Flow cytometer for analysis of general diagnostic factors in cells and body fluids

• US 20030119206A1
• Filed: 01/30/2003
• Published: 06/26/2003
• Est. Priority Date: 03/30/1998
• Status: Abandoned Application

• Current Assignee: Shafrira Shai
• Original Assignee: Shafrira Shai
• Inventors: Shai, Shafrira
• Application Number:
  US10/353,996
• Publication Number:
  US 20030119206A1
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 2333/295 from Chlamydiales (o)
• CPC Class Codes: G01N 2333/59 Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: G01N 2333/70589 CD45
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

The present invention relates to a system to analyse general diagnostic factors in cells and body fluids using a flow cytometer, and in particular to a system featuring a number of different fertility tests, in a simple, expedited format, in order to investigate factors affecting fertility, preferably in a semi or fully automated manner. Specifically, a preparative method has been developed to increase the success of in vitro fertilisation (I.V.F) and intrauterine insemination (I.U.I) in cases of immunoinfertility by removing sperm-bound antibodies from sperm cells. A special device has been designed to collect only motile sperm cells from semen samples. Thus, this invention provides improved methods for general diagnostic testing and infertility screening and enables gynecologists to obtain information from an infertile couple in a preliminary test, which until now has been time consuming and only possible to run in sophisticated laboratories.

Claims

1. A semi-automated fertility system for assessing the fertility of a couple, the couple consisting of a male partner and a female partner, comprising;

(a) a cervical smear including cervical mucus and at least one serum sample from the female partner;

(b) at least one semen sample and at least one serum sample from the male partner;

(c) a fertility kit for determining at least one fertility affecting factor, said fertility kit being used to perform a fertility test, said fertility kit including at least one reagent, such that said reagent is able to react with a sample selected from the group consisting of a cervical smear and serum sample from the female partner and, a semen sample and a serum sample from the male partner, to form a reaction product; and

(d) a flow cytometer, such that said flow cytometer is able to analyse said reaction product to determine said fertility factor.

2. The system of claim 1, wherein a plurality of tests can be performed on a single sample of the group consisting of, at least one female cervical smear, female cervical mucus, at least one female serum sample, at least one male semen sample and at least one male serum sample.

3. The system of claim 1, wherein said sample from the male partner is said semen sample, and said reagent is a viscous solution, such that motility of sperm in said sample is determined according to movement of said sperm through said viscous solution.

4. The system of claim 3, wherein said viscous solution includes a dye.

5. The system of claim 3 further comprising a device for measuring sperm motility in a sample of sperm, said device comprising:

(a) a sample compartment;

(b) at least one channel; and

(c) a barrier separating said sample compartment from said at least one channel, such that said sperm must cross over said barrier from said sample compartment to reach said at least one channel.

6. The system of claim 1, wherein said sample from the male partner is said semen sample, and said reagent is a dye to identify live cells, such that said fertility test determines a number of live cells.

7. The system of claim 6, wherein said dye includes dichlorfluorescein.

8. The system of claim 1, wherein said sample from the male partner is said semen sample, and said reagent is a morphology gate system comprising at least one gate such that said fertility test determines sperm cell morphology according to an ability of said sperm cells to enter through said at least one gate.

9. The system of claim 8, wherein said access is determined by geometry of said gate.

10. The system of claim 8, further comprising a dye.

11. The system of claim 10, wherein said dye is acridine orange.

12. The system of claim 1, wherein said sample from the male partner is said semen sample, and said reagent comprising:

(a) a solution including anti human antibodies conjugated with fluorescent dye, said anti human antibodies binding to an antibody present in cells of said semen sample; and

(b) a second solution including a dyed label, said dyed label binding to said anti human antibodies, such that antibodies bound to sperm are detected;

such that said fertility test is detection of sperm-bound antibodies.

13. The system of claim 12, wherein said reagent comprises:

(a) a solution to remove non-specific antibodies; and

(b) a second solution to block non-specific antibody binding sites on the sperm surface.

14. The system of claim 1, wherein said sample from the male partner is said semen sample, and said reagent is fluorescent micro sphere beads coated with zp-3 antibodies, and said fertility test is ability of said sperm to bind to said beads.

15. The system of claim 1, wherein said sample comprises said cervical smear of the female partner and said semen sample of the male partner, and said reagent comprises at least one antibody specific to at least one infectious agent of the genitalia, such that said fertility test is detection of said infectious agent in cervical smear and semen sample.

16. The system of claim 15 further comprising:

(a) polystyrene micro sphere beads coated with an antibody specific to an infectious agent;

(b) at least one biotin labeled antibody specific to said infectious agent, said biotin conjugate binding to said beads;

(c) a streptavidin protein, said protein binding to biotin; and

(d) a fluorescent labeled dye, said dye binding to said antibody.

17. The system of claim 15, wherein said sample comprises said cervical smear of the female partner and said semen sample of the male partner, and said reagent comprises at least one antibody specific to Clamydia trachomatis, such that said fertility test is detection of Chlamydia trachomatis in cervical smear and semen sample.

