45 replies to this topic

[Lucas]

Hi everybody, i had a great (not good) surprise today. I went to my doctors office and got my 5 months pcr (it was done in a different laboratorie) and it was 0.5% IS. What happened is: i had my 6 months results prior to the 5 months and it was 6.83% IS. My first pcr was 14.41%. There are 2 options: an error or i'm losing my response very quickly. anyone in this situation?

 

thanks in advance

[Trey]

This can be the result of several issues.  Some you can figure out, others you cannot.  The main issues are:

 

1) PCRs can vary by as much as .5 log even using the same equipment.

 

2) The labs may use a different "housekeeping gene" also called a "control gene" in their PCR testing.  If they are different, variations can occur.  You can ask the Onc to call the lab and find out which control gene they use (good luck with that).

 

3) The age of the sample when the PCR is done varies widely.  A PCR done within 12 - 24 hours of blood draw is more accurate than those done after 24 - 48 hours due to sample degradation.  PCR requires live cells.  Sample degradation starts immediately after sample draw and increases exponentially as the sample ages past 24 hours. (See added information later in this thread)

 

4) Makes of PCR equipment and the chemical reagents often vary in result.

 

5) Labs can make mistakes.  Errors can be introduced.

 

Overall, any single PCR should not be used to make a decision.  Only multiple PCRs and/or trends can tell the real story.

 

Better yet, FISH tells a more accurate story until CCyR is reached (approx 2 log reduction or 1% IS).  FISH should be done until CCyR is attained.

Edited by Trey, 07 August 2014 - 04:55 PM.

[Lucas]

Thanks, trey. 

 

They don't run PCR tests in my city, so they sent to another one far from here. My five month sample was taken in 05/10 and they ran the test in 05/17 (but i only got the results yesterday). My 6 month sample was taken on a monday and i got the results the next friday (5 days after). So, i think this 5 month pcr is totally out of the curve. I'm waiting for my cytogenetics now. Thanks again!

[Trey]

Let me expand a bit on the PCR sample degradation issue.  Most PCRs for CML BCR-ABL use ABL1 as the control gene.  ABL1 is different than the ABL in BCR-ABL, but is related in a way that makes it a relatively poor choice for control gene.  The biggest issue is that BCR-ABL degrades in a blood sample much faster than ABL1.  And since the PCR result is expressed in terms of BCR-ABL/ABL1, if the numerator and denominator do not degrade at similar rates, the result will vary widely.  In the attached article in Figure 4 under the "RESULTS" paragraph you can see the extreme disparity between BCR-ABL and ABL1 degradation from very early (within the first hour), and how at the 24 - 48 hour period the disparity is enormous.  So if the ABL1 degrades at a rate far slower than BCR-ABL, the final result will be a PCR result that is too low, getting lower as time goes on.  So the faster the sample is put through the PCR process, the more accurate the result will be.  This is one reason why it is important to rely on PCR trends rather than isolated PCR results.  And it is also important to keep the variables to a minimum, helping to ensure the PCR will be done under relatively similar sets of conditions each time.

 

http://www.ncbi.nlm....les/PMC1867593/

Edited by Trey, 07 August 2014 - 05:01 PM.

[Lucas]

Thanks again, trey. As i said, the sample was collected in 06/10 and the test was performed in 06/18 (so, 8 days after). I think that was the reason i had a low pcr. I'll do another pcr on monday (less than a month after the last one) just to see the trend.

 

Cheers!

[Lucas]

Trey, just another question: how is the process of the pcr test? how long does it take to complete the test? the report is done as soon as the test finished?

 

thanks in advance!!!

[LivingWellWithCML]

Let me expand a bit on the PCR sample degradation issue.  Most PCRs for CML BCR-ABL use ABL1 as the control gene.  ABL1 is different than the ABL in BCR-ABL, but is related in a way that makes it a relatively poor choice for control gene.  The biggest issue is that BCR-ABL degrades in a blood sample much faster than ABL1.  And since the PCR result is expressed in terms of BCR-ABL/ABL1, if the numerator and denominator do not degrade at similar rates, the result will vary widely.  In the attached article in Figure 4 under the "RESULTS" paragraph you can see the extreme disparity between BCR-ABL and ABL1 degradation from very early (within the first hour), and how at the 24 - 48 hour period the disparity is enormous.  So if the ABL1 degrades at a rate far slower than BCR-ABL, the final result will be a PCR result that is too low, getting lower as time goes on.  So the faster the sample is put through the PCR process, the more accurate the result will be.  This is one reason why it is important to rely on PCR trends rather than isolated PCR results.  And it is also important to keep the variables to a minimum, helping to ensure the PCR will be done under relatively similar sets of conditions each time.

