Assays of the

Immunocompetence
Objectives:
1. Describe how T and B cells are
enumerated, isolated and how
function is assessed
2. Describe the approaches used to
measure phagocyte and natural
killer cell function.
Assays of Immune Competence
▪ Analysis of :
1. Phagocytic cells
>nitroblue tetrazolium test, microbicidal assay and chemotaxis
assay
2. B cells
> enumeration, B cell function,
lymphoproliferation assay, EAC rosette test
3. T cells
> enumeration, DTH skin test, E-rosette test,
lymphoproliferation assay
Analysis of Phagocytic cells
1. Nitroblue Tetrazolium Test
• Evaluate the ability to generate toxic molecules to
kill microorganisms
• NBT dye : yellow → dark blue : cytoplasm of
phagocyte
Analysis of Phagocytic cells
2. Microbiocidal Assay or Intracellular Killing
Assay
• Evaluate functional integrity
• Tube : bacteria + neutrophils : incubated for 30
minutes
• Normal : neutrophils will kill 85-90% of the
bacteria
Analysis of Phagocytic cells
3. Chemotaxis Assay
• Ability of neutrophils to move or migrate toward a
stimuli
• Migration occurs through nitrocellulose membrane
in an agar medium
Analysis of T cells
1.Enumeration of T cell subsets
> sample+ monoclonal antibody (anti-Th)
with flurescent dye → fluorescence

2. Lymphocyte Proliferation assay

> T cell + mitogen→T cell proliferates
> measure mitosis : radioactive tritium
Analysis of T cells
3. Erythrocyte rosette assay
> T cell enumeration
> sample (lymphocytes) + sheep RBC →
rosette (mulberry shaped aggregates) : T
cells
Analysis of T cells
4. Delayed type hypersensitivity skin test
> T cell function
>Skin tests
>PPD skin test: for M. tuberculosis
> Diptheria toxoid: inactivated
toxin from diptheria
>intradermal injections of the material→
patient is observed for the first 3 days
>POSITIVE TEST: indicated by erythematous
(wheal/flare), indurated lesion which peaks at 48
hours
Analysis of B cells
1. Enumeration of B cells
> Sample (B cells) + gamma globulin
antibody with fluorescent dye
> incubation allows antibody to recognize
surface immunoglobulins of B cells
> bright green on fluorescence microscope
Analysis of B cells
2. Lymphocyte proliferation assay
> B cell + mitogen
> B cells proliferate rapidly
> radioactive tritium incorporated into
the B-cell DNA is measured
Analysis of B cells
3. Erythrocyte Antibody Complement Rosette
test
> sheep RBC coated with antibody and
complement + B cells → rosette
formation
Cell Flow Cytometry
• Automated system of identifying cells based
on the scattering of light as cells flow in a
single file through a laser beam
• Forward light scatter: measures cell size
• Side scatter: measure of cell granularity