Step 1

The sample for analysis is obtained by the physician during bronchoscopy. The region containing the suspicious lesion is rinsed with isotonic saline solution and the fluid aspirated (a bronchial lavage sample). After fixation of approximately 1 ml aspirated fluid with Saccomanno  reagent (50 % ethanol plus 2 % polyethylene glycol, "Carbowax"), the Epi proLung BL Reflex Assay can be performed in a molecular-diagnostic laboratory.

Step 2

The cellular fraction of the sample is used for cell lysis and subsequent chemical conversion of the cellular DNA using bisulfite. This chemical treatment converts unmethylated cytosines to uracils, resulting in a base-pair change (CpG → UpG) at each unmethylated CpG site while methylated CpG sites remain unaffected.

Step 3

Bisulfite-converted DNA is purified using a silica membrane-based nucleic acid extraction.

Step 4

The bisulfite-converted DNA is subject to PCR amplification specific for ^mSHOX2.  The primers, blockers and probes used are sensitive to the base-pair changes introduced by bisulfite conversion and can discriminate between methylated and unmethylated DNA.

Step 5

The test determines the relative DNA methylation of the SHOX2 gene in a background of unmethylated converted DNA. Relative quantitative values of SHOX2 methylation from the real-time PCR are subsequently converted into qualitative test results by dichotomizing to positive or negative test result using a cut-off value.

Epi proLung BL DNA Preparation Kit

for cell lysis followed by bisulfite conversion and purification of DNA from Saccomanno-fixed bronchial lavage samples.

Epi proLung BL real-time PCR Kit

for detection of ^mSHOX2 in bisulfite-converted DNA. The test is a duplex-PCR that amplifies methylated SHOX2 DNA as well as ACTB DNA (beta-actin) for DNA quantification. A valid ^mSHOX2 test result is either positive or negative provided valid control measurements are obtained.

Epi proLung BL Work Flow Control Kit

contains positive and negative control samples to establish the successful execution of the entire work flow from cell lysis to DNA purification and bisulfite conversion to amplification and detection.