Claims
- 1. A method for determining the number of cells of interest in a sample comprising:
(a) applying a sample comprising cellular material to a filter; (b) lysing the cells of interest to form a cell lysate; (c) immobilizing the released nucleic acid on or within the filter; (d) removing the remaining cell lysate from the filter and retaining the nucleic acid; (e) eluting the nucleic acid from the filter; (f) quantifying the amount of nucleic acid eluted from the filter; and (g) determining the number of cells of interest in the sample based on the determined quantity of nucleic acid eluted.
- 2. A method for determining the number of cells of interest in a sample comprising:
(a) applying a sample comprising cellular material to a filter; (b) lysing the cells of interest to form a cell lysate; (c) immobilizing the released nucleic acid on or within the filter; (d) removing the remaining cell lysate from the filter and retaining the nucleic acid; (e) quantifying the amount of nucleic acid retained on the filter; and (f) determining the number of cells of interest in the sample based on the determined quantity of nucleic acid retained.
- 3. A method for estimating the number of white blood cells in a blood sample comprising:
(a) isolating the nucleic acid from the blood sample; (b) determining the amount of nucleic acid isolated from the blood sample; and (c) determining the number of white blood cells in the blood sample based on the determined amount of nucleic acid.
- 4. The method according to claim 3, wherein the blood sample is a leukoreduced blood sample and wherein the nucleic acid measured is DNA.
- 5. The method according to claim 4, wherein the DNA is genomic DNA, double-stranded DNA, or single-stranded DNA.
- 6. The method according to claim 4, wherein the estimated number of white blood cells is at least 85% of the number of white blood cells as measured by flow cytometry.
- 7. The method according to claim 4, wherein the estimated number of white blood cells is at least 90% of the number of white blood cells as measured by flow cytometry.
- 8. The method according to claim 4, wherein the estimated number of white blood cells is at least 95% of the number of white blood cells as measured by flow cytometry.
- 9. The method according to claim 4, wherein the estimated number of white blood cells is at least 98% of the number of white blood cells as measured by flow cytometry.
- 10. The method according to claim 4, wherein the estimated number of white blood cells is at least 99% of the number of white blood cells as measured by flow cytometry.
- 11. A method for determining the amount of white blood cells in a blood sample comprising:
(a) applying a blood sample comprising cells to a filter in the absence of a chaotrope; (b) retaining the cells with the filter as a cellular retentate and removing contaminants; (c) lysing the cellular retentate from step (b) to form a cell lysate while retaining the cell lysate in the filter, the cell lysate retentate containing nucleic acid; (d) removing the remaining cell lysate from the filter and retaining the nucleic acid; (e) eluting the nucleic acid; (f) measuring the amount of nucleic acid eluted from the filter; and (g) determining the amount of nucleic acid measured to estimate the number of white blood cells in the sample based on an amount of approximately 7 pg of nucleic acid contained in each white blood cell.
- 12. The method according to claim 11, wherein the nucleic acid measured is DNA.
- 13. The method according to claim 12, wherein the DNA is genomic DNA, double-stranded DNA, or single-stranded DNA.
- 14. A method for isolating nucleic acid from a blood sample comprising:
(a) applying a blood sample comprising cells to a filter in the absence of a chaotrope; (b) retaining the cells with the filter as a cellular retentate and removing contaminants; (c) lysing the cellular retentate from step (b) to form a cell lysate while retaining the cell lysate in the filter, the cell lysate retentate containing nucleic acid; (d) removing the remaining cell lysate from the filter and retaining the nucleic acid; (e) heating the nucleic acid to an elevated temperature of 40° C. to 125° C. while retained by the filter; (f) eluting the nucleic acid; (g) quantifying the amount of nucleic acid eluted from the filter; and (h) determining the number of nucleic acid-containing cells in the sample based on the determined quantity of nucleic acid eluted.
- 15. The method according to claim 14, wherein steps (e) and (f) together are repeated at least once.
- 16. The method according to claim 14, wherein the elevated temperature is in the range of 80° C. to 95° C.
- 17. The method according to claim 14, wherein, prior to step (a), the filter is compressed to between 10% and 80% less than its original volume.
- 18. The method according to claim 17, wherein the filter is compressed to between 25% and 75% less than its original volume.
- 19. The method according to claim 17, wherein the filter is compressed to between 40% and 60% less than its original volume.
- 20. The method according to claim 14, wherein the cellular retentate comprises intact whole blood cells.
- 21. The method according to claim 14, wherein the cell lysate retentate comprises condensed material from the nucleus and blood cell debris.
