METHODS AND APPARATUS FOR STERILITY TESTING

• US 20090325220A1
• Filed: 06/08/2009
• Published: 12/31/2009
• Est. Priority Date: 08/19/2004
• Status: Active Grant

• Current Assignee: Blood Cell Storage, Inc.
• Original Assignee: Blood Cell Storage, Inc.
• Inventors: Barker, Lynn M., Reed, Michael W., Klip, Evert Jan
• Granted Patent: US 8,183,052 B2
• Time in Patent Office: Days
• US Class Current: 435/31
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 2021/6484 Optical fibres
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/80 Indicating pH value

Abstract

Methods and apparatus for testing the sterility of a product to be administered to a patient. The sterility of the product is tested by monitoring the pH change of a culture medium in contact with any culturable organisms (e.g., bacteria) that are collected by filtration of the product to be tested.

Claims

1. A method for testing the sterility of a sample comprising:

(a) providing a filter device having a chamber and a filter configured to collect culturable organisms;

(b) filtering a sample through the filter such that any collected culturable organisms are retained in the chamber;

(c) adding a culture medium to the chamber;

(d) irradiating a fluorescent species immobilized on a substrate with excitation light emanating from a probe physically isolated from the fluorescent species immobilized on the substrate,

wherein the fluorescent species immobilized on the substrate is in liquid communication with the culture medium,

wherein the excitation light has a wavelength sufficient to effect fluorescent emission from the fluorescent species, and

wherein the fluorescent species exhibits a first emission intensity at a first emission wavelength and a second emission intensity at a second emission wavelength, the ratio of the first and second emission intensities being dependent on pH;

(e) measuring the first and second emission intensities to determine the pH of the culture medium; and

(f) inferring from the determined pH of the culture medium the sterility of the sample.

2. The method of claim 1, wherein the filter comprises a membrane having a pore size of about 45 μ

m.

3. The method of claim 1, wherein the probe is physically isolated from the fluorescent species immobilized on the substrate by a window transparent to the excitation light and the fluorescent emission.

4. The method of claim 1, wherein the probe comprises one or more optical fibers.

5. The method of claim 1, wherein the fluorescent species is selected from the group consisting of a naphthofluorescein compound and a seminaphthorhodamine compound.

6. The method of claim 1, wherein the fluorescent species is a naphthofluorescein compound selected from the group consisting of a seminaphthofluorescein compound and a carboxynaphthofluorescein compound.

7. The method of claim 1, wherein the fluorescent species is a seminaphthofluorescein compound selected from the group consisting of 5′

(and 6′

)-carboxy-3,10-dihydroxy-spiro[7H-benzo[c]xanthene-7,1′

(3′

H)-isobenzofuran]-3′

-one and 2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoic acid.

8. The method of claim 1, wherein the fluorescent species comprises a conjugate of a fluorescent compound and a macromolecule.

9. The method of claim 8, wherein the macromolecule is an albumin.

10. The method of claim 1, wherein the fluorescent species comprises a seminaphthofluorescein/human serum albumin conjugate.

11. The method of claim 1, wherein the sample comprises blood or a blood product.

12. A method for testing the sterility of a sample, comprising:

(a) providing a filter device having a chamber and a filter configured to collect culturable organisms;

(b) filtering a sample through the filter such that any collected culturable organisms are retained in the chamber;

(c) adding a culture medium to the chamber;

(d) irradiating a fluorescent species in communication with the culture medium with excitation light having a wavelength sufficient to effect fluorescent emission from the fluorescent species,

wherein the fluorescent species exhibits a first emission intensity at a first emission wavelength and a second emission intensity at a second emission wavelength, the ratio of the first and second emission intensities being dependent on pH, and

wherein the fluorescent species comprises a 2-halo seminaphthofluorescein;

(e) measuring the first and second emission intensities to determine the pH of the culture medium; and

(f) inferring from the determined pH of the culture medium the sterility of the sample.

13. The method of claim 12, wherein the 2-halo seminaphthofluorescein is a 2-chloro seminaphthofluorescein.

14. The method of claim 12, wherein the 2-halo seminaphthofluorescein is 2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoic acid.

