Compositions and methods for stabilizing circulating tumor cells
Methods and compositions for stabilizing a biological sample for analysis, comprising the steps of obtaining in a sample collection device a biological sample from a subject, the biological sample including at least one circulating tumor cell from the subject. The methods may include a step of contacting the biological sample with a protective agent composition that includes a preservative agent, an optional anticoagulant, and a quenching agent to form a mixture that includes the protective agent composition and the sample.
1. A method for blood sample treatment comprising:
locating about 50 μl to about 400 μl of a protective agent into a tube, the protective agent including imidazolidinyl urea, EDTA and glycine;
drawing a blood sample having a first circulating tumor cell concentration into the tube, whereby it contacts the protective agent;
isolating circulating tumor cells from the contacted blood sample at least 24 hours after the blood draw, the contacted blood sample having a second circulating tumor cell concentration, wherein the second circulating tumor cell concentration is not lower or higher than the first circulating tumor cell concentration by any statistically significant value; and
wherein the concentration of the imidazolidinyl urea after the contacting step is greater than 5 mg/ml;
wherein the concentration of the glycine after the contacting step is below about 0.03 g/ml;
wherein the protective agent is present in an amount that is less than about 5% of an overall mixture volume of the protective agent and the drawn blood sample;
wherein the method is free of any step of refrigerating the contacted blood sample to a temperature below room temperature after it has been contacted with the protective agent composition; and
wherein as a result of metabolic inhibition of the circulating tumor cells in the treated blood sample, apoptotic and necrotic pathways are inhibited and the circulating tumor cells are protected from cell degradation.
2. The method of claim 1 , wherein a concentration of the imidazolidinyl urea prior to the contacting step is between about 0.1 g/ml and about 3 g/ml.
3. The method of claim 1 , wherein the circulating tumor cells are isolated from the contacted blood sample at least 3 days after the blood draw.
4. The method of claim 1 , wherein the circulating tumor cells are isolated from the contacted blood sample at least 7 days after the blood draw.
5. The method of claim 1 , wherein the circulating tumor cells are isolated from the contacted blood sample at least 14 days after the blood draw.
6. The method of claim 1 , wherein the protective agent contacts the circulating tumor cells so that after a period of at least 7 days from a time the blood sample is drawn, an amount of circulating tumor cells present in the contacted blood sample is at least about 90% of an amount of circulating tumor cells at a time the blood sample is drawn.
7. The method of claim 1 , wherein the protective agent contacts the circulating tumor cells so that after a period of at least 7 days from a time the blood sample is drawn, an amount of circulating tumor cells present in the contacted blood sample is about 100% of an amount of circulating tumor cells present in the blood sample at a time the blood sample is drawn.
8. The method of claim 1 , wherein the protective agent composition includes an amount of about 10 parts by weight of the imidazolidinyl urea per about 1 parts by weight of the glycine.
9. The method of claim 1 , wherein there is no significant degradation of EpCAM proteins in the contacted blood sample after 4 days at room temperature.
10. The method of claim 1 , wherein there is no significant degradation of CK proteins in the contacted blood sample after 4 days at room temperature.
11. The method of claim 1 , wherein at least 50% of the circulating tumor cells are recovered for isolation after day 1.
12. The method of claim 1 , wherein at least 50% of the circulating tumor cells are recovered for isolation after day 4.
13. The method of claim 1 , wherein CK protein in the contacted blood sample is stabilized.
14. The method of claim 1 , wherein EpCAM protein in the contacted blood sample is stabilized.
15. The method of claim 1 , wherein c-fos mRNA in the circulating tumor cells remains stable after 4 days at room temperature.
16. The method of claim 1 , wherein the protective agent is free of formaldehyde as detected by 13 C-NMR analysis.
17. The method of claim 1 , wherein DNA present in the treated blood samples exhibits ellipticity that is substantially similar to that of untreated native DNA as measured by circular dichroism spectroscopy.
18. The method of claim 1 , wherein DNA present in the treated blood samples exhibits DNA-dye fluorescence that is substantially similar to that of untreated native DNA as measured by fluorescence spectroscopy.
19. The method of claim 1 , wherein protective agent includes beta-mercaptoethanol.