Lab Methods

Question 1

What are the two main assay formats used to measure hormone levels?

Answer 1

Immunoassays and mass spectrometry

Question 2

How does a competitve immunoassay work?

Answer 2

Question 3

How does an immunometric (sandwich) immunoassay work?

Answer 3

Question 4

Name 4 types of signals that lab may use to detect analyte

Answer 4

1. Radioactive
2. Chemiluminescent
3. Colorimetric (ex. ELISA)
4. Flurorescent

Question 5

Name 2 advantages of sandwich assay over competitive assay

Answer 5

1. Greater analyte specificity
2. Greater analyte sensitivity
3. Ability to use monoclonal antibodies (more easily produced)

Question 6

Name 1 disadvantage of sandwich assay over competitive assay

Answer 6

Vulnerable to the hook effect

Question 7

What is the hook effect seen with sandwich assays?

Answer 7

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)

Question 8

How can a lab overcome the hook effect?

Answer 8

1. Analyte is allowed to bind to capture antibody, then washout, then addition of the detection antibody
2. Serial dilution of the sample
3. Consider a competitive assay instead

Question 9

What are heterophile antibodies

Answer 9

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.

Question 10

How can heterophile antibodies impact results?

Answer 10

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

Question 11

What should you do if heterophile antibody is suspected?

Answer 11

• Run sample on a different immunoassay platform
• Use heterophile antibody blocking agent
• Serial dilutions (you will see a nonlinear response to dilution)

Question 12

List 4 pre-analytical variables affecting hormone measurements

Answer 12

1. Episodic secretion (ex. pituitary hormones, cortisol)
2. Exercise (ex. ACTH, ADH, epinephrine)
3. Circadian rhythm/diurnal variation (ex. cortisol, ACTH)
4. Seasonal variation (estradiol, prolactin, testosterone)
5. Postural change (ex. renin, aldosterone, epi/norepi)
6. Nutrition (ex. insulin, IGF1, estradiol)

Question 13

What are the components of analytic validation done by the lab?

Answer 13

1. Linearity/reportable range: range of concentrations where assay is known to be reliable
2. Precision: reproducibility
3. Analyte sensitivity: how low a concentration can be measured (minimal detectable concentration)
4. Analyte specificity: ability to measure analyte without cross-reacting with other substances
5. Interference: ex. hemolysis and lipemia- impact on results
6. Accuracy
7. Sample types, matrix effects: ex. lavender vs green top
8. Stability: ex. room temperature, refridgerated, frozen
9. Carryover: cross-contamination between samples

Question 14

What is the minimum number of healthy people that should be used to generate a reference interval?

Answer 14

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.