15. The method of claim 12, wherein the fluorescent species comprises a conjugate of a 2-halo seminaphthofluorescein and a macromolecule.

16. The method of claim 15, wherein the macromolecule is an albumin.

17. The method of claim 15, wherein the macromolecule is a human serum albumin.

18. The method of claim 15, wherein the conjugate is a 2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoic acid/human serum albumin conjugate.

19. The method of claim 12, wherein the sample comprises blood or a blood product.

20. An apparatus for testing the sterility of a sample liquid comprising:

(a) a chamber having an inlet and an outlet;

(b) a filter disposed at the chamber outlet, the filter adapted to collect culturable organisms from a liquid passed through the filter; and

(c) a port assembly having a distal end extending into the chamber, the distal end comprising a fluorescent species immobilized on a substrate,

wherein the fluorescent species is in fluid communication with an interior of the chamber,

wherein the fluorescent species comprises a material that, when irradiated with an excitation light having a wavelength sufficient to effect fluorescent emission from the fluorescent species, exhibits a first emission intensity at a first emission wavelength and a second emission intensity at a second emission wavelength, the ratio of the first and second emission intensities being dependant on pH, and

wherein the port assembly further comprises a housing for receiving a probe.

21. The apparatus of claim 20, wherein the filter comprises a membrane having a pore size of about 45 μ

m.

22. The apparatus of claim 20, wherein the housing comprises an open first end and a second end terminating with a window.

23. The apparatus of claim 22, wherein the port assembly comprises a tip member reversibly connectable to the housing second end, wherein the tip member is adapted to receive liquid from a sample to be tested.

24. The apparatus of claim 20, further comprising:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emission intensity;

(c) a second emission detector for measuring the second emission intensity;

(d) an excitation lightguide for transmitting excitation light from the light source to the fluorescent species, wherein the excitation lightguide comprises a first terminus proximate to the light source and a second terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from the fluorescent species to the first emission detector, wherein the first emission lightguide comprises a first terminus proximate to the first emission detector and a second terminus;

(f) a second emission lightguide for transmitting emission from the fluorescent species to the second emission detector, wherein the second emission lightguide comprises a first terminus proximate to the second emission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second termini of the excitation lightguide, first emission lightguide, and second emission lightguide.

25. The apparatus of claim 24, wherein the light source is a light-emitting diode.

26. The apparatus of claim 24, wherein the first and second emission detectors are photodiodes.

27. The apparatus of claim 24, wherein the excitation lightguide, the first emission lightguide, and the second emission lightguide are optical fibers.

28. The apparatus of claim 22, wherein the port assembly comprises a tapered tube terminating with the window.

29. The apparatus of claim 20, wherein the fluorescent species comprises a seminaphthofluorescein/human serum albumin conjugate.

30. An apparatus for testing the sterility of a sample liquid comprising:

(a) a chamber having an inlet and an outlet;

(b) a filter disposed at the chamber outlet, the filter adapted to collect culturable organisms from a liquid passed through the filter, the filter having a fluorescent species immobilized thereon,

wherein the fluorescent species is in fluid communication with an interior of the chamber,

wherein the fluorescent species comprises a material that, when irradiated with an excitation light having a wavelength sufficient to effect fluorescent emission from the fluorescent species, exhibits a first emission intensity at a first emission wavelength and a second emission intensity at a second emission wavelength, the ratio of the first and second emission intensities being dependant on pH; and

(c) a port assembly having a distal end adjacent to the filter, the port assembly further comprising a housing for receiving a probe.

31. The apparatus of claim 30, wherein the filter comprises a membrane having a pore size of about 45 μ

m.

32. The apparatus of claim 30, wherein the housing comprises an open first end and a second end terminating with a window.

33. The apparatus of claim 30, further comprising:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emission intensity;

(c) a second emission detector for measuring the second emission intensity;

(d) an excitation lightguide for transmitting excitation light from the light source to the fluorescent species, wherein the excitation lightguide comprises a first terminus proximate to the light source and a second terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from the fluorescent species to the first emission detector, wherein the first emission lightguide comprises a first terminus proximate to the first emission detector and a second terminus;

(f) a second emission lightguide for transmitting emission from the fluorescent species to the second emission detector, wherein the second emission lightguide comprises a first terminus proximate to the second emission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second termini of the excitation lightguide, first emission lightguide, and second emission lightguide.

