A 38-year-old woman with no symptoms has had TSH between 38 and 45 mIU/L (reference 0.4-4.0) for a year, with free T4 16 pmol/L (1.2 ng/dL; reference 12-22), a normal free T3 and negative TPO antibodies. A trial of levothyroxine raised her free T4 to 21 pmol/L, but TSH stayed near 40 mIU/L. Laboratory checks: TSH after a heterophile blocking tube 41 mIU/L; corrected results at 1 in 2 and 1 in 4 dilutions 42 and 39 mIU/L; TSH recovery after polyethylene glycol (PEG) precipitation 11% (below 25% suggests a macro form; control serum 85%). What is the most likely explanation?

Macro-TSH: TSH bound to an autoantibody

A TSH-secreting pituitary adenoma

Subclinical hypothyroidism, undertreated

Heterophile antibody interference

In immunoassays, the term cross-reactivity means that:

The antibody also binds molecules of similar structure

Two analytes are measured in the same reaction cuvette

Human antibodies bind the assay's animal antibodies

Sample is carried over from one test to the next

A man on oral prednisolone has a morning serum cortisol of 780 nmol/L [28 µg/dL] by immunoassay, although his adrenal axis should be suppressed. LC-MS/MS on the same sample gives 45 nmol/L [1.6 µg/dL]. The best explanation is:

Cross-reaction of the antibody with prednisolone

Heterophile antibodies bridging the reagents

Hook effect from a very high cortisol

Steroid-induced rise in cortisol-binding globulin

The functional sensitivity of an immunoassay, used for example to grade TSH assays, is the lowest concentration that can be measured with:

A between-run CV of 20% or less

No cross-reactivity with related hormones

100% recovery of an added standard

A signal clearly above the zero calibrator

Human anti-mouse antibodies (HAMA) that interfere with immunoassays are most likely to develop after:

