Mass spectrometry techniques for determining the status of sepsis in an individual

Abstract

Mass spectrometry techniques for determining the status of sepsis in an individual are provided. A biomarker profile resolved from a biological sample, taken from the individual, using a mass spectrometry technique is compared to a reference biomarker profile. A single such comparison classifies the individual as belonging to or not belonging to a reference population. The individual's biomarker profile and the reference biomarker profile comprise a plurality of ions each having a mass-to-charge ratio of about 100 Daltons to about 1000 Daltons. The plurality of ions can be detected by electrospray ionization mass spectrometry in positive mode. The comparison uses a decision rule, such as a classification tree, that determines the status of sepsis in the individual without requiring knowledge of the identity of the biomarkers in the biomarker profile from the individual and without requiring knowledge of the identity of the biomarkers in the reference biomarker profile.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the progression of SIRS to sepsis. The condition of sepsis consists of at least three stages, with a septic patient progressing from severe sepsis to septic shock to multiple organ dysfunction.

FIG. 2 shows the relationship between sepsis and SIRS. The various sets shown in the Venn diagram correspond to populations of individuals having the indicated condition.

FIG. 3 shows the natural log of the ratio in average normalized peak intensities for about 400 ions for a sepsis-positive population versus a SIRS-positive population.

FIGS. 4A and 4B show the intensity of an ion having an m/z of 437.2 Da and a retention time on a C₁₈ reverse phase column of 1.42 min in an ESI-mass spectrometer profile. FIG. 4A shows changes in the presence in the ion in various populations of individuals who developed sepsis. Clinical suspicion of sepsis in the sepsis group occurred at “time 0,” as measured by conventional techniques. “Time—24 hours” and “time—48 hours” represent samples taken about 24 hours and about 48 hours, respectively, preceding the clinical suspicion of the onset of sepsis in the sepsis group. Individuals entered the study at “Day 1.” FIG. 4B shows the presence of the same ion in samples taken from populations of individuals who did not develop sepsis at time 0.

FIG. 5 is a classification tree fitted to data from time 0 in 10 sepsis patients and 10 SIRS patients, showing three biomarkers identified by electrospray mass spectrometry that are involved in distinguishing sepsis from SIRS.

FIG. 6 shows representative LC/MS and LC/MS/MS spectra obtained on plasma samples, using the configuration described in the examples.

FIGS. 7A and 7B show proteins that are regulated at higher levels in plasma up to 48 hours before conversion to sepsis.

FIGS. 8A and 8B show proteins that are regulated at lower levels in plasma up to 48 hours before conversion to sepsis.

Claims

1. A method of determining the status of sepsis in an individual using mass spectrometry, comprising: comparing (i) a first biomarker profile resolved from a first biological sample, taken from the individual, using a mass spectrometry technique and (ii) a reference biomarker profile; wherein a single such comparison classifies the individual as belonging to or not belonging to a reference population; wherein said individual's first biomarker profile and said reference biomarker profile comprise a plurality of ions each having a mass-to-charge ratio of about 100 Daltons to about 1000 Daltons; and wherein the comparison determines the status of sepsis in the individual.

2. The method of claim 1 wherein the comparing comprises applying a decision rule that determines the status of sepsis in the individual.

3. The method of claim 1 wherein the reference biomarker profile is obtained from biological samples from a reference population, wherein the reference population is selected from the group consisting of a normal reference population, a SIRS-positive reference population, an infected/SIRS-negative reference population, a sepsis-positive reference population, a reference population at a stage in the progression of sepsis, a SIRS-positive reference population confirmed as having sepsis after about 0-36 hours, a SIRS-positive reference population confirmed as having sepsis after about 36-60 hours, and a SIRS-positive reference population confirmed as having sepsis after about 60-84 hours.

4. The method of claim 1 wherein the reference biomarker profile is resolved by mass spectrometry from samples obtained from biological samples from a (i) SIRS-positive reference population that contracted sepsis and (ii) a SIRS-positive reference population that did not contract sepsis.

