BL 03-04-14 10-11am Immunodiagnosis - Cohen

Question 1

How the lab evaluates lymphocyte numbers

Answer 1

1. CBC + differential (done & IDed by robots)`

Question 2

How the lab evaluates B cell numbers

Answer 2

B cells in blood can be measured by

• counting cells with surface immunoglobulin.
• counting cells with markers CD19 or CD20 (most labs use these b/c they are more specific– a mac or PMN could have immune complexes on its surface, bound to its Fc receptors, & score as a false positive)

Question 3

How to count B cells using CD19/20

Answer 3

A fluorescent molecule is coupled to mAb to CD19 or 20, which is then mixed with a blood sample.

Question 4

How the lab evaluates T cell numbers

Answer 4

Can use…

• mAbs to CD3 (total T cells)
• mAbs to CD4 (helpers) or CD8 (killers)

Fluorescent cells can be counted under
microscope that has a UV light source & appropriate filters, or by flow cytometry

Question 5

Flow Cytometry - process

Answer 5

“Flow cytometers”

• Take cells in suspension & pump them through orifice so small that cells emerge in a single-file, very fine stream
• Lasers illuminate cells & light emitted / scattered by each cell is collected by photomultipliers
• Light scatter gives info about cell size & cytoplasmic granularity
• If cell has bound a fluorescent-tagged Ab, the fluorescent light emitted is quantified

—> Examines 10,000 individual cells / sec

Question 6

Tagging markers in Flow Cytometry

Answer 6

• mAbs to different cell surface molecules can be bought tagged w/ different dyes so that they can be used simultaneously

Other dyes can be used:

• Propidium iodide
• –> reacts quantitatively w/DNA, becoming fluorescent, so can tell where cell is in cell cycle by its DNA content

Question 7

Multiparameter cytometry

Answer 7

By using multiparameter cytometry, you can ask questions like:

• What % of cells in blood bears CD34 marker that is seen on hematopoietic stem cells?
• Are they cycling or resting?

Question 8

IDing Internal antigens via Flow cytometry

Answer 8

If cells are fixed & permeabilized, flow cytometry can be used to detect internal antigens
Internal antigens might include:
- cytokines (not yet secreted) 
- transcription factors
- Treg (how it is IDed)

Can distinguish whether a molecule is w/in a cell, or on its surface.

Question 9

Distinguishing pro-B, pre-B, immature B, & mature B cells

Answer 9

• distinguished by fluorescence microscopy using Abs to IgD, IgM, and H or L chains
• on both fixed (permeabilized) and intact cells, so you can distinguish whether a molecule is within a cell, or on its surface.

Total B cells: CD19, CD20
Naive B cells: CD19, IgD, CD10, IgM
Mature B cells: CD19, CD20, IgG
Memory B cells: CD19, CD27, IgM

Question 10

Serum protein electrophoresis - process / purpose / pros & cons

Answer 10

• to test the humoral arm of the immune system

1. Apply serum, turn on voltage, run.
2. Stain proteins.
3. Scan (—>peaks)

= cheap
= easily quantified
= NOT very sensitive to small abnormalities (could not pick up selective IgA deficiency b/c IgA runs pretty much together w/the much larger IgG/gamma globulin band)

Question 11

Fluids on which to perform Electrophoresis

Answer 11

Electrophoresis can be done on
- Urine (to look for “Bence Jones protein,” free Ig light chains seen in pts w/ multiple myeloma)
- Cerebrospinal fluid, where oligoclonal (a few clones) peaks in the IgG region are
sometimes seen in multiple sclerosis (see pic on handout if wish to)

Question 12

Single radial immunodiffusion

Answer 12

To measure levels of individual immunoglobulin classes or subclasses

To measure any other multivalent antigen (one that can form a ppt w/ an appropriate antibody)
- EX: individual complement or clotting components, if you have a specific antiserum

Gels of this type can be purchased ready-to-go.

