WO1991020009A1

WO1991020009A1 - Improved method and device for use of microspheres in microscopy and for quantifying the post coital test

Info

Publication number: WO1991020009A1
Application number: PCT/US1991/004128
Authority: WO
Inventors: Michael C. Doody
Original assignee: Doody Michael C
Priority date: 1990-06-15
Filing date: 1991-06-11
Publication date: 1991-12-26

Abstract

The use of microparticles (10) of a known uniform diameter allows uniform control of the thickness of the specimen layer (12), such as post coital cervical mucus, between a microscope slide (20) and a coverslip (25). The invention utilizes delivery of a dry microsphere (10) or similar microparticle into a specimen (12) that is to be microscopically examined. This allows control of the thickness of the specimen layer (12) during microscopic examination thereby preventing deformation of the sample (12) which is caused by pressure exerted by an unsupported coverslip (25) placed directly on the specimen (12). This also prevents dilution of the specimen (12) and alteration of certain physical characteristics of the fluid to be observed, i.e. viscosity, pH, osmolarity, ionic strength, and concentrations of solutes.

Description

Improved Method and Device for use of

Microspheres In Microscopy and for

Quantifying the Post Coital Test

Technical Field

This invention relates to the general field of microscopy and of supporting a coverslip during the microscopic examination of gelatinous specimens or of specimens in a liquid environment. More particularly it relates to the study, diagnosis and evaluation of cervical factor infertility and the diagnostic procedure known as the post coital test.

Background Art

One common cause of infertility that is easily diagnosed is cervical factor infertility. In order to reach the site of fertilization, spermatozoa must "swim" from the vagina through the mucus produced by the cervix. If the environment provided by the cervical mucus is incompatible with sperm motility or survivability, then insufficient numbers of sperm will survive, reach the uterus and eventually reach the site of fertilization. It is commonly known in the art that undesirable viscosity, antibodies or pH levels affect motility and survivability. It is estimated that 10-15% of infertile couples are incapable of conceiving because of cervical factor infertility. The method that is preferred by the medical community to diagnose and evaluate cervical factor infertility is the post coital test.

During the microscopic examination of certain types of specimens, such as cervical mucus, it is desirable to control the amount of pressure applied by the coverslip to the specimen or to prevent the coverslip from deforming the specimen. Certain prior art methods utilize microparticles delivered in a liquid carrier. This can create several problems. The use of a liquid carrier can result in the dilution of the specimen that may be undesirable in certain applications. The use of a liquid carrier can also disturb certain physical characteristics of the fluid to be observed, i.e. a liquid carrier can change the viscosity, pH, osmolarity, ionic strength, and concentrations of the solutes to name a few. Also the fluid carrier can and often fails to mix with the specimen. It is also often desirable to be able to accurately and reproducibly allow the inspection of a standard volume.

The post coital test has been in use for over one hundred years and is well known in the art. As originally intended, a quantity of post coital mucus would be inspected and the concentration of motile sperm assessed microscopically. As it has been implemented, a smear of mucus is sandwiched between a glass slide and a cover slip, flow and spreading of the mucus occurs, and the number of living and dead sperm in a high power field is then counted. There have been no changes or improvements in the post coital test since it was first introduced.

Due to the nature of cervical mucus, it is impossible using the traditional post coital test prior art to achieve uniformity of the thickness of the mucus layer across the entire specimen and between separately prepared specimens. No attempt has been made in the prior art to control or determine the average thickness of the mucus layer in the post coital test. Therefore, it is possible that the results could have been affected adversely affected by this variability. The medical community has accepted the limitation of inspecting a mucus layer of unknown thickness and has accepted a subjective analysis of the concentration of sperm found in post coital cervical mucus because there has not been a technically feasible alternative prior to the development of the present invention.

Difficulties in managing the spread of cervical mucus have prevented the use of fixed counting chambers such as those used for counting blood cells. Moreover, sperm cells are sensitive to mechanical shear forces generated by flow of liquid through narrow channels and the motility (percentage of living sperm) is decreased by the spread of mucus between a microscope slide and coverslip as practiced by the traditional post coital test prior art. Therefore, it is an object of this invention to deliver a dry microsphere or similar microparticle into a specimen that is to be microscopically examined and therefore provide a method to accurately and reproducibly quantify the post coital test.

It is another object of this invention to control the thickness of the specimen layer during microscopic examination by the use of plastic or other suitable solid material microspheres or microfibers acting as spacers between the microscope slide and the coverslip thus allowing the inspection of a standard volume.

It is another object of the present invention to deliver into the specimen a homogenous preparation of uniformly sized, inert and nontoxic plastic or other suitable solid material microspheres or microfibers which act as spacers to keep the microscope slide at a uniform distance from an overlying cover slip.

It is another object of this invention to control the thickness of the mucus layer in the post coital test by the use of plastic or other suitable solid material microspheres or microfibers acting as spacers between the microscope slide and the coverslip thus allowing the inspection of- a standard volume of post coital mucus regardless of location on the slide and regardless of which slide is being inspected. It is another object of the present invention to provide a technically feasible method of quantifying the results of the post coital test in volumetric terms, i.e. total or motile sperm per cubic centimeter of mucus.

