Practice Flashcards
PAS reaction
- Lymphoid cells - homogenous cytoplasm
- Myeloid cells - granulous color
- Can in some cases diagnose acute lymphoblastic leukemia (ALL) and also some subtypes of acute myeloid leukemia (AML)
Esterase reaction
- Distinguish between myeloid cells, monocytes and neutrophil granulocytes
- Used to identify monoblastic leukemias
Flow cytometry
Can characterize the differentiation and activation states of cell types by the determination of their surface and cytoplasmatic protein expression
Immunohistochemistry
Determination of antigens in tissue sections by the use of labeled antibodies
Indirect ELISA
- Add antigen to the plate to coat the wells
- Wash
- Add the biological sample (eg.blood serum)
- Wash
- Add labeled secondary antibodies
- Wash then add chromogenic substance for coloring
- Measure optical density
Sandwich ELISA
- Coat well with monoclonal Ab
- Blocking and washing
- Add antigen containing samples
- Add antibodies reactive with the same antigen (but different epitope)
- Wash and add chromogen -> measure OD
Competitive ELISA
- Unlabelled Ab are pre-incubated with bio samples containing antigen -> Ab-Ag complex formed
- Pipetted onto antigen coated ELISA plates
(The more Ag present in bio sample -> the less Ab molecules remain free and available to bind antigen on ELISA plates) - Apply enzyme-conjugated secondary Ab and chromogen
ELISPOT
- Indirect immunoassay
- Can detect cytokines (most often) secreted by individual cells. -> Determining number of positive cells even at a very low frequency
1. The wells have either nitrocellulose or PVDF membrane bottom with captured Ab
2. Pipetting live cells eg. lymphocytes into wells
3. Incubation at 37 degrees for 1-2 days
4. Wash cells out of wells
5. The secreted molecules bound to the capture Ab are visualized by using a labeled secondary Ab and a substrate that will form an insoluble precipitate.
Immuno blot (Western blot)
- Can determine the relative amount of a given protein within a biological sample using an antigen-specific primary Ab
1. Different molecular weight proteins are separated by SDS page
2. Blot (transfer) content of gel to a nitrocellulose or PVDF membrane
3. Incubate membrane with a primary Ab specific to the desired.
4. Incubation with secondary Ab (enzyme conjugated) that binds to primary
Radioimmunoassay (RIA)
- Can determine conc. of eg. hormones
- A radioactively labeled antigen competes for Ab binding sites with the sample antigen
Immunoradiometric assay (IRMA)
- Use monoclonal Ab bound to the inner surface of polystyrene tubes
- Samples of patients incubated with radioactive Ab
- Sample Ag will simultaneously bind to unlabelled Ab bound to the wall + radioactive Ab
- Unbound radioactive Ab removed by washing
Immunocytochemistry
- Can demonstrate specific proteins within cells/tissues using labeled Ab
Lateral flow test
- Immerse a test strip into a biological sample and fluid migrates in the strip by capillarity
- The sample is mixed with colored, specific Ab-coated microparticles (eg. latex)
- Ag content of sample binds to colored microparticles and migrates along test strip
- Accumulations of migrating colored microparticles
1st spot: If it contains Ag of interest it will be dyed
2nd spot: Anti-Ig Ab is dried onto the surface (serves as a positive internal control)
Serum electrophoresis
- Used to identify missing/overproduced proteins
- An electric field stimulates the movement of particles relative to a fluid
- Detected: albumin, alpha globulins, HDL, beta globulin, gamma globulins
Decreased albumin in electrophoresis
Nephrosis syndrome, liver disease
Increased gamma-globulin in electrophoresis
Autoimmune diseases and infections
Increased total protein conc. detected in electrophoresis
Multiple myeloma
Immunoelectrophoresis
- A special form og electrophoresis, for the separation and characterization of serum proteins
- After electrophoresis, Ab are applied next to the separated proteins and immunoprecipitates are formed after a period of diffusion
Turbidimetry and nephelometry
- Methods for determining the amount of cloudiness or turbidity in a solution based on measurment of the effect of this turbidity upon the transmission and scattering of light
- Turbidity in a liquid is caused by the presence of finely divided suspended particles.
What can be diagnosed with flow cytometry?
To diagnose hematological disorders and immunodeficiencies
Steps of flow cytometry
- Sample prep: Blood from subject, separate mononuclear cells using a Ficoll gradient, stain with fluorescent Ab conjugates
- Instrument setup: Optimize fluorescence detector sensitivity
- Data acquisition: Pass stained cell suspension through laser beam
- Data analysis: Gate cell populations of interest
What can a flow cytometer tell us about a cell?
- Its relative size (forward scatter)
- Its relative granularity or internal complexity (side scatter)
- Its relative fluorescence intensity
- Time-dependency (kinetics) of these parameters
Which parameters can be measured by complement tests?
- Conc. of individual complement factors
- Conc. of regulatory factors
- Activity of individual factors or overall activation
CH50 test
- The demnominator of the serum dilution that lyses 50% of sheep RBCs in a test tube
(eg. if we need 1/4 serum to lysis 50% of the RBCs then the CH50 will be 4) -> reciprocal
What does a normal CH50 result tell us?
- Indicates that all complement factors (C1-9) are present
- The level of some however might be reduced a lot without affecting the CH50