• fairly cheap
• slow for big hospital lab (which uses quicker tests that can be automated)

Question 13

Best overall test (for Th1 activity)

Answer 13

• skin test w/ common Ags to which most people
  will have DTH

A good set is: 
- Candida
- streptokinase/streptodornase (SK/SD,) trichophytin
- mumps
- tetanus
- tuberculin
Read at 24-48 hours

Question 14

Challenge DTH test

Answer 14

• to test T cell function
• over 98% of normals will become “sensitized” (immunized) to dinitrofluorobenzene in ~10 days if it’s painted on their skin
• This is like intentionally inducing poison ivy

Question 15

T cell mitogens PHA or Con A to test T cell function

Answer 15

• use to stimulate T cells in mononuclear leukocyte preps (lymphocytes + monocytes)
• observe either proliferation or IL-2, IL-4, or IFNgamma production

ConA & PHA (mitogens) both bind sugars associated w/ T cell receptor complex
—> fools T cells (all of them!) into thinking they are recognizing antigen.

Sometime useful to do b/c total numbers may be normal while function is impaired.

Question 16

Mitogens

Answer 16

= plant (usually) proteins that bind certain sugar sequences
= probably part of plant’s defense system against things like fungi
= Some of these sugar sequences are also found on human cells

Question 17

T cell testing in infants

Answer 17

Chest x-ray may be very useful

How does the thymus look??

Question 18

Lymphoid biopsy to test T cells

Answer 18

• may be necessary in suspected primary immunodeficiency

- biopsy of rectal mucosa is often less traumatic to the patient

Question 19

Autoimmune diseases - things to measure

Answer 19

• Antinuclear antibodies
• Rheumatoid factor
• Immune complexes
• Immunofluorescence
• Immunohistochemistry

Question 20

Antinuclear antibodies (ANA) - test

Answer 20

= Antibodies against autoantigens in nucleus
- best observed using human cells grown on a slide

To do the test:

1. Slide fixed w/ agent (alcohol or acetone) that makes cells’ plasma membranes permeable so Abs can penetrate to the interior
2. Drop pt’s serum on slide
3. After washing, add fluorescein-labeled goat anti-human IgG (occasionally, anti-IgA or –IgM)
4. A further wash & slide is examined under UV microscope

Experienced rheumatologists can tell much not only from the presence of fluorescence (indicating ANAs) but also from the pattern— speckled, diffuse, nucleolar, etc.

Question 21

Rheumatoid factor - test

Answer 21

= IgM anti-IgG
- detected by its ability to agglutinate latex particles if they have been coated w/ IgG
= “passive” agglutination, b/c the particle isn’t the antigen)
= simple & cheap test which anyone can do

Question 22

Immune complexes - test

Answer 22

• Immune complexes in serum often are insoluble in the cold
• If suspect Type III disease, put sample of serum in fridge & examine after 1-7 days for a precipitate
• this precipitate is called a mixed cryoglobulin (to distinguish it from the pure [monoclonal]
  cryoglobulin occasionally seen in multiple myeloma)

Question 23

Immunofluorescence

Answer 23

• used to ID antibody in a patient’s tissues (direct immunofluorescence) or in their blood (indirect)

Question 24

Immunohistochemistry

Answer 24

= very like immunofluorescence

• BUT uses a final Ab labeled instead w/ an enzyme (typically peroxidase), which produces brown or black product
• Can be observed w/ ordinary microscope & archived for a long time (fluorescence fades w/ time)

Question 25

Teating for specific antibody - why

Answer 25

Often want to measure Abto a particular antigen (rather than total levels of immunoglobulin) = as evidence of current or prior infection or immunization with a particular pathogen = in ppl w/ conditions like Common Variable Hypogammaglobulinemia (may have Ig in plasma but respond poorly to specific new antigen)

Question 26

Testing for specific antibody - types of tests

Answer 26

- Precipitation rxn (not done) - Simple ELISA - Immunofluorescence - Pass agglutination

Question 27

Precipitation rxn to test for specific antibody

Answer 27

Mix serum + antigen --> look for ppt - as in the quantitative precipitin test = Possible, but hopelessly insensitive, and never done for diagnosis.