34. The apparatus of claim 33, wherein the light source is a light-emitting diode.

35. The apparatus of claim 33, wherein the first and second emission detectors are photodiodes.

36. The apparatus of claim 33, wherein the excitation lightguide, the first emission lightguide, and the second emission lightguide are optical fibers.

37. The apparatus of claim 32, wherein the port assembly comprises a tapered tube terminating with the window.

38. The apparatus of claim 30, wherein the fluorescent species comprises a seminaphthofluorescein/human serum albumin conjugate.

39. A system for testing the sterility of a liquid sample, comprising:

(a) a plurality of stations, each station configured to receive a culture assembly;

(b) a plurality of culture assemblies, each culture assembly comprising;

(i) a chamber having an inlet and an outlet;

(ii) a filter disposed at the chamber outlet, the filter adapted to collect culturable organisms from a liquid passed through the filter;

(iii) a port assembly having a distal end extending into the chamber, the distal end comprising a fluorescent species immobilized on a substrate,

wherein the fluorescent species is in fluid communication with an interior of the chamber,

wherein the fluorescent species comprises a material that, when irradiated with an excitation light having a wavelength sufficient to effect fluorescent emission from the fluorescent species, exhibits a first emission intensity at a first emission wavelength and a second emission intensity at a second emission wavelength, the ratio of the first and second emission intensities being depending on pH; and

wherein the port assembly further comprises a probe housing.

40. The system of claim 39, wherein each culture assembly further comprises:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emission intensity;

(c) a second emission detector for measuring the second emission intensity;

(d) an excitation lightguide for transmitting excitation light from the light source to the fluorescent species, wherein the excitation lightguide comprises a first terminus proximate to the light source and a second terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from the fluorescent species to the first emission detector, wherein the first emission lightguide comprises a first terminus proximate to the first emission detector and a second terminus;

(f) a second emission lightguide for transmitting emission from the fluorescent species to the second emission detector, wherein the second emission lightguide comprises a first terminus proximate to the second emission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second termini of the excitation lightguide, first emission lightguide, and second emission lightguide.

41. A system for testing the sterility of a liquid sample, comprising:

(a) a plurality of stations, each station configured to receive a culture assembly;

(b) a plurality of culture assemblies, each culture assembly comprising;

(i) a chamber having an inlet and an outlet;

(ii) a filter disposed at the chamber outlet, the filter adapted to collect culturable organisms from a liquid passed through the filter, the filter having a fluorescent species immobilized thereon,

wherein the fluorescent species is in fluid communication with an interior of the chamber,

wherein the fluorescent species comprises a material that, when irradiated with an excitation light having a wavelength sufficient to effect fluorescent emission from the fluorescent species, exhibits a first emission intensity at a first emission wavelength and a second emission intensity at a second emission wavelength, the ratio of the first and second emission intensities being dependant on pH; and

(iii) a port assembly having a distal end adjacent to the filter, the port assembly further comprising a probe housing.

42. The system of claim 41, wherein each culture assembly further comprises:

(a) a light source for exciting the fluorescent species;

(b) a first emission detector for measuring the first emission intensity;

(c) a second emission detector for measuring the second emission intensity;

(d) an excitation lightguide for transmitting excitation light from the light source to the fluorescent species, wherein the excitation lightguide comprises a first terminus proximate to the light source and a second terminus distal to the light source;

(e) a first emission lightguide for transmitting emission from the fluorescent species to the first emission detector, wherein the first emission lightguide comprises a first terminus proximate to the first emission detector and a second terminus;

(f) a second emission lightguide for transmitting emission from the fluorescent species to the second emission detector, wherein the second emission lightguide comprises a first terminus proximate to the second emission detector and a second terminus; and

(g) a probe housing, wherein the probe housing houses the second termini of the excitation lightguide, first emission lightguide, and second emission lightguide.