Cellular labeling and quantification for nuclear magnetic resonance techniques

• US 20070253910A1
• Filed: 04/16/2007
• Published: 11/01/2007
• Est. Priority Date: 04/14/2006
• Status: Active Grant

• Current Assignee: Carnegie Mellon University
• Original Assignee: Carnegie Mellon University
• Inventors: Ahrens, Eric, Srinivas, Mangala
• Granted Patent: US 8,263,043 B2
• Time in Patent Office: Days
• US Class Current: 424/9.340
• CPC Class Codes: A61B 5/055 involving electronic [EMR] or nuclear [NMR] magnetic resonance, e.g. magnetic resonance imaging
• CPC Class Codes: A61B 5/411 Detecting or monitoring allergy or intolerance reactions to an allergenic agent or substance
• CPC Class Codes: A61B 5/413 Monitoring transplanted tissue or organ, e.g. for possible rejection reactions after a transplant
• CPC Class Codes: A61B 5/415 the glands, e.g. tonsils, adenoids or thymus
• CPC Class Codes: A61B 5/416 the spleen
• CPC Class Codes: A61B 5/417 the bone marrow
• CPC Class Codes: A61B 5/418 lymph vessels, ducts or nodes
• CPC Class Codes: A61B 5/4519 Muscles A61B5/389, A61B5/224 take precedence
• CPC Class Codes: A61B 5/7285 for synchronising or triggering a physiological measurement or image acquisition with a physiological event or waveform, e.g. an ECG signal A61B6/5205, A61B6/541, A61B8/5207, A61B8/543 take precedence
• CPC Class Codes: A61K 49/1896 not provided for elsewhere, e.g. cells, viruses, ghosts, red blood cells, virus capsides
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Abstract

The disclosure provides, in part, a method for quantifying cell numbers in vivo. The disclosure comprises a method of quantifying labeled cells by nuclear magnetic resonance techniques and a computer method for the same.

Claims

1. A method for quantifying cell number in vivo, the method comprising:

a. administering to the subject cells that are labeled with a fluorocarbon imaging reagent;

b. examining at least a portion of the subject by a nuclear magnetic resonance technique, thereby detecting labeled cells in the subject; and

c. quantifying the number of labeled cells in a region of interest (ROI).

2. The method of claim 1, wherein the fluorocarbon imaging reagent is a perfluoropolyether.

3. The method of claim 1, wherein the fluorocarbon imaging reagent is a perfluoro-crown ether.

4. The method of claim 1, wherein the imaging reagent is a perfluro-15-crown-5-ether.

5. The method of claim 1, wherein the fluorocarbon is a perfluorinated polyether having an average formula:

XO(Y—

O)_nZ

wherein Y is selected from the group comprising;

wherein n is an integer from 8 to 20;

wherein X and Z are the same and are selected from the group comprising;

perfluoroalkyls, perfluoroethers, fluoroalkyls terminated with fluoroacyl, carboxyl, amide or ester, methylols, acid chlorides, amides, amidines, acrylates and esters.

6. The method of claim 1, wherein the fluorocarbon imaging reagent is selected from the group comprising:

a linear perfluoropolyether, a cyclic perfluoropolyether and a mixture thereof.

7. The method of claim 1, wherein prior to administration to the subject, the cells are contacted with a composition comprising the fluorocarbon imaging reagent, whereby the cells are labeled with the fluorocarbon imaging reagent.

8. The method of claim 1, wherein prior to administration to the subject, the cells are labeled with perfluorocarbon emulsion particles using electroporation.

9. The method of claim 7, wherein the composition comprising the fluorocarbon imaging reagent further comprises an uptake enhancing reagent.

10. The method of claim 7, wherein the composition comprising the fluorocarbon imaging reagent further comprises:

a. a surfactant; and

b. a cationic lipid.

11. The method of claim 9, wherein the uptake enhancing reagent comprises a compound selected from the group consisting of:

a. cationic lipid; and

b. cationic polypeptide.

12. The method of claim 11, wherein the cationic peptide is a protamine.

13. The method of claim 7, wherein at least a portion of the fluorocarbon imaging reagent is internalized into the cell.

14. The method of claim 7, wherein the composition comprising the fluorocarbon imaging reagent further comprises Pluronic L-35.

15. The method of claim 7, wherein the fluorocarbon imaging reagent is formulated as an emulsion.

16. The method of claim 7, wherein the emulsion comprises particles having a mean diameter of between 30 and 500 nm.

17. The method of claim 1, wherein the cell is a mammalian cell.

18. The method of claim 1, wherein the cell is a cell of the immune system.

19. The method of claim 1, wherein the cell is a T cell.

20. The method of claim 1, wherein the cell is a dendritic cell.

21. The method of claim 1, wherein the cell is a stem cell.

22. The method of claim 1, wherein examining by a nuclear magnetic resonance technique comprises collecting a ¹⁹F data set.