5. The method claim 1, wherein the first biological sample is selected from the group consisting of blood, saliva, serum, plasma, urine, stool, cerebral spinal fluid, cells, a cellular extract, a tissue sample, and a tissue biopsy.

6. The method of claim 1, further comprising repeating the method at least once, wherein a separate biomarker profile is obtained from the individual from a separate biological sample taken each time the method is repeated.

7. The method of claim 6, wherein said separate biological sample is taken from the individual at 24 hour intervals.

8. The method of claim 1, wherein the determining the status of sepsis in the individual comprises predicting the onset of sepsis in the individual.

9. The method of claim 8, wherein the onset of sepsis is predicted at least about 24 hours prior to the determination of sepsis in the individual by way of clinical criteria.

10. The method of claim 8, wherein the onset of sepsis is predicted at least about 48 hours prior to the determination of sepsis in the individual by way of clinical criteria.

11. The method of claim 8, wherein the onset of sepsis is predicted at least about 96 hours prior to the determination of sepsis in the individual by way of clinical criteria.

12. The method of claim 1, wherein the determining the status of sepsis in the individual comprises determining the progression of sepsis in the individual.

13. The method of claim 1, wherein the determining the status of sepsis in the individual comprises diagnosing sepsis in the individual.

14. The method of claim 1, wherein the comparing step comprises applying a decision rule.

15. The method of claim 14, wherein applying the decision rule comprises using a data analysis algorithm.

16. The method of claim 15, wherein the data analysis algorithm is a classification tree.

17. The method of claim 15, wherein the data analysis algorithm is nonparametric.

18. The method of claim 17, wherein the nonparametric algorithm is a Wilcoxon Signed Rank Test.

19. The method of claim 15, wherein the data analysis algorithm is a multiple additive regression tree.

20. The method of claim 15, wherein the data analysis algorithm is a logistic regression.

21. The method of claim 15, wherein the data analysis algorithm comprises at least two input parameters.

22. The method of claim 21, wherein the data analysis algorithm comprises at least five input parameters.

23. The method of claim 21, wherein the data analysis algorithm comprises at least ten input parameters.

24. The method of claim 21, wherein the data analysis algorithm comprises at least twenty input parameters.

25. The method of claim 14, wherein the decision rule determines the status of sepsis in with an accuracy of at least about 60%.

26. The method of claim 14, wherein the decision rule determines the status of sepsis with an accuracy of at least about 70%.

27. The method of claim 14, wherein the decision rule determines the status of sepsis with an accuracy of at least about 80%.

28. The method of claim 14, wherein the decision rule determines the status of sepsis with an accuracy of at least about 90%.

29. The method of claim 25, wherein the determination of the status of sepsis in the individual is made at least about 48 hours prior to clinical confirmation of sepsis.

30. The method of claim 25, wherein the decision rule has been subjected to ten-fold cross-validation.

31. The method of claim 1, wherein the reference biomarker profile is obtained from a population comprising a single individual.

32. The method of claim 1, wherein the reference biomarker profile is obtained from a population comprising at least two individuals.

33. The method of claim 32, wherein the population comprises at least 20 individuals.

34. The method of claim 1, wherein the reference biomarker profile is obtained from a population selected from the group consisting of a normal reference population, a SIRS-positive reference population, an infected/SIRS-negative reference population, a sepsis-positive reference population, a reference population at a stage in the progression of sepsis, a SIRS-positive reference population confirmed as having sepsis after about 0-36 hours, a SIRS-positive reference population confirmed as having sepsis after about 36-60 hours, and a SIRS-positive reference population confirmed as having sepsis after about 60-84 hours.

35. The method of claim 1, further comprising comparing a second biomarker profile from the individual with a reference biomarker profile, wherein the second biomarker profile is obtained from a second biological sample taken from the individual.

36. The method of claim 35, wherein the second biological sample from the individual is taken about 24 hours after the first biological sample is taken from the individual.

37. The method of claim 35, wherein the second biomarker profile is compared to a different reference biomarker profile than the first biomarker profile.

38. The method of claim 1, wherein said plurality of ions are detected by electrospray ionization mass spectrometry in positive mode.