Question 28

Simple ELISA to detect specific antibody

Answer 28

= as done in HIV antibody screen 1. Antigen coupled to plate, 2. Test serum added If there is Ab to the Ag, it will bind ---> then IDed using an enzyme-coupled Ab. to the specific class of expected serum antibody

Question 29

Immunofluorescence to detect specific antibodies

Answer 29

- to detect antibodies to bacteria - quick, sensitive, and specific 1. Prepare smear of suspect bacterium, grown in culture 2. Layer patient’s serum over it & wash slide. 3. Add fluorescein-labeled goat anti-human Ig & wash again. 4. Look at it under microscope by UV light - If bugs fluoresce, there was Ab in the serum = indirect fluorescence test

Question 30

Passive agglutination

Answer 30

- The bigger the effective size of the antigen, the more sensitive the test - So, we couple small Ags to RBCs or latex beads, & add dilutions of patient’s serum, looking for an agglutination titer - Titer = reciprocal of highest dilution that will still do something (such as cause agglutination)

Question 31

Why measure antigen

Answer 31

- Often want to detect/measure a substance in blood or other fluid, or cells = could be a normal component (say, VEGF), or a pathogen (Chlamydia), or a drug (methamphetamine). - Clever immunologists can almost always find a way to produce Abs against substance of interest (except against really little ones like Na+ or glucose)

Question 32

Immunological techniques for measuring antigen

Answer 32

- capture ELISA - Fluorescent immunoassay (FIA) - Rapid screens - Reverse passive agglutination

Question 33

FIA, fluorescent immunoassays to detect antigen

Answer 33

= more sensitive version of capture ELISA test | - uses a substrate that becomes a fluorescent product

Question 34

Enzyme-linked immunosorbent assay, ELISA (capture or sandwhich ELISA) to detect antigen

Answer 34

- aka sandwich ELISA - If antigen is at least divalent, this is the favorite technique - Use enzyme-labeling technique b/c easy to detect & doesn’t involve radioactivity - Many Abs to viruses are measured by ELISA - Measure [Ag] in biological fluid or other solution

Question 35

Example of the use of ELISA to detect antigen

Answer 35

Measuring myocardial creatine kinase isoform MB (CK-MB) in a patient's serum = would be elevated shortly after a heart attack 1. Make/Buy 2 mAbs to human CK-MB, each to a different epitope 2. Put one mAb on a plate so that it’s stuck there. = the capture antibody 3. Add pt's serum & wash off anything not bound 4. Add 2nd Ab, which will stick to other epitope on antigen in proportion to how much antigen’s there. = 2nd Ab has enzyme coupled to it (usually peroxidase) & it completes the sandwich 5. Now add colorless peroxidase substrate (S) that produces a colored product (P) 6. Finally, measure intensity of product color in a plate spectrophotometer (aka ELISA reader)

Question 36

Competition assays to measure antigen

Answer 36

- to detect/measure small molecules, with only one epitope, which can’t be measured in a capture ELIS = various types of competition assays

Question 37

Rapid screens to detect antigen

Answer 37

- In the old days, a kid w/a sore throat got it cultured & had to wait 3 days before he found out if he was growing Streptococcus. - Now, using a kit, throat swab is extracted in a tube of buffer, & extract is passed through a membrane to which Strep antigens stick (b/c there’s a dot of anti-Strep antibody coupled to it) - To detect this binding, the kit provides another antibody against Strep to which liposomes (little fat droplets with a water interior) have been bound - The water contains a dye. - This preparation is also passed through the membrane, & it sticks if Ag has been trapped by the dot - Detergent is added to pop the liposomes, & so the spot turns color if there was Strep antigen in the extract.

Question 38

Reverse passive agglutination to detect antigen

Answer 38

For screening suspected bacterial meningitis 1. Obtain sets of tiny latex beads coated separately w/Abs to 4 common bacterial culprits 2. Add beads to a drop of pt's CSF 3. If any agglutinate, it’s b.c they have been cross-linked by bacterial antigen that was in the CSF