A grossly lipemic sample gives a falsely high result in an immunoturbidimetric CRP assay. The main reason is that the lipid particles:

Scatter light and add to the measured turbidity

Displace plasma water and dilute the CRP

Inhibit the enzyme label that makes the signal

Bind the antibody and block complex formation

Many analyzers read each reaction at a main wavelength and at a second wavelength where the product absorbs little. The second reading mainly corrects for:

Background absorbance from turbidity, color and optics

Errors in the volume of sample pipetted

Drift in the reaction temperature

Loss of enzyme activity during the reaction

In fluorometry, the emitted light has a longer wavelength than the exciting light because:

Some absorbed energy is lost before emission

Scatter in the cuvette shifts the color

The emission filter lengthens the wavelength

The molecule gains energy from the solvent

Spectrophotometry and photometric methods

8 DOH Abu Dhabi (HAAD) lab licensing practice questions on Spectrophotometry and photometric methods, part of Clinical Chemistry. The first 8 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 spectrophotometer, the part that picks out a narrow band of wavelengths from the lamp's light is the:
1. ALight source
2. BCuvette holder
3. CPhotomultiplier tube
4. DMonochromator
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Answer: D · Monochromator
The monochromator, a diffraction grating or prism with entrance and exit slits, spreads the light into its wavelengths and passes only a narrow band to the sample. The width of that band affects how closely the method follows Beer's law. Simple photometers use interference filters instead. The detector, such as a photodiode or photomultiplier tube, then converts the transmitted light into an electrical signal.
- A: The lamp emits a broad range of wavelengths.
- B: The cuvette holder only places the sample in the light path.
- C: The photomultiplier tube detects light; it does not select wavelengths.
Question 2 · Medium
A grossly lipemic sample gives a falsely high result in an immunoturbidimetric CRP assay. The main reason is that the lipid particles:
1. ADisplace plasma water and dilute the CRP
2. BInhibit the enzyme label that makes the signal
3. CBind the antibody and block complex formation
4. DScatter light and add to the measured turbidity
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Answer: D · Scatter light and add to the measured turbidity
Turbidimetry measures the fall in transmitted light as antigen-antibody complexes form and scatter light. Chylomicrons and large VLDL particles scatter light too, so they raise the apparent absorbance and can be read as extra CRP. Sample blanking and rate reading reduce the effect, and high-speed centrifugation or ultracentrifugation can clear the sample. The lipemia index shows when the manufacturer's limit is exceeded.
- A: Water displacement matters for indirect electrolyte methods and would lower the result.
- B: Immunoturbidimetric assays have no enzyme label; the signal is light scatter.
- C: Blocking the antibody would lower the result, not raise it.
Question 3 · Medium
Many analyzers read each reaction at a main wavelength and at a second wavelength where the product absorbs little. The second reading mainly corrects for:
1. AErrors in the volume of sample pipetted
2. BDrift in the reaction temperature
3. CBackground absorbance from turbidity, color and optics
4. DLoss of enzyme activity during the reaction
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Answer: C · Background absorbance from turbidity, color and optics
Lipemia, sample color, bubbles and cuvette scratches absorb or scatter light at both wavelengths, while the reaction product absorbs mainly at the main wavelength. Subtracting the second reading removes much of this background. The second wavelength is chosen where the product absorbs little and the interferent behaves much the same at both wavelengths. Bichromatic reading reduces but does not remove interference from heavy lipemia or hemolysis, so serum indices are still checked.
- A: A pipetting error changes the amount of product, which a second reading cannot correct.
- B: Temperature is controlled by the incubator, not corrected optically.
- D: Loss of enzyme activity is handled by reagent stability limits and calibration.
Question 4 · Hard
A spectrophotometer has stray light equal to 1.0% of the blank signal at 340 nm. A sample whose true absorbance is 2.00 will read approximately:
1. A2.30
2. B1.70
3. C1.99
4. D2.00
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Answer: B · 1.70
A true absorbance of 2.00 means 1% transmittance. The detector also receives stray light worth 1% of the blank signal, so it sees about 1% + 1% = 2% transmittance (more exactly 0.02 ÷ 1.01 = 0.0198). The measured absorbance is -log 0.0198 = about 1.70. Stray light therefore flattens the response at high absorbance and makes those results read falsely low, so samples above the verified linear range should be diluted.
- A: Stray light adds light, so the reading falls rather than rises.
- C: 1.99 subtracts 0.01 absorbance units, confusing absorbance with transmittance.
- D: 2.00 ignores the stray light that reaches the detector.
Question 5 · Medium
In fluorometry, the emitted light has a longer wavelength than the exciting light because:
1. AScatter in the cuvette shifts the color
2. BThe emission filter lengthens the wavelength
3. CThe molecule gains energy from the solvent
4. DSome absorbed energy is lost before emission
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Answer: D · Some absorbed energy is lost before emission
A molecule that absorbs light rises to an excited state, then loses some energy as heat before it falls back and emits a photon. The emitted photon carries less energy, so its wavelength is longer; this difference is the Stokes shift. It lets the emission filter pass the fluorescence while blocking the excitation light. Fluorometry is far more sensitive than absorbance photometry because the signal is measured against a dark background.
- A: Light scattered by the sample mostly keeps the excitation wavelength.
- B: Filters only select wavelengths; they cannot change them.
- C: Gaining energy would shorten the wavelength, not lengthen it.
Question 6 · Hard
With very concentrated samples, a fluorometric method reads lower than expected. One sample reads 40 units undiluted but 120 units after a 1 in 10 dilution (result multiplied by 10). Samples of lower concentration dilute in proportion. The most likely cause is:
1. AInner-filter effect at high concentration
2. BStray light reaching the detector
3. CPhotobleaching of the fluorophore
4. DRaman scatter from the solvent
Answer and explanation in LearnFree account
Question 7 · Easy
A method must read absorbance at 280 nm, in the ultraviolet range. Which light source suits this?
1. ADeuterium lamp
2. BHollow cathode lamp
3. CTungsten lamp
4. DRed light-emitting diode
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Question 8 · Easy
What is the purpose of a reagent blank in a photometric assay?
1. ATo increase the sensitivity of the assay
2. BTo correct for the reagents' own absorbance
3. CTo measure hemolysis in the sample
4. DTo calibrate the photometer's wavelength
Answer and explanation in LearnFree account

Spectrophotometry and photometric methods

In a spectrophotometer, the part that picks out a narrow band of wavelengths from the lamp's light is the:

A grossly lipemic sample gives a falsely high result in an immunoturbidimetric CRP assay. The main reason is that the lipid particles:

Many analyzers read each reaction at a main wavelength and at a second wavelength where the product absorbs little. The second reading mainly corrects for:

A spectrophotometer has stray light equal to 1.0% of the blank signal at 340 nm. A sample whose true absorbance is 2.00 will read approximately:

In fluorometry, the emitted light has a longer wavelength than the exciting light because:

With very concentrated samples, a fluorometric method reads lower than expected. One sample reads 40 units undiluted but 120 units after a 1 in 10 dilution (result multiplied by 10). Samples of lower concentration dilute in proportion. The most likely cause is:

A method must read absorbance at 280 nm, in the ultraviolet range. Which light source suits this?

What is the purpose of a reagent blank in a photometric assay?