39. The method of claim 1, wherein said biological sample is fractionated prior to resolving said individual's first biomarker profile.

40. The method of claim 1, wherein at least one separation method is used to obtain said individual's first biomarker profile from said biological sample.

41. The method of claim 1, wherein at least two separation methods are used to obtain said individual's first biomarker profile from said biological sample.

42. The method of claim 41, wherein said at least two separation methods include mass spectrometry.

43. The method of claim 1, wherein said mass spectrometry technique is selected from the group consisting of electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)n, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)n, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero.

44. The method of claim 41, wherein said at least two separation methods include at least one method selected from the group consisting of chemical extraction partitioning, ion exchange chromatography, reverse phase liquid chromatography, isoelectric focusing, one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), thin-layer chromatography, gas chromatography, liquid chromatography, and any combination thereof.

45. The method of claim 40, wherein said at least one separation method is LC/MS.

46. The method of claim 45, further comprising fractionating said biological sample.

47. The method of claim 46, wherein said fractionating comprises extracting said biological sample with ice cold methanol.

48. The method of claim 47, wherein said extracting comprises (i) adding said ice cold methanol to form a mixture having a final volume percent of methanol of about 67%, (ii) incubating the mixture at 4° C. for 20 minutes, (iii) precipitating protein by centrifugation at 12,000 rpm for 10 minutes, and (iv) removing the supernatant to obtain said individual's first biomarker profile.

49. A method of predicting the onset of sepsis in an individual, comprising: (a) measuring two features in a biomarker profile resolved from a first biological sample, taken from the individual, using a mass spectrometry technique; and(b) comparing the at least two features with the same at least two features in a reference populationwherein a single such comparison classifies the individual as belonging to or not belonging to the reference population, and wherein the comparison predicts the onset of sepsis.

50. The method of claim 49, wherein said prediction of the onset of sepsis is made about 12-36 hours prior to the onset of sepsis.

51. The method of claim 49, wherein said prediction of the onset of sepsis is made about 36-60 hours prior to the onset of sepsis.

52. The method of claim 49, wherein said prediction of the onset of sepsis is made about 60-84 hours prior to the onset of sepsis.

53. A biomarker profile comprising at least two features that contribute to the classification of an individual as belonging to a reference population with an accuracy of at least about 60%, based on a comparison with a reference profile of the reference population, wherein the reference population is selected from the group consisting of a normal reference population, a SIRS-positive reference population, an infected/SIRS-negative reference population, a sepsis-positive reference population, and a reference population at a stage in the progression of sepsis, and wherein the biomarker profile is resolved from a first biological sample, taken from the individual, using a mass spectrometry technique.

54. The method of claim 2 wherein the decision rule was trained using biomarker profiles from a reference population that includes sepsis-positive reference population and a SIRS-positive population.

55. The method of claim 54, wherein a feature used in the decision rule has a ratio of average normalized intensity in said sepsis-positive reference population versus said SIRS-positive reference population of about 3:1 or higher.

56. The method of claim 54, wherein a feature used in the decision rule has a ratio of average normalized intensity in said sepsis-positive reference population versus said SIRS-positive reference population of about 1:3 or lower.

57. The method of claim 14, wherein the decision rule determines the status of sepsis in an individual with an accuracy of at least about 60%.

58. The method of claim 14, wherein the decision rule determines the status of sepsis in an individual with an accuracy of at least about 70%.

59. The method of claim 14, wherein the decision rule determines the status of sepsis in and individual with an accuracy of at least about 80%.

60. The method of claim 14, wherein the decision rule determines the status of sepsis in an individual with an accuracy of at least about 90%.

61. The method of claim 14, wherein the decision rule determines the status of sepsis in a reference population with an accuracy of at least about 60%.

62. The method of claim 14, wherein the decision rule determines the status of sepsis in a reference population with an accuracy of at least about 70%.

63. The method of claim 14, wherein the decision rule determines the status of sepsis in a reference population with an accuracy of at least about 80%.

64. The method of claim 14, wherein the decision rule determines the status of sepsis in a reference population with an accuracy of at least about 90%.