23. The method of claim 1, further comprising collecting a IH data set.

24. The method of claim 1, further comprising generating and comparing a ¹⁹F image and a ¹H image.

25. The method of claim 1, wherein the nuclear magnetic resonance technique is magnetic resonance imaging (MRI).

26. The method of claim 1, wherein the nuclear magnetic resonance technique is magnetic resonance spectroscopy (MRS).

27. The method of claim 1, wherein the cell is administered to the subject as part of a cellular therapeutic regimen.

37. The method of claim 1, wherein quantifying comprises using a calibrated ¹⁹F signal in the ROI.

39. The method of claim 1, wherein examining comprises detecting a pre-calibrated ¹⁹F signal from which one can deduce a relationship between the ¹⁹F signal in the ROI and the representative number of ¹⁹F molecules or cell quantity.

40. The method of claim 1, wherein examining comprises detecting a contemporaneous signal in the ROI from which one can deduce a relationship between the signal and the representative number of ¹⁹F molecules or cell quantity.

41. The method of claim 1, wherein examining comprises detecting a post-calibrated ¹⁹F signal in the ROI from which one can deduce a relationship between the signal and the representative number of ¹⁹F molecules or cell quantity.

42. The method of claim 37, wherein the cellular dose of fluorocarbon imaging reagent is calculated prior to administration of cells to the subject.

44. The method of claim 1, wherein quantifying is done by comparison to a calibrated external ¹⁹F reference during the ¹⁹F MRI/MRS scan of the subject.

46. The method of claim 44, wherein the reference is a cell free reference.

48. The method of claim 1, wherein quantifying comprises calculating ratios of the intensity of ¹⁹F signal and the volume of labeled cells in a ROI compared to a reference.

50. The method of claim 1, wherein quantifying the number of labeled cells comprises using at least one of the parameters from the group consisting of:

(i) the cellular dose of labeling agent (i.e., F_c) measured in vitro;

(ii) in vivo ¹⁹F MRI/MRS data set taken in the subject at one or more time points following labeled cell administration;

(iii) the voxel volume;

(iv) the in-plane voxel area (i.e., area of the image pixel);

(v) the MRI/MRS data set from the ¹⁹F reference standard;

(vi) the measured Johnson noise of the ¹⁹F MRI/MRS data in the subject material;

(vii) the measured signal-to-noise ratio (SNR) of one or more voxels of the ¹⁹F MRI/MRS data set in the subject material;

(viii) the measured SNR of one or more voxels of the ¹⁹F MRI/MRS data set from the reference standard;

(ix) the ¹⁹F NMR relaxation times (T1, T2, and T2*) of the subject material; and

(x) the ¹⁹F NMR relaxation times (T1, T2, and T2*) of the reference standard.

51. The method of claim 1, wherein quantifying the number of labeled cells comprises using the formula:

28. A method for quantifying the number of cells labeled with a fluorocarbon imaging reagent in a recipient of a transplant that includes said labeled cells, the method comprising:

a. examining at least a portion of the subject by a nuclear magnetic resonance technique, thereby detecting labeled cells in the subject.

b. quantifying the number of labeled cells.

29. The method of claim 28, wherein the location and optionally the trafficking of labeled cells is detected in the transplant recipient.

30. The method of claim 28, wherein the nuclear magnetic resonance technique is selected from the group comprising:

magnetic resonance imaging and magnetic resonance spectroscopy.

31. The method of claim 28, wherein the transplant recipient is a bone marrow transplant recipient.

32. The method of claim 28, wherein the cells for transplant comprise hematopoietic stem cells.

33. The method of claim 28, wherein the cells for transplant are derived from bone marrow, cord blood or peripheral blood.

34. The method of claim 28, wherein the transplant recipient is the recipient of a donor organ.

35. The method of claim 28, wherein at least a portion of the cells of the donor organ are labeled with a fluorocarbon imaging reagent.

36. The method of claim 28, wherein the transplant recipient is a human.

38. The method of claim 28, wherein quantifying comprises using a calibrated 19F signal in the ROI.

43. The method of claim 38, wherein the cellular dose of fluorocarbon imaging reagent is calculated prior to transplantation.

45. The method of claim 28, wherein quantifying is done by comparison to a calibrated external ¹⁹F reference during the ¹⁹F MRI/MRS scan of the subject.

47. The method of claim 45, wherein the reference is a cell free reference.

49. The method of claim 28, wherein quantifying comprises calculating ratios of the intensity of ¹⁹F signal and the volume of labeled cells in a ROI compared to a reference.

52. The method of claim 28, wherein quantifying the number of labeled cells comprises using at least one of the parameters from the group consisting of:

(i) the cellular dose of labeling agent (i.e., F_c) measured in vitro;

(ii) in vivo ¹⁹F MRI/MRS data set taken in the subject at one or more time points following labeled cell administration;

(iii) the voxel volume;

(iv) the in-plane voxel area (i.e., area of the image pixel);

(v) the MRI/MRS data set from the ¹⁹F reference standard;

(vi) the measured Johnson noise of the ¹⁹F MRI/MRS data in the subject material;

(vii) the measured signal-to-noise ratio (SNR) of one or more voxels of the ¹⁹F MRI/MRS data set in the subject material;

(viii) the measured SNR of one or more voxels of the ¹⁹F MRI/MRS data set from the reference standard;

(ix) the ¹⁹F NMR relaxation times (T1, T2, and T2*) of the subject material; and

(x) the ¹⁹F NMR relaxation times (T1, T2, and T2*) of the reference standard.

