Compositions and methods for stabilizing circulating tumor cells
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, especially blood, 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 device comprising:
an evacuated tube containing less than 10 mL of a mixture, the mixture including:
a. a blood sample including one or more tumor-based nucleic acid fragments present within the blood sample in a first concentration;
b. a protective agent in an amount of from about 50 to about 400 μl comprising imidazolidinyl urea and glycine, the protective agent present in an amount that is less than 5% by volume of the mixture, but greater than 1 part by volume protective agent to 300 parts by volume mixture, and the glycine being present in an amount of 1% to 10% by weight of the protective agent;
wherein at least 24 hours after contact between the protective agent and the blood sample, the one or more tumor-based nucleic acid fragments are present in a second concentration that is not lower or higher than the first concentration by any statistically significant value.
2. The device of claim 1 , wherein the protective agent includes an anticoagulant in an amount of from 1% to 70% by weight of the protective agent.
3. The device of claim 1 , wherein the concentration of the imidazolidinyl urea in the protective agent is between about 0.1 g/ml and about 3 g/ml.
4. The device of claim 2 , wherein the concentration of the imidazolidinyl urea in the protective agent is between about 0.1 g/ml and about 3 g/ml.
5. The device of claim 1 , wherein the concentration of the imidazolidinyl urea in the protective agent is about 0.1 g/ml.
6. The device of claim 2 , wherein the concentration of the imidazolidinyl urea in the protective agent is about 0.1 g/ml.
7. The device of claim 1 , wherein the mixture is essentially free of detectable covalent modifications.
8. The device of claim 5 , wherein the mixture is essentially free of detectable covalent modifications.
9. The device of claim 1 , wherein the protective agent composition includes:
a. imidazolidinyl urea in an amount of about 0.1 to about 1.0% by weight of the total mixture; and
b. ethylenediaminetetraacetic acid in an amount of at least 5% by weight of the protective agent.
10. The device of claim 7 , wherein the protective agent composition includes:
a. imidazolidinyl urea in an amount of about 0.1 to about 1.0% by weight of the total mixture; and
b. ethylenediaminetetraacetic acid in an amount of at least 5% by weight of the protective agent.
11. The device of claim 10 , wherein the protective agent composition includes an amount of about 10 parts by weight of the imidazolidinyl urea to about 1 parts by weight of the glycine.
12. The device of claim 1 , wherein the blood sample includes EpCAM proteins and there is no significant degradation of the EpCAM proteins after 4 days at room temperature.
13. The device of claim 5 , wherein the blood sample includes EpCAM proteins and there is no significant degradation of the EpCAM proteins after 4 days at room temperature.
14. The device of claim 1 , wherein the blood sample includes CK proteins and there is no significant degradation of the CK proteins after 4 days at room temperature.
15. The device of claim 5 , wherein the blood sample includes CK proteins and there is no significant degradation of the CK proteins after 4 days at room temperature.
16. The device of claim 1 , wherein c-fos mRNA levels in the blood sample remains stable after 4 days at room temperature.
17. The device of claim 2 , wherein c-fos mRNA levels in the blood sample remains stable after 4 days at room temperature.
18. The device of claim 5 , wherein c-fos mRNA levels in the blood sample remains stable after 4 days at room temperature.
19. The device of claim 1 , wherein cyclin D1 mRNA levels in the blood sample remains stable after 4 days at room temperature.
20. The device of claim 1 , wherein cyclin D1 mRNA levels in the blood sample remains stable after 4 days at room temperature.
21. The device of claim 10 , wherein cyclin D1 mRNA levels in the blood sample remains stable after 4 days at room temperature.
22. A device comprising:
an evacuated tube containing less than 10 mL of a mixture, the mixture including:
a. a blood sample including one or more tumor-based nucleic acid fragments present within the blood sample in a first concentration;
b. a protective agent in an amount of from about 50 to about 400 μl comprising imidazolidinyl urea, EDTA, and glycine, the protective agent present in an amount that is less than 5% by volume of the mixture, but greater than 1 part by volume protective agent to 300 parts by volume mixture, the glycine present in an amount of 1% to 10% by weight of the protective agent and the imidazolidinyl urea present in an amount of about 0.1 g/ml of the protective agent;
wherein at least 24 hours after contact between the protective agent and the blood sample, the one or more tumor-based nucleic acid fragments are present in a second concentration that is not lower or higher than the first concentration by any statistically significant value.