fluorescence
the desired antigen
• Look for fluorescence
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Immunofluorescence: Interpretation
Fluorescence = patient has the antigen
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Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA
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Enzyme-Linked Immunosorbent Assay: Purpose
• Detection of antibodies in patient specimen • Examples: •
home pregnancy tests
•
HIV tests
•
tests for some coagulation factors, cytokines, and autoantibodies
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ELISA: Method
• Add patient specimen to well coated with ligand • Add AHG with enzyme attached • Add substrate • Measure color change
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ELISA: Interpretation
Color change = patient has the antibody
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ELISA: Variations • Sandwich immunoassay •
detects antigen (not antibody)
•
coat well with antibody
•
rest is like ELISA
• Radioimmunoassay •
detects antibody or antigen
•
detector is a radioactive substance
•
otherwise like ELISA or sandwich immunoassay www.indiandentalacademy.com
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Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot
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Western Blot: Purpose
• Detection of antibodies in patient specimen • Most common example: HIV test
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Western Blot: Method • Make a protein suspension of the target of the antibody you’re looking for (e.g., HIV)
• Electrophorese the suspension onto a little gel strip • Apply the patient’s specimen (containing antibodies) to the strip
• Add AHG that has an enzyme attached • Add substrate and look for bands
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Western Blot: Interpretation
Bands on strip = patient has antibodies to corresponding proteins
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Enough bands = patient is “positive� www.indiandentalacademy.com
Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot • Flow cytometry
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Flow Cytometry: Purpose
• Characterization of cell size, complexity, antigens
• Examples: •
diagnosis of leukemia and lymphoma
•
determination of CD4/CD8 counts in patients with HIV
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Flow Cytometry: Interpretation
Complicated! Combine size, complexity and antigen expression data to come up with meaningful description of cells.
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Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot • Flow cytometry
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