Time-resolved fluoroimmunoassays use europium chelate labels because their fluorescence:

Lasts long after background fluorescence fades

Needs no excitation light source

Increases when the label is bound to antibody

Is emitted at the same wavelength as excitation

A clinically well 34-year-old woman takes 10 mg of biotin daily for her hair. TSH 0.02 mIU/L (0.4-4.0), free T4 3.4 ng/dL [44 pmol/L] (0.8-1.8 [10-23]), TSH receptor antibody positive. All three assays use streptavidin-biotin. The best next step is:

Repeat the tests after a biotin-free period

Order a radioactive iodine uptake scan

Start antithyroid drug treatment

Retest with a heterophile blocking tube

Endocrinology: test procedures (immunoassay, fluorescence)

9 ASCP MLS practice questions on Endocrinology: test procedures (immunoassay, fluorescence), part of Chemistry. The first 9 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Easy
In a one-step sandwich immunoassay, a falsely low result caused by an extremely high analyte concentration is called the:
1. AHeterophile antibody effect
2. BCarryover effect
3. CHigh-dose hook effect
4. DSample matrix effect
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Answer: C · High-dose hook effect
In a one-step assay, the capture antibody, labeled antibody and sample are added together. When the analyte is extremely high, it saturates both antibodies separately, so few sandwiches form and the signal falls. The result can be falsely low or only mildly raised. Repeating on a diluted sample reveals the true value, and two-step assays with a wash between steps are much less prone to the effect.
- A: Heterophile antibodies usually cause falsely high results by bridging the antibodies.
- B: Carryover from a high sample raises, not lowers, the next result.
- D: Matrix effects come from sample composition, not from excess analyte.
Question 2 · Medium
Time-resolved fluoroimmunoassays use europium chelate labels because their fluorescence:
1. ANeeds no excitation light source
2. BIncreases when the label is bound to antibody
3. CLasts long after background fluorescence fades
4. DIs emitted at the same wavelength as excitation
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Answer: C · Lasts long after background fluorescence fades
Serum proteins, drugs and plastics give background fluorescence that decays within nanoseconds. Lanthanide chelates such as europium keep fluorescing for hundreds of microseconds and have a large Stokes shift. The reader waits until the background has died away and then measures the label, which greatly improves the signal-to-noise ratio. This gives the sensitivity needed for hormones present at very low levels.
- A: Fluorescence always needs excitation light; chemiluminescence does not.
- B: A change on binding is the basis of fluorescence polarization, not time resolution.
- D: Lanthanide chelates have a large Stokes shift, so emission differs from excitation.
Question 3 · Hard
A 45-year-old man with a 4-cm pituitary mass has prolactin 48 ng/mL [48 µg/L] (reference < 20) on a one-step assay. On a 1:100 dilution the analyzer reads 52 ng/mL. What prolactin should be reported?
1. A520 ng/mL
2. B5,200 ng/mL
3. C52 ng/mL
4. D48 ng/mL
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Answer: B · 5,200 ng/mL
The undiluted result is falsely low because of the high-dose hook effect. The diluted reading is multiplied by the dilution factor: 52 ng/mL x 100 = 5,200 ng/mL [5,200 µg/L]. A level this high with a large mass fits a macroprolactinoma, whereas the undiluted value could be mistaken for stalk compression by a nonfunctioning tumor. Labs should dilute prolactin whenever a large pituitary tumor is reported.
- A: 520 ng/mL uses a dilution factor of 10 instead of 100.
- C: 52 ng/mL reports the diluted reading without the dilution factor.
- D: 48 ng/mL is the hooked, falsely low undiluted result.
Question 4 · Hard
A clinically well 34-year-old woman takes 10 mg of biotin daily for her hair. TSH 0.02 mIU/L (0.4-4.0), free T4 3.4 ng/dL [44 pmol/L] (0.8-1.8 [10-23]), TSH receptor antibody positive. All three assays use streptavidin-biotin. The best next step is:
1. ARepeat the tests after a biotin-free period
2. BOrder a radioactive iodine uptake scan
3. CStart antithyroid drug treatment