- 22. The method according to claim 14, wherein step (b) comprises lysing the cellular retentate to form a cell lysate containing the nucleic acid by the addition of a low salt buffer capable of lysing the cells.
- 23. The method according to claim 22, wherein step (b) comprises:
(a) rupturing the intact whole cells retained by the filter to leave condensed material from the nucleus retained by the filter; and (b) lysing the condensed material from the nucleus to form the cell lysate containing the nucleic acid.
- 24. The method according to claim 23, wherein the intact whole cells are ruptured to form condensed material from the nucleus by addition of detergent.
- 25. The method according to claim 14, wherein the filter composition and dimensions are selected so that the nucleic acid is retained by the filter in step (c) in the absence of ionic interaction.
- 26. The method according to claim 14, wherein the retaining step is further defined as physically retarding the movement of the nucleic acid through the filter.
- 27. The method according to claim 14, wherein the filter composition and dimensions are selected so that the nucleic acid is retained by the filter in step (c) in the form of a web.
- 28. The method according to claim 14, wherein the filter comprises a plurality of fibers and has a disordered structure.
- 29. The method according to claim 28, wherein the fiber diameters are in the range of 1 μm to 10 μm.
- 30. The method according to claim 14, wherein the cells are white blood cells.
- 31. The method according to claim 30, which method further comprises applying blood to the solid phase, lysing the red blood cells therefrom, washing the solid phase to remove contaminants and obtaining the cell lysate from the white blood cells.
- 32. The method according to claim 31, wherein the blood is leukoreduced blood.
- 33. The method according to claim 32, wherein the estimated number of white blood cells is at least 85% of the number of white blood cells as measured by flow cytometry.
- 34. The method according to claim 32, wherein the estimated number of white blood cells is at least 90% of the number of white blood cells as measured by flow cytometry.
- 35. The method according to claim 32, wherein the estimated number of white blood cells is at least 95% of the number of white blood cells as measured by flow cytometry.
- 36. The method according to claim 32, wherein the estimated number of white blood cells is at least 98% of the number of white blood cells as measured by flow cytometry.
- 37. The method according to claim 32, wherein the estimated number of white blood cells is at least 99% of the number of white blood cells as measured by flow cytometry.
- 38. The method according to claim 14, wherein the nucleic acid comprises DNA.
- 39. The method according to claim 38, wherein the DNA is genomic DNA, double-stranded DNA, or single-stranded DNA.
- 40. The method according to claim 14, which is carried out without any centrifugation steps.
- 41. The method according to claim 14, which is carried out in the absence of a chaotrope.
- 42. The method according to claim 14, wherein the presence of nucleic acid is detected using a fluorescent signal, color indicator, photometric indicator, enzymatic indicator, or radioactive indicator.
- 43. The method according to claim 14, wherein the presence of nucleic acid is detected using an indicator specific for double-stranded DNA.
- 44. The method according to claim 14, wherein the presence of nucleic acid is detected using an indicator from the group consisting of PicoGreen, a labeled nucleic acid probe specific for genomic DNA, ethidium bromide, or an indicator specific for double-stranded DNA.
- 45. The method for estimating the amount of white blood cells in a leukoreduced blood sample comprising:
(a) compressing a filter to between 10% and 80% less than its original volume; (b) applying a leukoreduced blood sample comprising cells to the filter in the absence of a chaotrope; (c) retaining the cells with the filter as a cellular retentate comprising whole cells, condensed nuclear material, and cell debris; (d) removing non-cellular contaminants from the retentate; (e) lysing the cells in the cellular retentate from step (b) while the retentate is entrapped within the filter to form a cell lysate containing the nucleic acid; (f) retaining the nucleic acid while removing remaining cell lysate; (g) heating the nucleic acid to an elevated temperature of 40° C. to 125° C. while retained by the filter; (h) eluting the nucleic acid; (i) measuring the amount of genomic DNA eluted from the filter; (j) repeating steps (g) and (h); and (k) determining the amount of nucleic acid measured to estimate the number of white blood cells in the sample based on an amount of approximately 7 pg of nucleic acid contained in each white blood cell.
- 46. The method according to claim 45, wherein the estimated number of white blood cells is at least 85% of the number of white blood cells as measured by flow cytometry.
- 47. The method according to claim 45, wherein the estimated number of white blood cells is at least 90% of the number of white blood cells as measured by flow cytometry.
- 48. The method according to claim 45, wherein the estimated number of white blood cells is at least 95% of the number of white blood cells as measured by flow cytometry.
