Immunologic Laboratory TestsKristine Krafts, M.D.

• Agglutination reactions• DAT• IAT• Immunofluorescence• ELISA• Western blot• Flow cytometry

Immunologic Lab Tests Outline

• What does it measure?• Where does the Ag-Ab interaction occur?• How is the Ag-Ab complex detected?

Things to Remember About Each Test

• Agglutination reactions

Immunologic Lab Tests Outline

• Detection of Ag or Ab in patient specimen• Examples:

• testing for antibodies to infectious agents• testing for Hemophilus influenzae type B capsular

antigen in CSF

Agglutination Reactions: Purpose

• Use particles coated with Ag or Ab• Add patient’s serum (containing Ab or Ag)• See if particles clump

Agglutination Reactions: Method

Clumping = patient has the antibody (or antigen)

Agglutination Reactions: Interpretation

negative positive

• Agglutination reactions• DAT

Immunologic Lab Tests Outline

• Detection of Ab (or complement) on patient’s red cells

• Also called the direct Coombs Test• Performed in patients with hemolytic anemia

DAT: Purpose

• Use patient’s red cells (coated with Ab)• Add anti-human globulin (AHG) (Coombs reagent)• Look for agglutination

DAT: Method

patient red cells + AHG = agglutination

Clumping = patient red cells are coated with antibody and/or complement

DAT: Interpretation

• Agglutination reactions• DAT• IAT

Immunologic Lab Tests Outline

• Detection of antibodies to red cell antigens• Also called the indirect Coombs Test• Performed as part of pre-transfusion testing

• antibody screen• cross-match

IAT: Purpose

• Use patient serum (containing Ab)• Add donor RBCs (coated with Ag)• Add anti-human globulin (Coombs reagent)• Look for agglutination

IAT: Method

patient Ab

+ AHG =agglutinationdonor RBC

=Ab-coateddonor RBC

+

patient serum(without red cell

Ab)

AHG

reagent RBC(with red cell Ag)

patient serum(with red cell

Ab)

AHG

reagent RBC(with red cell Ag)

ANTIBODY SCREENING

no agglutination(negative test)

agglutination(positive test)

Clumping = patient has an antibody to the donor (or reagent) red cells

IAT: Interpretation

• Agglutination reactions• DAT• IAT• Immunofluorescence

Immunologic Lab Tests Outline

• Detection of a specific antigen or antibody in a histologic specimen

• Examples: • detection of bacterial organisms• detection of antigen-antibody complexes

Immunofluorescence: Purpose

• Fix specimen on slide

• Add antibody specific for the desired antigen

• Look for fluorescence

• Fix specimen on slide

• Add antibody specific for the desired antigen

• Add second antibody

• Look for fluorescence

Direct Indirect

Immunofluorescence: Methods

Fluorescence = patient has the antigen

Immunofluorescence: Interpretation

• Agglutination reactions• DAT• IAT• Immunofluorescence• ELISA

Immunologic Lab Tests Outline

• Detection of antibodies in patient specimen• Examples:

• home pregnancy tests• HIV tests• tests for some coagulation factors, cytokines,

and autoantibodies

ELISA: Purpose

• Add patient specimen to well coated with ligand• Add AHG with enzyme attached• Add substrate• Measure color change

ELISA: Method

Color change = patient has the antibody

ELISA: Interpretation

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

ELISA: Variations

• Agglutination reactions• DAT• IAT• Immunofluorescence• ELISA• Western blot

Immunologic Lab Tests Outline

• Detection of antibodies in patient specimen

• Most common example: HIV test

Western Blot: Purpose

• 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

Western Blot: Method

Bands on strip = patient has antibodies to corresponding proteins

Western Blot: Interpretation

Enough bands = patient is “positive”

• Agglutination reactions• DAT• IAT• Immunofluorescence• ELISA• Western blot• Flow cytometry

Immunologic Lab Tests Outline

• Characterization of cell size, complexity, antigens• Examples:

• diagnosis of leukemia and lymphoma• determination of CD4/CD8 counts in patients

with HIV

Flow Cytometry: Purpose

Complicated! Combine size, complexity and antigen expression data to come up with

meaningful description of cells.

Flow Cytometry: Interpretation