 

http://www.ncbi.nlm....les/PMC1867593/

 

Trey - as always, this is very useful information.  Thank you for sharing it!  Just jumping on this thread since I got my latest PCR results and I registered a pretty big drop (0.05% IS to "undetectable" in 4 months), and I have to say that I'm a bit skeptical of the result for a number of reasons.  Could I ask a few questions about this (adding a bit to the questions that Lucas has)?

 

• How long does it take to run an RQ-PCR for BCR-ABL/ABL1 and obtain a result? (I assume 2 - 4 hours)


• If we had a choice for a control gene, what should it be? (My lab uses ABL1, which I guess isn't desirable)


• In looking at my report, I *think* Emory ran the PCR on the same day as the blood draw [I made the mistake of getting my most recent blood draw on a Friday morning.  I should know better!].  The report has three data fields: Collected (8/8/14), Received (8/8/14), and Signed Out (8/15/14, so one week after Collected/Received).  I don't know if Received is the date when the test was actually run?  I'm even thinking the unthinkable ... is it possible that the sample just sat around for a week on-site before the test was run?


• You would think that a responsible research institution that's capable of handling Ebola patients would have good controls in place to limit sample degradation and get the test underway swiftly.  I always stress to the nurse the importance of getting my PCR started and please rush that blood where it needs to go - they get very annoyed with me.  My blood draw was at 9am on a Friday, so I have to think that the RQ-PCR was run on the same day.  But I have no direct evidence ... for all I know, it might've sat in a vault all weekend.  In your opinion, how important is it to look into this further?


• And my most important question <smile> -- Trey, would you mind if I asked if your own PCR tests are run on-site at your treatment center or are they shipped to a separate lab, and which control gene  is used?  You are such a calming force on this board for me (and pretty much everyone else), so I am curious what your routine is, since it appears to be effective at monitoring your "undetectable" disease burden.

I know I'm enamored a bit with the details, but I hope that the feedback it generates will be helpful for others!

[Lucas]

Hi, dan. how are you?

 

I found this topic in another forum. hope it's usefull:

 

https://www.mail-arc...m/msg09427.html

[Trey]

Dan,

 

PCR takes a couple hours after set-up is done.  The report is ready soon after but sits idle in several Inboxes waiting for review as it works its way to you.  No good reason for the usual 2 - 3 week report lag.

 

Regarding choice of control gene, GUSB (or GUS) has the best correlation with BCR-ABL degradation, and is recommended by the article I cited above. 

 

Trey's First Law of PCRs: Never draw PCR sample on Friday!!!! 

Trey's Second Law of PCRs: Most labs do batch processing one or two days per week [NOTE: Edited]  To get a valid result provide the sample the day before batch processing.

https://www.testmenu...ry/Tests/252798

 

I found out what specific day my lab does PCRs (Thursday) and I go the day before to give the sample.  Most labs do PCR batching one or two days per week to save money.  So they do them all at one time on a specific day.  But batching on Monday is ridiculous since all samples will be degraded unless you sneek one in early Monday AM.  Is that calming?  Probably not.

 

 

Lucas,

 

That link was quoting me from a post I had done when I posted on that site years ago, so same story.

Edited by Trey, 19 August 2014 - 01:21 PM.

[LivingWellWithCML]

Wow, that's really interesting and, of course, unnerving!  Ugh.  But thanks Trey - your insights are always helpful, even if the insights aren't always positive, LOL.

 

So I'm trying to reconcile this testing protocol with my past PCR results (all peripheral blood), so I think it's safe to assume that the Monday batching has been normal course of business for at least the last couple of years.  I looked at two past results that were 0.1% IS back in 2012 where both blood draws were taken on a Tuesday.  If they were testing only on Mondays, then it would mean that the sample sat (refrigerated it appears) for an entire week before the PCR test, yet they registered a meaningful positive ratio right at MMR.  How is this possible?

[tiredblood]

My initial PCR was drawn on a Friday and performed on a Monday evening (3 days).

the second one was drawn on a Thursday morning and performed on Tuesday morning (5 days).

The third was drawn on a Friday evening and performed on a Wednesday afternoon (5 days).

Each was performed at Quest Diagnostic lab located in the hospital where I go to see the doctor at the adjoining clinic and lab.  Each report has "medium importance" listed on the report sheet.

 

When I went to Quest's website and searched for the CPT code 91065, listed on the report, I learned that they only check for the originally abnormal mutation in subsequent PCRs, in my case, the P210.  I also read that the lavender tube is preferred, but they will accept the yellow-top citrate or green-top heparin tubes.

 

I think I will try to get the labs drawn on a Monday.  If it is still going to set around for 5 days, I guess they are examined as the specimens are accessioned.  I know what questions to ask now.

[tiredblood]

Now that I've dug a little bit further on Quest's website, it says that specimens should be at room temperature or refrigerated with specimen stability of 72 hours.  Now, I'm wondering what's up with two of mine taking 5 days.

[Trey]

I wrote an article about this a couple years ago called "PCR Confusion". 

http://www.cancernet...t's-perspective

Even degraded samples will often give a result because of the control gene unless the sample is "rotten" (all cells DNA unzipped).  The issue is the variation in the rate of degradation.  If both degraded on the same slope, then doing a PCR after several days of refrigeration would be OK.  But since the control gene degrades more slowly, the result is not accurate enough to be useful after a couple days.

 

No matter what any lab says the laws of nature regarding rapid degradation of blood samples cannot be overcome except by freezing the sample.  But most labs will not accept frozen samples because -- wait for it -- they don't want to unfreeze them because it takes too much time to do it properly.  So any sample over 48 hours old at the absolute maximum is suspect even if refrigerated.  But if kept at room temperature, the max time is likely about 24 - 36 hours.  But batch processing of samples is an industry standard, so many samples sit around way too long.  The only solution is to know when the batch processing occurs, and have the sample drawn to match.  Grrrrrr....

Edited by Trey, 18 August 2014 - 08:50 AM.

[tiredblood]

LOL. How would you go about finding out when the batch processing begins?

[Lucas]

Nice to know, trey!! Thanks again!!!

[Trey]

LOL. How would you go about finding out when the batch processing begins?

 

Ask your Onc to find out (unlikely to work), or ask the Onc where the sample is processed and call that lab (more likely to work).

[LivingWellWithCML]

Ask your Onc to find out (unlikely to work), or ask the Onc where the sample is processed and call that lab (more likely to work).

 

Great idea, Trey.  For my case, I ended up calling Emory Lab this morning.  For anyone on the board who is interested in these details, here is what I learned.  BTW, huge thanks to the friendly and incredibly-patient staff at the lab for connecting me with someone who actually processes the blood draws and runs the PCR test.  That was very reassuring to speak to someone right at the source!  The individual I spoke to was incredibly helpful and explained the process to me in reasonable detail.  Note that this process could be different for other labs, but this is how my lab works:

 

1. Blood draw is taken at the clinic, and the sample is sent over to the lab across the street ... usually within an hour or so.  

2. The RNA sample to be used for the PCR test is augmented with an RNA stabilization solution called RNAlater.  Then they freeze (not refrigerate ... -freeze-) the sample.

3. She was very clear in saying that RNAlater and the freezing of samples preserves the integrity and will give just as good of a result whether it's run the next day, or a week later, or maybe even longer.  She reinforced that with me several times during our conversation.

4. This particular lab runs PCRs on Tuesdays.  The time of day varies depending on staffing and workload, but she said that they never have a problem processing the backlog of tests on that day.  Pathology does their reviews & sign-offs on Fridays.

5. She told me that I could get my labs drawn on Monday "if it would make me feel better", but she assured me that their PCR testing methods would give essentially the same result regardless of the day I have my labs drawn.

 

Personally, I found this reassuring.  I also had a conversation with a family friend who works at Emory and has three PCR machines in his office.  He gave me the same explanation about RNAlater, and he said that it was especially effective for his work in bacterium analysis, because live bacteria deteriorates very quickly.  Same situation though ... in fact, he stores his RNAlater-treated samples at ambient room temp and said that they preserve quite well.

 

For the most part, this is settled my stomach about my own "undetectable" result, but I am planning to take Trey's advice and schedule all future blood draws on Mondays.

 

I hope this is helpful for others ...

[gerry]

So Dan,

Does this mean you can at least tap your toes as part of a happy dance for PCRU? Full happy dance to come if the next result is the same? 

[LivingWellWithCML]

Absolutely -4.2 log PCRU (ha ha) is a tap-your-toes moment.  Thanks for reminding me (and others) to cherish the moments when we have a good response to treatment.  It's definitely a big deal. :-)

[Trey]

Dan,

That is good to hear they freeze the sample, and it is very unusual for labs to do that since it increases the PCR process complexity.  Using RNAlater would not be necessary given they are freezing samples, especially since RNAlater also requires its own set of special procedures.  I would recommend the Monday blood draw since Emory is now doing Tuesday batch processing.  This would ensure the most accurate result.