PCR Testing

By Anjana

Patients from lists are interested in having a basic knowledge about PCR testing so I thought that I would put together a post based on my own meager knowledge. Now that many patients achieve CCR, it is unsurprising that patients want to know about the technique recommended to monitor post-CCR status. Hope folks find this post
useful.

PCR or Polymerase Chain Reaction test is done in CML to detect minimal residual disease (MRD).

Patients are in MRD when the high
leukemic burden is lowered and they are in complete cytogenetic remission.(CCR) They still have the leukemia so this state is called MRD. When in MRD state, the leukemia is undetectable by conventional cytogenetics and FISH and PCR is the best method to monitor MRD.

It is the most sensitive test for CML capable of up to 10(-8) sensitivity levels and therefore can detect 1 leukemic cell in up to 1 million or more good cells. PCR testing can be done from the blood or the marrow.

Detecting one leukemic cell in a million is like looking for a needle in a haystack. So, how exactly is it done? A very layman explanation is given taken mostly from Dr. Goldman's excellent
article, "Cytogenetic and Molecular Monitoring of Residual Disease
in Chronic Myeloid Leukemia".

PCR is a molecular technique used to identify genes using RNA and DNA. The PCR reaction allows millions of copies of a single gene to be produced by the process of amplification. That is how one needle is found in the haystack- by amplification to produce many copies which can then be seen.

m-RNA (messenger RNA) is extracted from the blood of patients and reverse transcripted into cDNA (copy DNA). That is why the test is called RT-PCR or reverse transcriptase polymerase chain reaction.

The cDNA is subjected to a rapid cycle of heating and cooling aided by enzymes called polymerases. Each cycle takes 1-3 minutes, so repeating the process for 45 minutes can result in a chain reaction that creates millions of copies of a specific strand of DNA. Therefore if 1 leukemic cell containing the BCR-ABL gene is present in a million cells looked at, it can be detected by this amplification procedure used in PCR.

The level of sensitivity can be increased by 2-3 logs by subjecting the DNA to nested primers. This uses a second round of PCR using a new set of primers internal to those used in the first
amplification. Since two rounds of amplifications are done, nested
RT-PCR is more sensitive than normal RT-PCR.

Contamination can give rise to false positive results and false negative results can also be obtained due to lack of RNA and DNA integrity. Thus, an internal control or housekeeping gene is used such as ABL, BCR or G6PDH (glucose â€"6-phosphate  ehydrogenase). RT-PCR can be qualitative or quantitative.

Qualitative RT-PCR will detect the presence or absence of BCR-ABL transcripts whereas quantitative RT-PCR will give a percent of BCR-ABL present and thus provides a good way of monitoring for MRD.

Usually, a competitive RT-PCR is used which involves the co-amplification of an internal control (the competitor) at graded concentrations with the target gene. The BCR-ABL copy number is normalized to the number of endogenous control gene transcripts and expressed as a percentage ratio.

However, competitive PCR is very time-consuming so another quantitative method was determined which was more rapid and efficient. This is the real-time quantitative PCR or RQ-PCR. The precision and speed occurs because the quantification is done during
the exponential phase of PCR amplification.  RQ-PCR minimizes post-PCR manipulation and so in theory minimises the risk of contamination. Again, the BCR-ABL transcripts are expressed as a percentage ratio comparing the leukemic gene to normal control gene used. For example: BCR-ABL/ABL as a percentage or BCR-ABL/G6PDH as a percentage.

RQ-PCR is rapid, efficient and can give the results as a number which can be tracked over time. However, according to the abstract below, the sensitivity of a nested RT-PCR may be higher so once a patient tests positive on RQ-PCR, he can then test on the more sensitive nested PCR. Look at the difference between real-time PCR and a nested PCR this way. A leukemic cell may not be picked up at the sensitivity level of real-time quantitative PCR but when you subject the sample to a second round of amplification as done in a
nested PCR (which gives greater sensitivity), the leukemic cell gets
detected.

It is a good idea always to ask the sensitivity of the PCR being used as some are as sensitive as detecting 1 leukemic cell in 1 million cells and others as insensitive as detecting 1 leukemic cell in 1000 cells. Obviously, one should try to be tested on the more sensitive techniques. Some of the big centers may first test you on a less sensitive PCR technique and then graduate you to a more sensitive PCR technique when you have tested negative on the less sensitive one.

This post on PCR testing is a very layman version attempting to explain the various types of PCR testing and I am happy to be corrected by others more knowledgeable

Abstract on nested and RQ-PCR

Other pages on PCR
  CML Bouys
  CML Primer
  From Ambion

CML Prisms--Literature

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