Guest guest Posted June 12, 2007 Report Share Posted June 12, 2007 Tracey wrote: > The issue that I'm interested in is this; although our FISH and BMB > tests on diagnosis would have us believe that every single one of our > stem cells is leukemic (or at least more than 95% of them), I > personally don't believe this to be the case because I don't feel > that a sample of 20 or 200 cells is representative of all the stem > cells we have. This is the issue that Marcos and I have been batting > around as he believes the BMB and FISH ARE representative of the > whole picture. > I just thought I'd chime in since I have some experience with the common tests for CML. The FISH test involves 2 DNA probes that have a fluorescent dye attached. One probe for BCR (green) and the other for ABL (orange). The test can be performed on blood or marrow but peripheral blood access is easier and the cells are pretty hardy and can sit around for a couple days if needed. You take the blood and add a chemical that puts small holes in the cell surface thus allowing the probes to get insidewhere they bind to the DNA. Turn off the lights and look under a microscope. Only nucleated cells (all the white cells and hematopoietic progenitors) will have dye in them so the tech starts counting with a clicker. Cells that appear to have orange and green in proximity or appear yellow (fusion of color) are Ph+. If dyes are apart, the Ph-. Counting takes 15 minutes and the FISH takes about 2 hours in total to perform. The cells that are seen as Ph+ would be any blast cells but also many of the white blood cells also. The disease often presents as proliferation of myeloid cells (neutrophils, basophils, eosinophils) but lymphocytes and monocytes can be Ph+ also. As I recall, in a newly diagnosed CML patient, 100% of the the myeloid lineage cells are Ph+. That's about 70-80% of the white blood cell population right off the bat. The lymphocytes tend to be Ph- and and the moncytes are about hafl Ph+. To Tracey's comment, I have seen high FISH counts (90+%) but never 100%. There is always a few normal cells (probably lymphocytes) in the sample. Now the Conventional karyotyping Cytogenetic (CC) test is similar in sensitivity and provides similar results as the FISH test but there is a big difference. The CC has to be done on a marrow since you are only counting the cells in metaphase. This is a step in the cell division process just before the cell divides and these cells are only in the marrow. Why a metaphase? Because its easier to discern the different chomosomes banding patterns and shape in a cell in metaphase (short chromosome 22 = Ph+) than the other steps in the cell cycle. After the marrow is drawn, the sample is sent to the lab where they incubate the cells in a culture media. The next day the techs remove the cells with many of the mature cells having died off. Dark room again with a microscope and a tech counts 20 metaphases in a 3-4 hours. Quality of the sample and lab time limits the number of metaphase that can be counted in a fairly routine manner to 20. I've seen samples (non-CML) where we struggled to find 5 metaphases to count. A cell in metaphase is likely a normal Ph- Hematopoietic progenitor or a Ph+ leukemia cell. Its impossible to tell right now if the metaphase is a progenitor or a HSC. I'm not sure if it matters. Most new patients are 20/20 positive thus 100%. Tracey is right that in reality the marrow is not 100% leukemic. If it was, a patient responding to Gleevec would become dysplastic (empty marrow) so the normal HSC must still be there to later regenerate normal blood cells. The reason the CC test is often 100% Ph+ is probably due to the fact that the leukemic HSC are dividing more often and the resulting progenitors are also dividing faster than there normal counterparts. By the time a person is diagnosed with CML, more than 95% of the cells dividing in the marrow are PH+ and since the sensitivity of the CC test is 5%, the normal metaphases don't get picked up so the test result is 100% PH+. To use the analogy of an assembly line, the CC test is counting the cells at the begining of the line and the FISH test counts the end product. Still, they are linked so that as a person responds to a therapy, the results for both test decline. I hope this helps. I think that many of the posters on this board already have a fantastic knowledge of CML. Quote Link to comment Share on other sites More sharing options...
