Frequently asked questions in clonality testing
- What is the clonality detection rate of the BIOMED-2 / EuroClonality primers
- How can I learn clonality testing?
- Which Taq polymerase should I use?
- Can agarosegels be used as read-out for clonality-testing?
- DNA extraction from formalin-fixed-paraffin-embedded tissues (FFPE)
- Does the use of unbuffered or buffered formalin affect molecular clonality testing?
- Is the 3730 DNA analyzer (Applied Biosystems) suitable for clonality testing?
- Which software can be used?
- Protocols and literature about clonality.
- Control samples.
- Why can skin biopsies be problematic for clonality testing?
- Commercially available BIOMED-2 EuroClonality primers (Invivoscribe) versus own primer mixes
- What are the non-specific products in the EuroClonality/BIOMED-2 PCRs?
1. What is the clonality detection rate of the BIOMED-2 / EuroClonality primers
The high detection rate of clonality of the BIOMED-2 primers is due to the primers and the readouts, and also to complementarity of the approach. For testing B-cell clonality, we highly recommend testing IGK next to IGH PCRs. Inclusion of IGK testing increases the detection rate for clonality in B-cell lymphomas to 99%. Note that using the FR3 PCR as a single clonality target, the detection rate of clonality is only 63%. Similarly, we recommend testing TCRB next to TCRG, because TCRBis a valuable tool for high detection of clonality of T-cell lymphomas. Further details can be found in the Leukemia 2007 publications (see question 9).
2. How can I learn clonality testing?
There is always the possiblility to participate in the EuroClonality/BIOMED-2 workshop: “Clonality Assessment in Pathology”, which is annually held in Nijmegen, The Netherlands. For more information you can look at the EuroClonality website.
3. Which Taq polymerase should I use?
The BIOMED-2 multiplex PCRs are designed using AmpliTaq Gold, which for that reason is the enzyme we recommend to use in the diagnostic setting. Other Taq polymerases have not been tested or approved for clonality testing in the consortium, but migth work as well.
4. Can agarosegels be used as read-out for clonality-testing?
No, agarose gel electrophoresis can never be used as a readout for clonality testing, simply because the resolution of the system is not sufficient to discern a monoclonal band from a polyclonal smear.
Either Genescanning or Heteroduplex analysis using polyacrylamide gels are required. Information on the readouts, as on the PCR conditions is provided in the Leukemia paper: “Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations” by van Dongen et al., 2003. (vol 17:2257-2317). At page 2265 you will find the protocols for heteroduplex analysis of PCR products, including the denaturation conditions of the PCR products. At page 2263, you will find the specific PCR conditions.
5. DNA extraction from formalin-fixed-paraffin-embedded tissues (FFPE)
A fixed DNA input is necessary for clonality assessment not only for reasons of standardization, but also to be able to exclude pseudoclonality.
A reliable concentration measurement of DNA is therefore essential. A reliable measurement of DNA derived from paraffin-embedded tissue is only possible after purification of the DNA-sample (to get rid of small oligonucleotides, and debris) by affinity-based micro colums as provided by Qiagen for example.
There are also protocols in which a crude DNA-extract (so Proteinase-K treatment, followed by a Prot K inactivation step) is used, without further purification of the DNA. In those DNA- extracts, the measured concentration does not reflect the real DNA quantity because of RNA co-presence and protein debris, which will also be apparent from the A260/280nm measurement.
Some protocols use DNA-spin columns, either the QIAGEN-kit protocol or a related one recommended for FFPE samples. Although the DNA is purified, there still is the probability of PCR-inhibiting factors in the DNA-sample, therefore two concentrations: 50 and 200 ng DNA per PCR (50 ul), are used for input in each PCR. Adjustment of the DNA-input may be necessary when there is a low percentage (5-10%) of representative B or T-cells present in the tissue sample.
6. Does the use of unbuffered or buffered formalin affect molecular clonality testing?
We strongly recommend the use of buffered formalin (4%) in the routine pathology setting. The use of buffered formalin (and small tissue biopsy specimens, and standard fixation times 8 hours – 16 hours) will improve the quality of the diagnostic specimen and will result in PCR- amplification of fragments around 300 or 400 bp, which allows proper clonality testing.
Unbuffered formalin is an aggressive fixative, which results in more extensive degradation of DNA and produces very small fragments in the quality control PCR. For overview see Groenen et al (in press)
7. Is the 3730 DNA analyzer (Applied Biosystems) suitable for clonality testing?
The 3730 DNA analyzer from Applied Biosystems can be used for clonality testing. The 3730 DNA analyzer is an instrument having 48 capillaries with a length of 50 cm. The polymer is: POP7 .
Different fluorochromes can be detected. The following fluorochromes can be run simultaneously:
|Dye Set D||FAM-HEX-NED-ROX||(ROX is used as size standard)|
|Dye Set G5||FAM-VIC-NED-PET-LIZ||(LIZ is used as size standard)|
|Dye SET Z||FAM-VIC-NED-ROX||(ROX is used as size standard)|
8. Which software can be used?
Genotyper version 3.7
Genemapper version 4.0
Genemarker version 2.4.1
Peakscanner (free download via Applied Biosystems)
9. Protocols and literature about clonality
10. Control samples
Testing of each clonality -target should be performed in duplicate and a polyclonal control is essential for interpretation of patient -data. In addition, a monoclonal control can be added to check the different rearrangements. Possible types of polyclonal controls include tonsil DNA (esp. for Ig targets) and mononuclear cell fraction (MNC; esp. for TCR targets).
11. Why can skin biopsies be problematic for clonality testing?
A reason why skin biopsies can be problematic is that these biopsies often contain quite scanty lymphoid populations in combination with a high proportion of keratin etc. This can result in over-interpretation of incidentally dominant peaks. These patterns are typical of skin lesions. It is important to run duplicate reactions, to increase the amount of DNA analyzed, and to assess additional biopsies if these are available to look for consistently expanded peaks. It may also be helpful to run the products on a heteroduplex gel, which can overcome the problem of over-interpretation of incidentally dominant peaks.
12. Commercially available BIOMED-2 / EuroClonality primers (Invivoscribe, IVS) versus own primer mixes
Commercial BIOMED-2 / EuroClonality primers are tested and produced under standardized conditions and delivered with optimized reagents. In contrast, the use of BIOMED-2 primers that are ordered and mixed locally (based on the description of primers in Leukemia 2003; 17:2257-2317) bears the risk of missing certain types of rearrangements and to produce false negative results. Therefore in-house primer mixes should only be used when sufficient expertise is available within your laboratory to collect appropriate controls and QC the primers in individual pairs. Without own extensive primer testing, we would recommend to use commercially produced and quality controlled primermixes.
13. What are the non-specific products in the EuroClonality/BIOMED-2 PCRs?
Since the EuroClonality?BIOMED-2 PCRs are multiplex PCRs there are some non-specific bands, which are listed in the table below.