Treatment with mouse monoclonal antibodies

Long-term high-dose biotin intake

Immunoassay principles and interferences

13 DOH Abu Dhabi (HAAD) lab licensing practice questions on Immunoassay principles and interferences, part of Clinical Chemistry. The first 13 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 · Hard
A 38-year-old woman with no symptoms has had TSH between 38 and 45 mIU/L (reference 0.4-4.0) for a year, with free T4 16 pmol/L (1.2 ng/dL; reference 12-22), a normal free T3 and negative TPO antibodies. A trial of levothyroxine raised her free T4 to 21 pmol/L, but TSH stayed near 40 mIU/L. Laboratory checks: TSH after a heterophile blocking tube 41 mIU/L; corrected results at 1 in 2 and 1 in 4 dilutions 42 and 39 mIU/L; TSH recovery after polyethylene glycol (PEG) precipitation 11% (below 25% suggests a macro form; control serum 85%). What is the most likely explanation?
1. AMacro-TSH: TSH bound to an autoantibody
2. BA TSH-secreting pituitary adenoma
3. CSubclinical hypothyroidism, undertreated
4. DHeterophile antibody interference
Show answerHide answer
Answer: A · Macro-TSH: TSH bound to an autoantibody
The unchanged result after the blocking tube and the linear dilutions argue against heterophile antibodies, but they do not exclude a large TSH-immunoglobulin complex, which both assay antibodies still recognize. PEG precipitates immunoglobulins, and only 11% of the TSH stayed in the supernatant, so most of the measured TSH is bound to IgG. This macro-TSH has little biological activity and is cleared slowly, which is why it stayed high when levothyroxine raised her free T4. Gel filtration chromatography confirms it, and the report should warn against raising the dose to normalize the TSH. True hypothyroidism would show a falling TSH on treatment, and a TSH-secreting adenoma gives a high free T4.
- B: A TSH-secreting adenoma gives a high free T4 with an unsuppressed TSH.
- C: In true hypothyroidism, TSH falls when levothyroxine raises the free T4.
- D: The blocking tube did not lower the result, which argues against heterophile antibodies.
Question 2 · Easy
In immunoassays, the term cross-reactivity means that:
1. ATwo analytes are measured in the same reaction cuvette
2. BHuman antibodies bind the assay's animal antibodies
3. CSample is carried over from one test to the next
4. DThe antibody also binds molecules of similar structure
Show answerHide answer
Answer: D · The antibody also binds molecules of similar structure
Cross-reactivity is the binding of the assay antibody to substances that resemble the analyte, such as metabolites, related hormones or drugs. It is a matter of antibody specificity and is usually worst in competitive assays for small molecules, where one antibody must tell apart very similar structures. Manufacturers list the cross-reactivity of known compounds in the package insert. LC-MS/MS avoids most of these problems because chromatography and mass separate the compounds.
- A: Measuring two analytes in one cuvette is multiplexing, not cross-reactivity.
- B: Antibodies against the reagent animal antibodies are heterophile antibodies.
- C: Carryover is an analyzer error between samples.
Question 3 · Medium
A man on oral prednisolone has a morning serum cortisol of 780 nmol/L [28 µg/dL] by immunoassay, although his adrenal axis should be suppressed. LC-MS/MS on the same sample gives 45 nmol/L [1.6 µg/dL]. The best explanation is:
1. AHeterophile antibodies bridging the reagents
2. BCross-reaction of the antibody with prednisolone
3. CHook effect from a very high cortisol
4. DSteroid-induced rise in cortisol-binding globulin
Show answerHide answer
Answer: B · Cross-reaction of the antibody with prednisolone
Prednisolone differs from cortisol only by one extra double bond, so many cortisol immunoassay antibodies bind it. The immunoassay therefore counts prednisolone as cortisol, while LC-MS/MS separates the two and shows that true cortisol is suppressed. Prednisone, which is converted to prednisolone, 6-methylprednisolone and 11-deoxycortisol (high after metyrapone) also cross-react in some assays. Cortisol should be measured after the steroid has been withheld, or by mass spectrometry.
- A: Heterophile antibodies rarely affect competitive cortisol assays, and the known steroid explains the result.
- C: Cortisol is measured by competitive assays, which do not show a hook effect.
- D: Glucocorticoids lower, not raise, cortisol-binding globulin.
Question 4 · Medium
The functional sensitivity of an immunoassay, used for example to grade TSH assays, is the lowest concentration that can be measured with:
1. ANo cross-reactivity with related hormones
2. BA between-run CV of 20% or less
3. C100% recovery of an added standard
4. DA signal clearly above the zero calibrator
Show answerHide answer
Answer: B · A between-run CV of 20% or less
Functional sensitivity is the lowest concentration that an assay measures with a between-run coefficient of variation of 20% or less, so it reflects clinically useful precision. The limit of detection, or analytical sensitivity, is only the lowest level that can be told apart from zero and is usually much lower. Third-generation TSH assays reach a functional sensitivity of about 0.01 to 0.02 mIU/L, which separates suppressed from low-normal TSH. Results below the functional sensitivity should not be reported as exact numbers.
- A: Cross-reactivity concerns specificity, not sensitivity.
- C: Recovery studies check accuracy, not the lowest reliable concentration.
- D: A signal above the zero calibrator defines the limit of detection.
Question 5 · Easy