18. The system of claim 17 further comprising:

(a) polystyrene micro sphere beads coated with an antibody specific to Clamydia trachomatis;

(b) at least one biotin labeled antibody specific to Chlamydia trachomatis, said biotin labeled antibody binding to said beads;

(c) a streptavidin protein, said protein binding to biotin; and

(d) a fluorescent labeled dye, said dye binding to said antibody.

19. The system of claim 1, wherein said sample comprises said serum sample of the female partner and said serum sample of the male partner such that said fertility test is detection of hormone levels in serum sample.

20. The system of claim 19 further comprising:

(a) at least one polystyrene micro sphere bead coated with antibodies specific for the hormone to be tested;

(b) at least one biotin labeled antibody, said antibody binding to said hormone;

(c) a streptavidin protein, said protein binding to biotin; and

(d) a fluorescent labeled dye, said dye binding to said antibody.

21. The system of claim 1, wherein said fertility test is the ability of sperm cells to undergo acrosome reaction.

22. The system of claim 1, wherein said fertility test is sperm cell count and white blood cell count.

23. A semi-automated system for assessing diagnostic factors, comprising;

(a) at least one cell and body fluid sample;

(b) a kit for determining at least one diagnostic factor, said kit being used to perform a diagnostic test, said kit including at least one reagent, such that said reagent is able to react with at least one cell and body fluid sample to form a reaction product; and

(c) a flow cytometer, such that said flow cytometer is able to analyse said reaction product to determine said diagnostic factor.

24. The system of claim 23, wherein said diagnostic factor is hormone level.

25. The system of claim 23, wherein said diagnostic factor is the identification of antigens of any component of an infectious agent.

26. The system of claim 23, wherein said diagnostic factor is a fertility factor.

27. A method for detecting sperm-binding antibodies in cervical mucus of the female partner comprising the steps of:

(a) washing semen sample of the male partner in a solution of low pH to remove specific and non specific antibodies;

(b) incubating the semen sample of the male partner in a solution to block non specific binding sites in the semen sample;

(c) incubating treated semen sample of the male partner with cervical mucus of the female partner;

(d) incubating mixture of said treated semen sample of the male partner and cervical mucus of the female partner with anti human antibodies bound to fluorescent dye; and

(e) detecting results in flow cytometer.

28. A method for predicting success of IVF and IUI treatment, comprising the steps of:

(a) washing and capacitation of sperm sample;

(b) incubating said sperm sample with fluorescently labeled beads coated with peptides of the oocyte- membrane;

(c) washing said sperm cells; and

(d) detecting sperm cells bound to oocyte membrane peptide to predict success of IVF and IUI treatment.

29. The method of claim 28, wherein said prediction of success of IVF and IUI treatment is determined by visual observation of a dye.

30. A method of collecting motile sperm cells from a sample of sperm, comprising the steps of:

(a) providing a device for measuring sperm motility in a sample of sperm, said device including;

(i) a sample compartment;

(ii) at least one channel; and

(iii) a barrier separating said sample compartment from said at least one channel, such that said sperm must cross over said barrier from said sample compartment to reach said channel;

(b) filling said channels of said device with a viscous solution;

(c) putting said sample in said sample compartment of said device; and

(d) collecting motile sperm cells from said channels of said device.

31. The method of claim 30, further comprising separating white blood cells by magnetic separation with magnetic beads coated with anti CD-45 antibodies.

32. A method of removal of sperm bound antibodies from semen comprising the steps of:

(a) forming a cell pellet by centrifugation of the semen;

(b) adding an acidic solution to said cell pellet to remove antisperm antibodies; and

(c) resuspending cell pellet in a mixture of washing solution, reagent to increase cell motility and a reagent to prevent free radical production to obtain semen without sperm bound antibodies.

33. The method of claim 32, wherein said reagent to increase cell motility includes hyaluronic acid.

34. The method of claim 32, wherein said reagent to prevent free radical production includes ferulic acid.

35. A method for increasing success of IVF treatment and IUI treatment, comprising the steps of:

(a) removing white blood cells and separating motile sperm cells from semen by;

(i) providing a device, for separation of motile sperm cells from non-motile material, said non-motile material including white blood cells, in a sample of sperm, said device comprising;

(I) a sample compartment;

(II) at least one channel; and

(III) a barrier separating said sample compartment from said at least one channel, such that said sperm must cross over said barrier from said sample compartment to reach said channel;

(ii) filling said channels of said device with a viscous solution;

(iii) mixing semen with magnetic beads coupled with anti CD45;

(iv) putting said sample in said sample compartment and incubating; and

(v) collecting motile sperm cells from said channels;

(b) removing sperm bound antibodies by;

(i) forming a cell pellet by centrifugation;

(ii) adding an acidic solution to remove antisperm antibodies; and

(iii) resuspending cell pellet in a mixture of washing solution, reagent to increase cell motility and a reagent to prevent free radical production.

36. A device for measuring sperm motility in a sample of sperm, comprising;

(a) a sample compartment;

(b) at least one channel; and

(c) a barrier separating said sample compartment from said at least one channel, such that said sperm must cross over said barrier from said sample compartment to reach said at least one channel.

37. The device of claim 36, wherein said at least one channel contains a viscous fluid.

38. The device of claim 37, wherein said viscous fluid contains at least one dye, such that the sperm are able to contact said dye upon reaching said at least one channel.