Hi y’all, I am due for my 3 month blood work. My Doc has ordered a Fish instead of a PCR. When questioned over the phone through a nurse the Doctor said Fish is the same as PCR. With all my reading and studying I do not think this is accurate and I am disturbed about her knowledge. Please help and advise. Thanks soo much, Dawn
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PCR versus Fish
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I am, too, at the 3-month point and awaiting. I had my bloods taken at 12 weeks after the start of imatinib and I am awaiting results that take 6 weeks to produce.
My doctor, knowing that I like detail, produced the following slide on the screen of her computer which she tells me is the prime reference used for CML:
https://www.leukemia-net.org/content/leukemias/cml/recommendations/e8078...
There are indeed two tests, FISH and PCR which she describes as requiring:
The slide is very helpful
That slide is indeed very good - though can be hard to read at times for some people. We like it very much here, and you'll also find it under "Patient Info" then "Treatment Guidelines" in the main site menu.
There, you'll also find the NCCN guidelines in use in the USA. They are quite similar to the European Leukaemia Net (ELN) ones.
The ELN guidelines are due a refresh, which has been in the mix for a while and it's a bit frustrating that they haven't finalised them yet. When done, this will give guidance around cessation / reduction etc., which are "standards" many patients are eager to see.
ESMO (European Society for Medical Oncology) have published their own updated clinical guidelines in absence of an update from ELN. It's a more technical read, but for the more sciencey patient it's a good read.
https://www.esmo.org/Guidelines/Haematological-Malignancies/Chronic-Myel...
Hi Dawn,
It looks like the ELN guidelines are recommending PCR and CBA (Chromosome Banding Analysis) at the three month checkup. I think my doctor had all of them (PCR, FISH, CBA) performed at my three month check. It seems that there wouldn't be a downside to doing all of them, except that the FISH and CBA were performed on bone marrow. 
FISH is a measure of actual CML cells observed under the microscope. It is expressed as a percentage - "how many cells with CML out of 200 cells counted". Most patients at diagnosis will have 100% CML cells observed which means 200 out of 200 counted showed the bcr-abl gene (which is dyed to fluoresce so they can see it). This means FISH = 100%.
The most important milestone achieved during TKI treatment is for FISH to reduce to zero (i.e. no CML cells observed under the microscope). FISH = zero is labeled cytogenetic remission (i.e. CCyR) and is the single most important indicator of no CML disease progression. Once you achieve FISH = zero and keep it for six or more months chances of disease advancing is near zero. This is very good.
Once FISH goes to zero - it does not mean you no longer have CML cells - just that they are so few that they are not easily observed anymore in samples. This is where PCR comes in.
PCR is a molecular measure of CML "activity". CML cells produce proteins unique to the cell. PCR is a process where the CML gene is amplified to produce the bcr-abl protein which is then measured and calibrated against a control gene (which makes different proteins that are normal). This ratio - is also a percentage - expresses the residual amount of CML left in the body. It is not very precise, but is very sensitive to the bits of CML that are left. PCR acts like a "canary in the mineshaft" in revealing trends - CML getting less or CML coming back. It has an error range of one log - that is a factor of 10 - this is why PCR reporting to 15 million decimal places is stupid.
Once FISH gets to zero - PCR is approximately less than 1.0%. PCR < 0.1% is VERY VERY LOW residual level of CML. PCR <0.01% is functionally equivalent to no detection and is at the limit of the technology.
Hope this is useful.
Hi Dawn,
You might want to give our Q-PCR Primer for Patients a read.... see link below.
Re: cytogenetics/FISH and qualitative PCR testing: (used to confirm the presence of BCR/ABL1 and thus diagnose Ph+CML)
Genetic Tests used at diagnosis and in the first months of TKI therapy
Some sort of genetic testing will be done to look for the Philadelphia chromosome and/or the BCR-ABL1 gene. The following types of tests can confirm or deny a diagnosis of CML.
Cytogenetics: also called Karyotyping
Chromosomes can only be seen when cells are in the dividing phase. Blood or marrow samples are cultured in the lab so that the cells begin to grow and divide, although this is not always successful. The dividing cells are looked at under a microscope to assess the number of immature vs mature cells as well as changes to chromosomes (pieces of DNA) and, in the case of CML, to detect the Philadelphia chromosome. Sensitivity is limited, typically detecting 1 out of 20 cells tested. Even when the Philadelphia chromosome is not seen, other tests can confirm the presence of the BCR-ABL1 gene.
Qualitative PCR:
The polymerase chain reaction based qualitative test is used to diagnose Ph+CML by confirming whether or not BCR-ABL1 gene transcripts (copies) are present in a blood and/or bone marrow sample. It can detect very small amounts of BCR-ABL1, even when the Philadelphia chromosome is not detected in bone marrow cells with cytogenetic testing.
FISH: Fluorescence in situ hybridization
A more sensitive method than cytogenetics, testing upwards of 50 to 200 cells. FISH uses probes labelled with fluorescent dyes which ‘light up’ the fused BCR-ABL1 gene sequence. Fluorescent probes are sections of single strands of DNA complementary to the specific portions of the DNA of interest, in this case, the ABL1 and BCR-ABL1 genes. When slides are examined using a special microscope, the genes that match the DNA probe appear as bright spots on a dark background. The test determines the percentage of cells in a sample containing the BCR-ABL1 gene. It can be used on either blood or bone marrow samples without the need to culture the cells, so results are available more quickly than with conventional cytogenetics.
Q RT-PCR -Quantitative Reverse Transcriptase Polymerase Chain Reaction: What the test measures and its relationship to other tests
At diagnosis, virtually every white cell in a blood or marrow sample will be leukaemic (Ph+) so the result should, in theory, be 100% Ph+. However, because there are higher levels of Ph+ cells present at diagnosis, q-PCR (quantitative) testing is not accurate, which is why Ph positivity varies between 50% and 100%. This test may be used to establish a baseline value of Ph+ cells at diagnosis.
After the start of therapy, q-PCR is used at specific time points after cytogenetic/FISH tests. Once tests show that the Ph+ cell population has reduced to less than 10%, q-PCR testing can more accurately quantify the amount of residual disease left in the marrow. The goal of TKI therapy is to reduce the abnormal BCR-ABL1 gene to a deep molecular level, preferably to at least 0.1% (MMR/MR3).
During the first 3, 6, 9 and 12 months of therapy, Ph+ cells should reduce significantly. When the level of Ph+ cells falls below 1%, q-PCR testing is extremely accurate and will be used to monitor the stability of a molecular response. Under ideal conditions, this test can detect 1 Ph+ cell in every 100,000 cells, although more commonly it detects 1 Ph+ cell in every 10,000.
How Q-PCR testing works in practice
Q-PCR testing extracts the available mRNA in a blood or marrow sample. A test result is expressed as a percentage showing the ratio between of mRNA from normal control gene transcripts, for example, ABL1 or BCR, compared to mRNA from the abnormal BCR-ABL1 gene transcripts present in a sample.
To perform the test, samples of blood or bone marrow are sent to the molecular pathology laboratory where mRNA is extracted from the white cells. There are a variety of ways of doing this and methods vary to a greater or lesser degree between laboratories.
To ensure results that more accurately reflect the number of Ph positive cells present in an individual, samples taken from patients must contain adequate numbers of copies of a control (normal) gene. The control genes most commonly used are ABL1, BCR or GUSB. There is no consensus as to which of these normal genes is the best control to use. The choice lies entirely with the laboratory performing the test.
Further reading from our Q-PCR booklet