Input and sample collection for the Illumina Protein Prep (IPP)

Which sample inputs and input amounts are supported by the assay?

• 55 µl of human plasma with EDTA or 55 µl of human serum, collected according to the guidance in the Product Documentation.

• Samples collected using unsupported methods not specified for this assay have poor results.

Is it possible to run the assay with pooled samples (for CV calculations, etc.)?

Yes, pooled samples can be run. The Calibrators and QC controls used in this assay are also pooled samples.

If collecting multiple samples over a period of time before performing the assay, is there a way to store samples before assay preparation?

Processing and freezing of plasma and serum samples must be completed within 2 hours of collection. Processed samples can be stored at -80°C, and freeze-thawed a maximum of three times.

Is there any guidance on serum and plasma QC prior to input into the assay?

Illumina does not have any formal guidance for plasma and serum QC prior to running the assay. However, it is recommended that samples that look compromised are documented so that they can be cross referenced against assay QC metrics after processing. Examples of compromised samples could be hemolyzed (pink) or highly lipemic (milky white) samples.

Is there any QC that can be performed on the input to assess in advance whether or not a questionable sample will be successful in the Illumina Protein Prep assay?

Not at this time. Generally, Illumina recommends testing samples of questionable origin (eg, very old or not collected according to Illumina specifications) before proceeding with large numbers. Performance is not guaranteed.

Are there any commercially available plasma/serum samples that can be used as positive controls?

The Illumina Protein Prep assay contains commercial plasma/serum controls which are used in the normalization process. If additional controls are needed, they can be purchased here.

Is there more data regarding comparison between serum and plasma?

Illumina generally recommends plasma over serum whenever possible as it provides a better reflection of the secreted proteome in the absence of clotting.

Table summary of expected performance metrics (Median CV calculated using samples from healthy donors). Table is from this datasheet. The standard deviation table above is described for 10 plates.

Can labs use existing plasma samples which have protease inhibitor added?

Illumina does not expect to see any assay inhibition due to protease inhibitors. It would rather preserve signatures and stabilize proteins.

What are some known interfering substances?

What is known about different sample types and how they will perform in this assay?

How can hemolyzed, lipemic, or icteric samples be identified? How would these samples perform in Illumina Protein Prep?

• Hemolyzed blood is blood in which red blood cells have been broken down or lysed. Hemolyzed blood appears reddish or pinkish, while normal blood plasma or serum is clear and straw-colored. Hemolyzed samples can significantly alter the concentrations of various analytes.

• Lipemic samples appear cloudy or milky, which is caused by elevated levels of lipids in the blood. Lipemic samples can interfere with the Illumina Protein Prep assay.

• Icteric samples appear yellow or brownish-yellow, which is caused by high levels of bilirubin in the blood. Icteric samples can interfere with the Illumina Protein Prep assay.

• The image below shows hemolyzed, lipemic, normal, and icteric samples, respectively.