A 69-year-old woman has back pain and a total protein of 116 g/L (11.6 g/dL) with albumin 29 g/L (2.9 g/dL). Serum protein electrophoresis shows one tall, sharp M-spike of 64 g/L (6.4 g/dL) in the mid-gamma region. On serum immunofixation, run at the kit's standard dilutions, the kappa lane shows a dense band at the position of the spike. At the same position the IgG lane shows a band with darkly stained edges and a pale, almost unstained center. The IgA, IgM and lambda lanes show no bands, and the kit controls are acceptable. What is the best next step?

Repeat the IgG lane at a higher dilution

Add anti-IgD and anti-IgE antisera

Pretreat the serum with 2-mercaptoethanol

Report a free kappa light chain protein

In the precipitin curve shown, increasing amounts of antigen are added to tubes holding the same amount of antibody. Zone A is the:

Prozone, with antibody in excess

Prozone, with antigen in excess

Equivalence zone, with optimal ratio

Postzone, with antigen in excess

In nephelometry, the detector measures:

Light scattered at an angle by immune complexes

The fall in light passing straight through

Light given off by a chemical reaction

Fluorescence emitted after excitation

A patient with IgG myeloma has serum IgG 11 g/L (1,100 mg/dL; reference 7-16 g/L) by nephelometry at the standard dilution, although total protein is 118 g/L (11.8 g/dL) and electrophoresis shows a large M-spike. At a tenfold higher dilution, the corrected IgG is 72 g/L. The most likely cause of the first result is:

Antigen excess at the standard dilution

Antibody excess (prozone) at the standard dilution

A dilution error in the repeat test

A monoclonal IgG that the antiserum cannot detect

Agglutination, precipitation and nephelometry

10 DOH Abu Dhabi (HAAD) lab licensing practice questions on Agglutination, precipitation and nephelometry, part of Immunology and Serology. The first 10 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 69-year-old woman has back pain and a total protein of 116 g/L (11.6 g/dL) with albumin 29 g/L (2.9 g/dL). Serum protein electrophoresis shows one tall, sharp M-spike of 64 g/L (6.4 g/dL) in the mid-gamma region. On serum immunofixation, run at the kit's standard dilutions, the kappa lane shows a dense band at the position of the spike. At the same position the IgG lane shows a band with darkly stained edges and a pale, almost unstained center. The IgA, IgM and lambda lanes show no bands, and the kit controls are acceptable. What is the best next step?
1. ARepeat the IgG lane at a higher dilution
2. BAdd anti-IgD and anti-IgE antisera
3. CPretreat the serum with 2-mercaptoethanol
4. DReport a free kappa light chain protein
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Answer: A · Repeat the IgG lane at a higher dilution
A precipitin band forms only where antigen and antibody are close to equivalence. With 64 g/L of monoclonal IgG, the anti-IgG in the middle of the band is swamped by antigen, so soluble complexes wash out and leave a 'hollow' band, stained only at its edges, where the proportions are nearer equivalence; antisera differ in strength, so the excess can show in one lane only. Repeating the IgG lane at a higher sample dilution gives a solid IgG band at the kappa position and identifies an IgG kappa M-protein. Anti-IgD and anti-IgE are needed only when a light-chain band has no matching heavy-chain band, and here the IgG lane does react. 2-Mercaptoethanol is used to break up IgM or IgA polymers and does not correct antigen excess.
- B: The IgG lane does react, so there is no need to look for IgD or IgE.
- C: 2-Mercaptoethanol breaks up IgM or IgA polymers and does not correct antigen excess.
- D: The pale-centered IgG band shows that the kappa belongs to an intact IgG.
Question 2 · Easy
In the precipitin curve shown, increasing amounts of antigen are added to tubes holding the same amount of antibody. Zone A is the:
Figure: MedExams
1. AProzone, with antigen in excess
2. BEquivalence zone, with optimal ratio
3. CPostzone, with antigen in excess
4. DProzone, with antibody in excess
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Answer: D · Prozone, with antibody in excess
On the left of the curve, little antigen has been added, so each antigen molecule is covered by many antibody molecules and small soluble complexes form instead of a lattice. This is the prozone, the zone of antibody excess, where precipitation is weak. In zone B, antigen and antibody are in the best proportions and the most precipitate forms (equivalence). In zone C, antigen is in excess (postzone), which can cause falsely low results in nephelometry and immunoassays.
- A: The prozone is the zone of antibody excess, not antigen excess.
- B: The equivalence zone is at the peak, in zone B.
- C: The postzone of antigen excess is on the right, in zone C.
Question 3 · Easy
A test uses latex beads coated with a soluble antigen, which clump when the patient's serum contains the matching antibody. What type of agglutination is this?
1. APassive agglutination
2. BAgglutination inhibition
3. CReverse passive agglutination
4. DDirect agglutination
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Answer: A · Passive agglutination
In passive (indirect) agglutination, a soluble antigen is fixed to a carrier such as latex, gelatin or red cells, and patient antibody links the carriers into visible clumps. Direct agglutination uses antigens that are a natural part of a particle, such as whole bacteria or red cells. In reverse passive agglutination the carrier holds antibody and the test detects antigen. The carrier makes the reaction visible within minutes, which is why slide tests are quick.
- B: In an inhibition test, a positive sample stops the clumping.
- C: Reverse passive tests coat the carrier with antibody to detect antigen.
- D: Direct agglutination uses antigens native to the particle, such as whole bacteria.
Question 4 · Easy
In nephelometry, the detector measures:
1. AThe fall in light passing straight through
2. BLight given off by a chemical reaction
3. CLight scattered at an angle by immune complexes
4. DFluorescence emitted after excitation
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Answer: C · Light scattered at an angle by immune complexes
A nephelometer measures light scattered by antigen-antibody complexes, with the detector placed at an angle to the incident beam. Turbidimetry instead measures the decrease in light that passes straight through the cuvette. Both methods are used for serum proteins such as immunoglobulins, complement, CRP and free light chains. Because scatter is read against a dark background, nephelometry is more sensitive at low concentrations.
- A: That describes turbidimetry, which reads transmitted light.
- B: Light from a chemical reaction is chemiluminescence.
- D: Fluorescence is read in fluorescent immunoassays, not in nephelometry.
Question 5 · Hard
A patient with IgG myeloma has serum IgG 11 g/L (1,100 mg/dL; reference 7-16 g/L) by nephelometry at the standard dilution, although total protein is 118 g/L (11.8 g/dL) and electrophoresis shows a large M-spike. At a tenfold higher dilution, the corrected IgG is 72 g/L. The most likely cause of the first result is:
1. AAntigen excess at the standard dilution
2. BAntibody excess (prozone) at the standard dilution
3. CA dilution error in the repeat test
4. DA monoclonal IgG that the antiserum cannot detect
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Answer: A · Antigen excess at the standard dilution
With a very high IgG, the patient antigen overwhelms the reagent antibody, so the complexes stay small, scatter little light and the result falls on the descending side of the curve (antigen excess, also called the postzone or hook effect). Diluting the sample brings it back to the side of the curve where scatter rises with concentration. The total protein of 118 g/L and the large M-spike agree with the repeat value. A nephelometric result that does not fit the electrophoresis should always be rerun at a higher dilution.
- B: The reagent antibody is meant to be in excess; the very high patient antigen is the problem.
- C: The high total protein and large M-spike support the repeat, not the first result.
- D: The repeat measured the IgG well, so the antiserum does recognize it.
Question 6 · Medium
In Ouchterlony double diffusion, the precipitin lines from two adjacent antigen wells meet, but one line continues past the junction as a short spur. What does this show?
1. APartial identity: shared and extra epitopes
2. BAntigen excess in one of the two wells
3. CNon-identity: no epitopes in common
4. DIdentity: exactly the same epitopes
Answer and explanation in LearnFree account
Question 7 · Medium
A grossly lipemic serum gives an unexpectedly high C3 result by nephelometry, while the controls in the same run are in range. What is the most likely cause, and the best action?
1. AAntigen excess; report the result unchanged
2. BAntibody excess; dilute the reagent antiserum
3. CLight scatter by lipid; clear the sample and retest
4. DCalibration drift; recalibrate and rerun the batch
Answer and explanation in LearnFree account
Question 8 · Medium
A latex slide agglutination test is read 10 minutes after mixing, although the kit says to read it at 2 minutes. The main risk is:
1. AA false negative as clumps dissolve
2. BNo change, as agglutination is permanent
3. CA false positive as the mixture dries
4. DA false negative from prozone
Answer and explanation in LearnFree account
Question 9 · Hard
For a tube agglutination test, 0.1 mL of serum is added to 0.9 mL of saline in tube 1, and twofold serial dilutions are made from tube 1 through tube 6. Tubes 1 to 4 show agglutination; tubes 5 and 6 do not. What is the titer?
1. A16
2. B40
3. C80
4. D160
Answer and explanation in LearnFree account
Question 10 · Easy
In a precipitation reaction, a false-negative result caused by a large excess of antigen is called:
1. AThe zone of equivalence
2. BThe prozone
3. CThe postzone
4. DSensitization
Answer and explanation in LearnFree account

Agglutination, precipitation and nephelometry

In the precipitin curve shown, increasing amounts of antigen are added to tubes holding the same amount of antibody. Zone A is the:

A test uses latex beads coated with a soluble antigen, which clump when the patient's serum contains the matching antibody. What type of agglutination is this?

In nephelometry, the detector measures:

In Ouchterlony double diffusion, the precipitin lines from two adjacent antigen wells meet, but one line continues past the junction as a short spur. What does this show?

A grossly lipemic serum gives an unexpectedly high C3 result by nephelometry, while the controls in the same run are in range. What is the most likely cause, and the best action?

A latex slide agglutination test is read 10 minutes after mixing, although the kit says to read it at 2 minutes. The main risk is:

For a tube agglutination test, 0.1 mL of serum is added to 0.9 mL of saline in tube 1, and twofold serial dilutions are made from tube 1 through tube 6. Tubes 1 to 4 show agglutination; tubes 5 and 6 do not. What is the titer?

In a precipitation reaction, a false-negative result caused by a large excess of antigen is called: