Lab Methods Flashcards
What are the two main assay formats used to measure hormone levels?
Immunoassays and mass spectrometry
How does a competitve immunoassay work?
How does an immunometric (sandwich) immunoassay work?
Name 4 types of signals that lab may use to detect analyte
- Radioactive
- Chemiluminescent
- Colorimetric (ex. ELISA)
- Flurorescent
Name 2 advantages of sandwich assay over competitive assay
- Greater analyte specificity
- Greater analyte sensitivity
- Ability to use monoclonal antibodies (more easily produced)
Name 1 disadvantage of sandwich assay over competitive assay
Vulnerable to the hook effect
What is the hook effect seen with sandwich assays?
Falsely low values in the presence of large amounts of analyte (analyte saturates BOTH the capture and detection antibodies prior to formation of a sandwich complex)
How can a lab overcome the hook effect?
- Analyte is allowed to bind to capture antibody, then washout, then addition of the detection antibody
- Serial dilution of the sample
- Consider a competitive assay instead
What are heterophile antibodies
Human antianimal antibody or human antimouse antibody.
Antibodies present in human serum that may bind to and interfere with animal antibodies used for a particular immunoassay.
How can heterophile antibodies impact results?
Sandwich assay:
- bridge the capture and detection antibody in the absence of analyte –> falsely elevated result
- attach at binding site of capture antibody, prevent binding of analyte –> falsely low result
Competitive assay:
- Outcompete the assay antibody for tracer –> falsely high result
- Bind preferentially to the analyte over tracer –> falsely low result
What should you do if heterophile antibody is suspected?
- Run sample on a different immunoassay platform
- Use heterophile antibody blocking agent
- Serial dilutions (you will see a nonlinear response to dilution)
List 4 pre-analytical variables affecting hormone measurements
- Episodic secretion (ex. pituitary hormones, cortisol)
- Exercise (ex. ACTH, ADH, epinephrine)
- Circadian rhythm/diurnal variation (ex. cortisol, ACTH)
- Seasonal variation (estradiol, prolactin, testosterone)
- Postural change (ex. renin, aldosterone, epi/norepi)
- Nutrition (ex. insulin, IGF1, estradiol)
What are the components of analytic validation done by the lab?
- Linearity/reportable range: range of concentrations where assay is known to be reliable
- Precision: reproducibility
- Analyte sensitivity: how low a concentration can be measured (minimal detectable concentration)
- Analyte specificity: ability to measure analyte without cross-reacting with other substances
- Interference: ex. hemolysis and lipemia- impact on results
- Accuracy
- Sample types, matrix effects: ex. lavender vs green top
- Stability: ex. room temperature, refridgerated, frozen
- Carryover: cross-contamination between samples
What is the minimum number of healthy people that should be used to generate a reference interval?
120
In practice, the numbers are often lower AND this makes it very difficult to generate age- or tanner stage-specific intervals. Therefore many labs rely on the literature for this.