Human anti-mouse antibodies (HAMA) that interfere with immunoassays are most likely to develop after:
1. ALong-term high-dose biotin intake
2. BA course of oral antibiotics
3. CTreatment with mouse monoclonal antibodies
4. DRepeated red cell transfusions
Show answerHide answer
Answer: C · Treatment with mouse monoclonal antibodies
Mouse monoclonal antibodies given for treatment or imaging, and close contact with animals, can make a patient produce antibodies against mouse immunoglobulin. Because most immunoassay reagent antibodies are mouse monoclonals, HAMA can bridge the capture and detection antibodies to give a falsely high result, or block them to give a falsely low one. Manufacturers add animal immunoglobulin or blocking agents to reduce the effect. A result that does not fit the clinical picture should prompt the laboratory to look for interference.
- A: Biotin interferes through streptavidin binding, not through antibodies.
- B: Antibiotics do not cause antibodies against animal immunoglobulin.
- D: Transfusions cause red cell alloantibodies, not antibodies to mouse immunoglobulin.
Question 6 · Easy
In a competitive immunoassay with a labeled antigen, a higher analyte concentration in the patient sample gives:
1. AMore bound label and a higher signal
2. BNo change in the bound label signal
3. CLess bound label and a lower signal
4. DA higher signal only after dilution
Answer and explanation in LearnFree account
Question 7 · Hard
A 34-year-old woman has had serum hCG between 40 and 60 IU/L for 3 months with no pregnancy on ultrasound, and chemotherapy for a suspected trophoblastic tumor is planned. Her urine hCG is negative. Neat, 1 in 2 and 1 in 4 dilutions of her serum give 52, 20 and 8 IU/L after correction for dilution. What is the most likely explanation?
1. AHigh-dose hook effect
2. BAn occult ectopic pregnancy
3. CHeterophile antibodies
4. DPituitary hCG secretion
Answer and explanation in LearnFree account
Question 8 · Medium
In a sandwich immunoassay that holds a biotinylated antibody on streptavidin-coated particles, high-dose biotin in the sample gives a falsely low result because free biotin:
1. ABridges the capture and the labeled antibodies
2. BQuenches the light given off by the label
3. CBlocks streptavidin, so fewer sandwiches are captured
4. DBinds the analyte and masks its two epitopes
Answer and explanation in LearnFree account
Question 9 · Hard
A well 50-year-old woman has TSH 12.4 mIU/L (0.4-4.0) and free T4 13 pmol/L (10-23); interference is queried. After a heterophile blocking tube, TSH is 12.1 mIU/L. Corrected results on 1 in 2 and 1 in 4 dilutions are 12.6 and 12.2 mIU/L, and a second manufacturer's assay gives 11.8 mIU/L. What do these results show?
1. AA high-dose hook effect
2. BInterference from biotin intake
3. CNo evidence of assay interference
4. DHeterophile antibody interference
Answer and explanation in LearnFree account
Question 10 · Medium
In a patient with thyroglobulin autoantibodies, what result does a sandwich (immunometric) thyroglobulin assay most often give?
1. AA falsely high result
2. BA result the antibodies do not affect
3. CA result that rises on dilution
4. DA falsely low result
Answer and explanation in LearnFree account
Question 11 · Easy
Why are small molecules such as digoxin, cortisol and testosterone usually measured by competitive rather than sandwich immunoassays?
1. AThey are too small to bind two antibodies at once
2. BCompetitive assays are always more sensitive
3. CTheir levels are too high for sandwich assays
4. DThey lose their structure in sandwich reagents
Answer and explanation in LearnFree account
Question 12 · Easy
Which assay design is most prone to the high-dose hook effect?
1. ATwo-step sandwich assay with a wash
2. BIon-selective electrode measurement
3. CCompetitive assay with labeled antigen
4. DOne-step sandwich assay
Answer and explanation in LearnFree account
Question 13 · Medium
In chronic kidney disease, a second-generation 'intact' PTH assay reads higher than a third-generation (whole PTH) assay on the same sample, because the second-generation assay:
1. AAlso detects C-terminal fragments such as PTH 7-84
2. BUses a competitive instead of a sandwich design
3. CIs affected by the high phosphate level
4. DCross-reacts with PTH-related peptide
Answer and explanation in LearnFree account

Immunoassay principles and interferences

In immunoassays, the term cross-reactivity means that:

A man on oral prednisolone has a morning serum cortisol of 780 nmol/L [28 µg/dL] by immunoassay, although his adrenal axis should be suppressed. LC-MS/MS on the same sample gives 45 nmol/L [1.6 µg/dL]. The best explanation is:

The functional sensitivity of an immunoassay, used for example to grade TSH assays, is the lowest concentration that can be measured with:

Human anti-mouse antibodies (HAMA) that interfere with immunoassays are most likely to develop after:

In a competitive immunoassay with a labeled antigen, a higher analyte concentration in the patient sample gives:

In a sandwich immunoassay that holds a biotinylated antibody on streptavidin-coated particles, high-dose biotin in the sample gives a falsely low result because free biotin:

In a patient with thyroglobulin autoantibodies, what result does a sandwich (immunometric) thyroglobulin assay most often give?

Why are small molecules such as digoxin, cortisol and testosterone usually measured by competitive rather than sandwich immunoassays?

Which assay design is most prone to the high-dose hook effect?

In chronic kidney disease, a second-generation 'intact' PTH assay reads higher than a third-generation (whole PTH) assay on the same sample, because the second-generation assay: