Can plasma, hemolytic, lipemic and icteric samples

be used in ImmunoCAP for IgE testing?

Studies have been performed of EDTA-, heparin-, and citrate-plasma, as well as of hemolytic,
icteric, and lipemic samples. No significant difference in the results was found using these
types of samples, compared to serum.

Specimen Collection, Handling and Preparation:

ImmunoCAP assays have been validated for diagnostic use for serum and plasma. Serum and
plasma (EDTA-, Heparin- or Citrate-plasma) from venous or capillary blood can be used in
all ImmunoCAP instruments and there are no significant differences in results using samples
prepared using these different methods.
Venous or capillary blood should be collected and the serum or plasma should be separated by
centrifugation. Specimens may be kept at room temperature for shipping purposes only,
otherwise stored at +2-8°C if assayed within one week after collection or at -20°C if assayed
later. Avoid repeated freezing and thawing.

Other body fluids have not been validated but may be used for research purposes.

All samples should be handled as potentially hazardous.

Sodium Azide is routinely used in Phadia controls and may also be used as preservatives in
samples, if desired.

No interference from gels in primary sample tubes has been observed.

Tot IgE:Plasma vs. serum samples

There are no significant differences in results between plasma and serum samples in
ImmunoCAP Total IgE. Neither icteric, lipemic nor hemolytic samples do interfere with
results in ImmunoCAP Total IgE.

24 samples were tested as serum and as EDTA, Heparin, and Citrate plasma.

Each sample was tested in 3 replicates in one assay. The quotitient between the results
obtained in serum samples and those obtained in the respective type of plasma samples was
calculated.

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The following mean quotitients for the samples were obtained:

EDTA- Heparin- Citrate-

plasma plasma plasma
Mean
quotitient,
0.95 1.00 1.03
positive
samples

Conclusion: There are no significant differences in results between plasma and serum samples
in ImmunoCAP Total IgE.

sIgE: Plasma vs. serum samples

There are no significant differences in results between plasma and serum samples in
ImmunoCAP Specific IgE.

24 samples (1 negative) were tested as serum and as EDTA-, Heparin- and Citrate-plasma.

Each sample was tested in 3 replicates in one assay. The quotient between the results obtained
in serum samples and those obtained in the respective type of plasma sample was calculated.

The following mean quotients for the samples were obtained:

EDTA-plasma Heparin-plasma Citrate-plasma

Mean quotient, 1,00 1,03 0,99
positive samples
The negative serum sample gave a negative result in all types of plasma:
EDTA-plasma Heparin-plasma Citrate-plasma
Result of negative Negative Negative Negative
sample

Conclusion: There are no significant differences in results between plasma and serum samples
in ImmunoCAP Specific IgE.

Tot IgE: Interference of icteric and lipemic samples

3 icteric and 3 lipemic samples were tested. Each sample was analyzed in 4 replicates in one
assay. The quotitient between the results obtained in non-icteric and non-lipemic serum
samples, and those obtained in the icteric and lipemic serum samples was calculated.

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The following mean quotitients for the samples were obtained:

Lipemic samples Icteric

samples
Mean quotitient 1.02 1.01

Conclusion: Icteric or lipemic samples do not interfere with results in ImmunoCAP Total IgE.

Tot IgE: Interference of hemolytic serum

6 samples were tested for low and high degree of hemolysis.

Each sample was analyzed in 4 replicates in one assay.

The quotitient between the results obtained in non-hemolytic serum samples, and those
obtained in the serum samples with high and low degree of hemolysis was calculated.

Low hemolytic High hemolytic

degree degree
Mean quotitient 1.02 0.99

Conclusion: There is no interference of hemolytic samples on results in ImmunoCAP Total

IgE.

sIgE: Interference of icteric, lipemic and hemolytic and samples

Icteric, lipemic or hemolytic samples do not interfere with results in ImmunoCAP Specific
IgE.

Interference with other Immunoglobulines

No cross-reaction with IgG1, IgG2, IgG3, IgG4, IgA, IgM and IgD in the ImmunoCAP
Specific IgE.

Assay was detectable using physiological concentrations of the Immunoglobulins:

• IgG 8-16 mg/ml

• IgM 0.5-1.9 mg/ml
• IgA 1.4-4,2 mg/ml
• IgD < 0.4 mg/ml

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Interference with high Total IgE levels in serum
Influence of high total IgE (e.g. 5000 -10000 kU/l) in individual samples is normally very
low. However, very low levels of allergen specific IgE antibodies should be evaluated with
caution when total IgE values are above 1000 kU/l.

Heat-treatment of patient sera and influence of the IgE test results

When samples are heat-treated, some proteins undergo steric changes that can be irreversible
or partly irreversible. Very high temperatures (boiling) can destroy the proteins completely.

It is known that some parts of the IgE molecule (e.g. the receptor binding part) change their
conformation during heat-treatment (56° C for 30 minutes).

In ImmunoCAP tests for total IgE (tIgE) and allergen specific IgE (sIgE) stable epitopes of
the IgE molecule are used for the anti-IgE antibody conjugate attachment. Mild heat treatment
therefore has no influence on the IgE test results.
Stability of IgE antibodies in serum
Samples are stable at +2 - +8 °C for at least a week. Samples can be frozen and thawed at
least twice without any effect on results (IR 6) Studies have also shown that IgE antibodies
are stable in serum samples after storage at -20º C for years (IR11-13).

This has been verified by testing the same samples in Pharmacia CAP System in 1987,again
in ImmunoCAP100 in 1995 and over again in ImmunoCAP 1000 in 2004. The results are
shown in Figure 1. This is an example of truly impressive reproducibility over time (IR 11).

Figure 1. Consistency over time. Comparison between results obtained in Pharmacia CAP
System™ in 1987, in ImmunoCAP®100 in 1995 and in ImmunoCAP™1000 in 2004.

Notes:
Venoms and drugs
Blood samples for testing with drugs and venom ImmunoCAP should be collected during or
close to the event, preferably not later than 6 months after exposure. If the test result is
negative and an IgE-mediated reaction is still strongly suspected, it is advisable to draw a new
sample and repeat the test at 5 to 6 weeks (1,2).

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