Guest guest Posted June 13, 2007 Report Share Posted June 13, 2007 Thanks for this explanation . It clarifies what the FISH test does. My FISH was 100% at dx (7% 6 months later, 0% at 12 months). I think I have read a few posts with people reporting 100% FISH results. I do not think 100% on a test (FISH ot cytogenetics) means 100% of the cells are leukemic, I argued enough with my first oncologist about that, and I actually served her the same argument you are giving, I wouldn't have had a normal cbc 3 weeks after starting gleevec if it was the case. Just from a statistical point of view, a limited sample is unlikely to show rare events. The same applies for PCRU not being equivalent to being disease free. To come back to the question about how many HSCs are ph+ at dx (assuming 100% FISH, 100% cc), what you mention about the cc looking at progenitors or HSCs seems to me be the core of it. If it looks only at progenitors, assuming the ph+ HSCs are producing more progenitors than normal HSCs, the cc result about how many progenitors are ph+ is overestimating the number of ph+ HSCs. If the cc looks only at HSCs, then it should be a fair indicator of how many HSCs are ph+, assuming cml is diffuse and the bone marrow sample is representative. No wonder I can't get straight answers from my different drs Marcos. On 6/12/07, <timothyfarley16@...> wrote: > > > > > > > Tracey wrote: > > > The issue that I'm interested in is this; although our FISH and BMB > > tests on diagnosis would have us believe that every single one of > our > > stem cells is leukemic (or at least more than 95% of them), I > > personally don't believe this to be the case because I don't feel > > that a sample of 20 or 200 cells is representative of all the stem > > cells we have. This is the issue that Marcos and I have been > batting > > around as he believes the BMB and FISH ARE representative of the > > whole picture. > > > > I just thought I'd chime in since I have some experience with the > common tests for CML. > > The FISH test involves 2 DNA probes that have a fluorescent dye > attached. One probe for BCR (green) and the other for ABL (orange). > The test can be performed on blood or marrow but peripheral blood > access is easier and the cells are pretty hardy and can sit around > for > a couple days if needed. You take the blood and add a chemical that > puts small holes in the cell surface thus allowing the probes to get > insidewhere they bind to the DNA. Turn off > the lights and look under a microscope. Only nucleated cells (all > the > white cells and hematopoietic progenitors) will have dye in them so > the > tech starts counting with a clicker. Cells that appear to have > orange > and green in proximity or appear yellow (fusion of color) are Ph+. > If > dyes are apart, the Ph-. Counting takes 15 minutes and the FISH > takes about 2 hours in total to > perform. The cells that are seen as Ph+ would be any blast cells but > also many of the white blood cells also. The disease often presents > as > proliferation of myeloid cells (neutrophils, basophils, eosinophils) > but lymphocytes and monocytes can be Ph+ also. As I recall, in a > newly > diagnosed CML patient, 100% of the the myeloid lineage cells are > Ph+. > That's about 70-80% of the white blood cell population right off the > bat. The lymphocytes tend to be Ph- and and the moncytes are about > hafl Ph+. > > To Tracey's comment, I have seen high FISH counts (90+%) but never > 100%. There is always a few normal cells (probably lymphocytes) in > the > sample. > > Now the Conventional karyotyping Cytogenetic (CC) test is similar in > sensitivity and provides similar results as the FISH test but there > is > a big difference. The CC has to be done on a marrow since you are > only > counting the cells in metaphase. This is a step in the cell division > process just before the cell divides and these cells are only in the > marrow. Why a metaphase? Because its easier to discern the > different > chomosomes banding patterns and shape in a cell in metaphase (short > chromosome 22 = Ph+) than the other steps in the cell cycle. After > the marrow is drawn, the sample > is sent to the lab where they incubate the cells in a culture media. > The next day the techs remove the cells with many of the mature cells > having died off. Dark room again with a microscope and a tech > counts 20 metaphases in a 3-4 hours. Quality of the sample and lab > time limits the number of metaphase that can be counted in a fairly > routine manner to 20. I've seen samples (non-CML) where we struggled > to find 5 metaphases to count. > > A cell in metaphase is likely a normal Ph- Hematopoietic progenitor > or > a Ph+ leukemia cell. Its impossible to tell right now if the > metaphase > is a progenitor or a HSC. I'm not sure if it matters. Most new > patients are 20/20 positive thus > 100%. Tracey is right that in reality the marrow is not 100% > leukemic. If it was, a patient responding to Gleevec would become > dysplastic (empty marrow) so the normal HSC must still be there to > later regenerate normal blood cells. The reason the CC test is often > 100% Ph+ is probably due to the fact that the leukemic HSC are > dividing > more often and the resulting progenitors are also dividing faster > than > there normal counterparts. By the time a person is diagnosed with > CML, > more than 95% of the cells dividing in the marrow are PH+ and since > the > sensitivity of the CC test is 5%, the normal metaphases don't get > picked up so the test result is 100% PH+. > > To use the analogy of an assembly line, the CC test is counting the > cells at the begining of the line and the FISH test counts the end > product. Still, they are linked so that as a person responds to a > therapy, the results for both test decline. > > I hope this helps. I think that many of the posters on this board > already have a fantastic knowledge of CML. > > -- Marcos Perreau Guimaraes Suppes Brain Lab Ventura Hall - CSLI Stanford University 220 Panama street Stanford CA 94305-4101 650 329 9920 x 305 650 630 5015 (cell) marcospg@... montereyunderwater@... www.stanford.edu/~marcospg/ Quote Link to comment Share on other sites More sharing options...
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