Troubleshooting guide  

This guide is intended to provide some practical hints during the test procedure and for the correct interpretation of results

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Low Actin value?  

Sometimes customers report low values (below 4ng/µl) for the housekeeping gene actin when analyzing samples with the AdnaTest Breast, Colon or Prostate Cancer. What does that mean?

In most cases this indicates problems with blood  sample collection or blood sample transportation.  A common problem during  blood sample collection is the blood volume. The blood volume in the  AdnaCollect tubes must be close to 7.5 ml. If the blood volume is <6.5 ml, the sample should be discarded because the stabilizer reagent is toxic to the cells. The blood samples must not be stored or transported outside of the specified temperature range of 4-10°C. Also note that the blood sample tubes should never come into direct contact with ice-packs during storage and transportation because partial freezing of the blood sample may occur. The transportation time for the blood samples must not exceed 24h in AdnaCollect tubes or 4 hours in EDTA tubes. 

Another problem that can occur is Rnase contamination. This is critical if it happens during the last two washing steps in the mRNA purification procedure. During RNA purification one should always make sure that the workflow is performed rapidly and in an RNase-free environment. Purified mRNA samples should always be immediately processed and reverse transcribed. Do not store them frozen or wait too long for the RT procedure.

Hence, sample results that have low actin values and are negative for tumor markers must be disregarded because they may represent false negatives due to loss of sample integrity. However, sample results that have low actin values but are positive for tumor markers are acceptable as positives as long as the control samples that are tested concurrently are valid.

Weak positive control sample?  

All or some of the bands in the positive control sample appear very weak or are undetectable.

This indicates degradation of the positive control sample due to contamination or multiple freeze-thaw cycles. Hence, it is recommended to divide and freeze aliquots of the positive control. Contact QIAGEN Hannover for a replacement if needed.

What are critical steps during CTC enrichment?  

Before use in blood samples,the beads must be pre-washed as described in the manual to remove the Sodium azide preservative because it is toxic to cells. Insufficient pre-washing of the beads will likely lead to a loss in sensitivity.

Clotting samples  

Even if EDTA is used as anticoagulant, some sample may show clotting or fiber like aggregates attached to the beads that become visible after the washing steps. This may be observed particularly in late stage metastatic patient samples and could be due to increased thrombotic -conditions in such patients. The results obtained from such samples are dubious and should be discarded.

Beads are "slippery" during mRNA purification

This phenomenon is generally observed during the washing step using “Washbuffer B”.  This phenomenon may depend upon the brand of Eppendorf tubes used.  If this problem is observed, extra care should be taken during pipetting to avoid bead loss.

Are the Oligo-dT beads included in the PCR reaction ?

Indeed the beads should be included in the PCR. During reverse transcription, the oligo-dT onthese beads is used as a primer for the reaction. Therefore, the resulting cDNA remains attached to the beads. Before transferring the cDNA sample to the PCR Master-Mix, it is essentialto re-suspend the beads carefully.

Bands in the RT or negative control samples

If bands appear in the C- negative control, a contamination mayhave occurred during the PCR step. In most cases this can be fixed by repeating the PCR. However, if bands appear in the RT control the contamination may have occurred during earlier steps in the workflow and the sample result must be regarded as invalid.

In the AdnaTest EMT/StemCell the fragment sizes of the markers is false.

This problem is sometimes observed  when the Agilent Bioanalyzer 2100 is used for fragment analysis. It is due to a misinterpretation of the lower control Marker and can be cured by using the “manually set lower marker” option in the Agilent software. This will not only correct the sizes, but also the fragment concentrations, because the integrated lower marker is also used to normalize the calculated concentrations.

Can TAQ-Polymerase or Reverse Transcriptase different from what is recommended in the manual be used ?

This should be definitively avoided. The whole test procedures have been optimized with the use of the recommended enzymes. The use of alternatives will lead to changes in sensitivity and/or specificity since the given cut-off values will not be valid anymore.

How pure are the CTCs enriched from blood samples using The AdnaTest ?

After the enrichment process for the CTCs, about 1000-1500 leucocytes remain present (100-150 when using the AdnaTest EMT/SC). However, these low numbers of leukocytes do not interfere with  CTC detection in the  test procedures since the markers utilized  are usually not expressed in leucocytes at detectable levels.