53. The method of claim 28, wherein quantifying the number of labeled cells comprises using the formula:

63. The method of claim 28, wherein examining comprises detecting a pre-calibrated ¹⁹F signal from which one can deduce a relationship between the ¹⁹F signal in the ROI and the representative number of ¹⁹F molecules or cell quantity.

64. The method of claim 28, wherein examining comprises detecting a contemporaneous signal in the ROI from which one can deduce a relationship between the signal and the representative number of ¹⁹F molecules or cell quantity.

65. The method of claim 28, wherein examining comprises detecting a post-calibrated ¹⁹F signal in the ROI from which one can deduce a relationship between the signal and the representative number of ¹⁹F molecules or cell quantity.

54. A calculating system for the quantification of ¹⁹F labeled cells, the system comprising:

a computer;

a computer readable medium, operatively coupled to the computer, the computer readable medium programs codes performing functions comprising;

quantification of ¹⁹F labeled cells in a ROI examined by a magnetic resonance technique.

55. The system according to claim 54, wherein quantification comprises relating a calibrated NMR signal to a cellular dose or cell quantity.

56. The system according to claim 54, wherein the functions are comprising:

calculating the number of labeled cells by ratios of the intensity of ¹⁹F signal and the volume of labeled cells in a ROI compared to a reference.

57. The system according to claim 54, wherein the functions are comprising:

calculating the number of labeled cells using at least one of the parameters from the group consisting of;

(i) the cellular dose of labeling agent (i.e., F_c) measured in vitro;

(ii) in vivo ¹⁹F MRI/MRS data set taken in the subject at one or more time points following labeled cell administration;

(iii) the voxel volume;

(iv) the in-plane voxel area (i.e., area of the image pixel);

(v) the MRI/MRS data set from the ¹⁹F reference standard;

(vi) the measured Johnson noise of the ¹⁹F MRI/MRS data in the subject material;

(vii) the measured signal-to-noise ratio (SNR) of one or more voxels of the ¹⁹F MRI/MRS data set in the subject material;

(viii) the measured SNR of one or more voxels of the ¹⁹F MRI/MRS data set from the reference standard;

(ix) the ¹⁹F NMR relaxation times (T1, T2, and T2*) of the subject material; and

(x) the ¹⁹F NMR relaxation times (T1, T2, and T2*) of the reference standard.

58. The system according to claim 54, wherein the functions are comprising:

calculating the number of labeled cells according to the formula;

$N_c=\frac{\left[F_R\right]<br><br>\text{\hspace{1em}<br><br>}<br><br>v}{I_R}<br><br>\frac{1}{F_c}<br><br>\underset{i=1}{\overset{N_{\mathrm{ROI}}}{\sum <br><br>}}<br><br>I_c^{\left(i\right)}$

59. A computer readable medium having computer readable program codes embodied therein for performing in vivo quantification of ¹⁹F labeled cells, the computer readable medium program codes performing functions comprising:

calculating the number of ¹⁹F labeled cells in a ROI detected by a magnetic resonance technique.

60. The system according to claim 59, wherein the functions are comprising:

calculating the number of labeled cells by ratios of the intensity of ¹⁹F signal and the volume of labeled cells in a ROI compared to a reference.

61. The system according to claim 59, wherein the functions are comprising:

calculating the number of labeled cells using at least one of the parameters from the group consisting of;

(i) the cellular dose of labeling agent (i.e., F_c) measured in vitro;

(ii) in vivo ¹⁹F MR/MRS data set taken in the subject at one or more time points following labeled cell administration;

(iii) the voxel volume;

(iv) the in-plane voxel area (i.e., area of the image pixel);

(v) the MRI/MRS data set from the ¹⁹F reference standard;

(vi) the measured Johnson noise of the ¹⁹F MRI/MRS data in the subject material;

(vii) the measured signal-to-noise ratio (SNR) of one or more voxels of the ¹⁹F MRI/MRS data set in the subject material;

(viii) the measured SNR of one or more voxels of the ¹⁹F MRI/MRS data set from the reference standard;

(ix) the ¹⁹F NMR relaxation times (T1, T2, and T2*) of the subject material; and

(x) the ¹⁹F NMR relaxation times (T1, T2, and T2*) of the reference standard.

62. The system according to claim 59, wherein the functions are comprising:

calculating the number of labeled cells according to the formula;

$N_c=\frac{\left[F_R\right]<br><br>\text{\hspace{1em}<br><br>}<br><br>v}{I_R}<br><br>\frac{1}{F_c}<br><br>\underset{i=1}{\overset{N_{\mathrm{ROI}}}{\sum <br><br>}}<br><br>I_c^{\left(i\right)}$