- 49. The method according to claim 45, wherein the estimated number of white blood cells is at least 98% of the number of white blood cells as measured by flow cytometry.
- 50. The method according to claim 45, wherein the estimated number of white blood cells is at least 99% of the number of white blood cells as measured by flow cytometry.
- 51. A kit for quantifying the amount of genomic DNA in a blood sample comprising cells containing nucleic acid comprising:
(a) a filter supported on a support, said filter comprising matrix means for physically retarding movement of (cells and) nucleic acid herethrough in the absence of a chaotrope; (b) one or more solutions selected from the group consisting of a red cell lysis solution, a solution for rupturing intact whole cells to leave condensed nuclear material, a lysis solution for lysing nuclear material and an elution solution; and (c) an indicator capable of providing quantitative measurement of double-stranded or genomic DNA in a concentration as low as 1000 ng/ml.
- 52. The kit according to claim 51, wherein the indicator is capable of providing quantitative measurement of double-stranded or genomic DNA in a concentration as low as 200 μg/ml.
- 53. The kit according to claim 51, wherein the blood sample is a leukoreduced blood sample.
- 54. A method for estimating the amount of white blood cells in a blood sample, comprising:
(a) applying a blood sample to a substrate consisting of a coating enabling cellular lysis and release of nucleic acids; (b) immobilizing the released nucleic acid to a matrix, the substrate physically capturing the nucleic acid; (c) bonding the nucleic acid to the substrate; (d) generating a signal when the nucleic acid bonds to the substrate; (e) analyzing the amount of nucleic acid captured by quantifying the generated signal; (f) using the amount of nucleic acid measured to estimate the number of white blood cells in the blood sample.
- 55. The method according to claim 54, wherein the generating step is further defined as generating a fluorescent signal, color indicator, photometric indicator, enzymatic indicator, or radioactive indicator.
- 56. The method according to claim 54, wherein the blood sample is a leukoreduced blood sample.
- 57. The method according to claim 56, wherein the estimated number of white blood cells is at least 85% of the number of white blood cells as measured by flow cytometry.
- 58. The method according to claim 56, wherein the estimated number of white blood cells is at least 90% of the number of white blood cells as measured by flow cytometry.
- 59. The method according to claim 56, wherein the estimated number of white blood cells is at least 95% of the number of white blood cells as measured by flow cytometry.
- 60. The method according to claim 56, wherein the estimated number of white blood cells is at least 98% of the number of white blood cells as measured by flow cytometry.
- 61. The method according to claim 56, wherein the estimated number of white blood cells is at least 99% of the number of white blood cells as measured by flow cytometry.
Priority Claims (2)
Number |
Date |
Country |
Kind |
9918193.5 |
Aug 1999 |
GB |
|
9822141.9 |
Oct 1998 |
GB |
|
CROSSREFERENCE TO RELATED APPLICATIONS
[0001] This is a Continuation-in-Part of U.S. Ser. No. Utility patent application Ser. No. 09/736,659, filed Dec. 14, 2000, which is a Continuation-in-Part of PCT application No. PCT/US00/10230, filed Apr. 14, 2000, which claims the benefit of priority under 35 USC Section 119(e) of U.S. Provisional Patent Application No. 60/129,191, filed on Apr. 14, 1999; U.S. Provisional Patent Application No. 60/180,353, filed on Feb. 4, 2000; and U.S. Provisional Patent Application No. 60/193,556, filed on Mar. 31, 2000, all of which are incorporated herein by reference.
[0002] This is also a Continuation-in-Part of a CPA application filed Mar. 21, 2001, which is a CPA of U.S. Ser. No. Utility patent application Ser. No. 09/414,533, filed Oct. 8, 1999, which claims the benefit of priority of United Kingdom application 9918193.5, filed on Aug. 2, 1999, and United Kingdom application 9822141.9, filed on Oct. 9, 1998, all of which are incorporated herein by reference.
[0003] This also claims priority of U.S. Provisional Patent Application No. 60/226,650, filed Aug. 21, 2000, which is incorporated herein by reference.
Continuation in Parts (3)
|
Number |
Date |
Country |
Parent |
09414533 |
Oct 1999 |
US |
Child |
09908452 |
Jul 2001 |
US |
Parent |
09736659 |
Dec 2000 |
US |
Child |
09908452 |
Jul 2001 |
US |
Parent |
PCT/US00/10230 |
Apr 2000 |
US |
Child |
09736659 |
Dec 2000 |
US |