4. DRetest with a heterophile blocking tube
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Answer: A · Repeat the tests after a biotin-free period
Biotin blocks streptavidin capture: sandwich assays such as TSH read falsely low, while competitive assays such as free T4 and TSH receptor antibody read falsely high. Together these mimic Graves disease in a patient who is clinically well. Repeating after stopping biotin for at least 2 days (longer for very high doses, per the manufacturer), or testing on a platform without streptavidin, gives the true results. Treating on these numbers could cause real harm.
- B: Imaging is premature until the results are confirmed.
- C: Treatment should wait until biotin interference is excluded.
- D: Blocking tubes remove heterophile antibodies, not biotin.
Question 5 · Medium
In a competitive fluorescence polarization immunoassay, how does polarization change as the patient's analyte concentration rises?
1. AIt increases
2. BIt decreases
3. CIt rises, then falls
4. DIt stays the same
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Answer: B · It decreases
A small fluorescein-labeled analyte (tracer) rotates quickly in solution and emits depolarized light. When a large antibody binds it, it rotates slowly and the emitted light stays polarized. Patient analyte competes with the tracer for antibody, so more patient analyte leaves more tracer free and lowers polarization. FPIA is homogeneous, with no separation step, and has been used for cortisol, T4 and many drugs.
- A: Polarization is highest when most tracer is bound, which happens at low analyte levels.
- C: A rise then fall describes a hook pattern, not FPIA.
- D: Patient analyte displaces tracer, so polarization does change.
Question 6 · Medium
A TSH result is suspected to be falsely raised by heterophile antibodies. Which finding supports this?
1. ALinear recovery on serial dilution
2. BA matching low free T4 result
3. CSame result on a different platform
4. DA large drop after a blocking reagent
Answer and explanation in LearnFree account
Question 7 · Easy
High-dose biotin supplements interfere with immunoassays that use:
1. AStreptavidin-biotin capture
2. BAcridinium ester labels
3. CHorseradish peroxidase labels
4. DAlkaline phosphatase labels
Answer and explanation in LearnFree account
Question 8 · Medium
A competitive immunoassay for free T4 uses streptavidin-biotin separation. A sample with a very high biotin level will most likely give:
1. AA result flagged as hemolyzed
2. BA falsely low result
3. CA falsely high result
4. DAn unaffected result
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Question 9 · Easy
How do heterophile antibodies most often cause a false result in a two-site sandwich immunoassay?
1. AThey compete with labeled analyte for antibody
2. BThey bridge the capture and labeled antibodies
3. CThey block streptavidin on the solid phase
4. DThey saturate both antibodies like excess analyte
Answer and explanation in LearnFree account

Endocrinology: test procedures (immunoassay, fluorescence)

In a one-step sandwich immunoassay, a falsely low result caused by an extremely high analyte concentration is called the:

Time-resolved fluoroimmunoassays use europium chelate labels because their fluorescence:

A 45-year-old man with a 4-cm pituitary mass has prolactin 48 ng/mL [48 µg/L] (reference < 20) on a one-step assay. On a 1:100 dilution the analyzer reads 52 ng/mL. What prolactin should be reported?

A clinically well 34-year-old woman takes 10 mg of biotin daily for her hair. TSH 0.02 mIU/L (0.4-4.0), free T4 3.4 ng/dL [44 pmol/L] (0.8-1.8 [10-23]), TSH receptor antibody positive. All three assays use streptavidin-biotin. The best next step is:

In a competitive fluorescence polarization immunoassay, how does polarization change as the patient's analyte concentration rises?

A TSH result is suspected to be falsely raised by heterophile antibodies. Which finding supports this?

High-dose biotin supplements interfere with immunoassays that use:

A competitive immunoassay for free T4 uses streptavidin-biotin separation. A sample with a very high biotin level will most likely give:

How do heterophile antibodies most often cause a false result in a two-site sandwich immunoassay?