It is another object of the present invention to allow for the establishment of true ranges of normal and abnormal tests based on the volumetric expression of the test results.

It is another object of the present invention to deliver dry microspheres or similar microparticles into a specimen that is to be microscopically examined without resulting in the dilution of the specimen.

It is still another object of the present invention to deliver a dry microsphere or similar microparticle into a specimen that is to be microscopically examined without disturbing certain physical characteristics of the fluid to be observed, i.e. viscosity, pH, osmolarity, ionic strength, and concentrations of solutes.

A further object of the present invention is to deliver dry microspheres or similar microparticles into a specimen that is to be microscopically examined in a manner that allows thorough mixing of the microspheres with the specimen.

Other objects and advantages over the prior art will become apparent to those skilled in the art upon reading the detailed description together with the drawings as described as follows.

Disclosure of the Invention

The method of the present invention comprises fixing the distance between a microscope slide and a coverslip such that each microscopic field of view will inspect a reproducible volume. The fixed distance contains a sample of a specimen to be examined. The specimen can be suspended in a liquid environment or can be a liquid or gelatinous sample.

Fixing the distance between the microscope slide and a coverslip comprises applying a gelatinous or liquid specimen, such as cervical mucus, or a specimen in a liquid environment to a microscope slide; disposing a plurality of uniformly sized symmetrical microparticles upon the slide such that the microparticles are interspersed within the specimen to be microscopically examined; and applying a cover slip over the microparticle laced specimen. The practitioner then exerts pressure on the coverslip overlying the specimen causing the gelatinous or liquid specimen to safely expand thus filling the space between the microscope slide and the coverslip defined by the microparticles.

Accordingly, one practising the various features of this invention, may quantify the post coital test such that the results may be accurately and reproducibly stated in terms of total or motile sperm per cubic centimeter. The distance fixed between the coverslip and the microscope slide by the microparticles contains a sample of post coital cervical mucus for microscopic quantitative analysis. The microspheres or microfibers allow for the application of external pressure sufficient to spread the mucus layer while preventing turbulence injury to the sperm cell which would decrease their motility. The sperm cells are then counted and the number of living and dead sperm per high powered field is mathematically converted to living or dead sperm per volume of post coital mucus. Thus, 87% of the observed variation in motility in the traditional post coital test is eliminated by this invention. Those skilled in the art will recognize that microspheres or microfibers are being used to prevent injury to sperm cells during dynamic flow and spreading of post coital mucus.

The device comprises a reservoir for containing a microparticle suspension under pressure; a volatile fluid carrier is contained in the reservoir to serve as a dry propellant; uniformly sized microparticles of uniform diameter in suspension in the fluid carrier; a mechanical means for discharging the microparticles and a discharge port in the discharge means selectively sized such that the microspheres or microfibers of uniform diameter can be discharged, without clogging, onto a microscope slide in an aerosol fashion.

The device alternatively consists of a reservoir for containing the dry microparticles; one or more orifices in the reservoir to allow the exit a controlled amount of the microparticles and an optional perforated plate(s) or screen(s) to disperse the microparticles in order to randomize the sprinkling of the microparticles.

Brief Description of the Drawings

The above mentioned features of the invention will become more clearly understood from the following detailed description of the invention read together with the drawings in which:

Figure 1 illustrates a cross sectional view of the traditional post coital test prior art method. It will be understood that these figures are for illustrative purposes and are not drawn to scale.

Figure 2 illustrates a cross sectional view of the method of conducting the post coital test taught by the present invention.

Figure 2-b illustrates a cross sectional view of the method of conducting the post coital test taught by the present invention utilizing microfibers as spacers.

Figure 2-c illustrates a cross sectional view of the method of using microspheres as spacers for microscopy taught by the present invention.

Figure 2-d illustrates a cross sectional view of the method of using microfibers as spacers for microscopy taught by the present invention.

Figure 3 illustrates an elevational view of the method of conducting the post coital test taught by the present invention.

Figure 4 illustrates an elevated view of the device for disposing the microspheres on the microscope slide in the present invention.

Figure 5 illustrates a cross sectional view of the device for disposing the microspheres on the microscope slide in the present invention showing the pressure on the inside of the reservoir and the microspheres being dispensed in aerosol fashion.

Figure 6 illustrates a cross sectional view of an alternative device for disposing the microspheres on the microscope slide in the present invention in a sprinkling fashion.

Figure 7 illustrates an inverted cross sectional view of an alternative device for disposing the microspheres on the microscope slide in the present invention in a sprinkling fashion showing the sprinkling of the microparticles. Best Mode for Carrying out the Invention

A slide prepared in accordance with the present invention is illustrated generally at 2 in the figures. A sample of a gelatinous specimen 12' in Figure 2-c and 2-d is disposed on a glass slide 20. The slide 20 is sprayed with a dry aerosol application of microspheres 10 of uniform diameter such that the application produces a selected density of microspheres per square centimeter. The slide is then covered with a coverslip 25 applying pressure until no further spread of specimen is possible. A definite "stop point" of the expansion of the specimen is observed as the coverslip 25 engages the microsphere 10 beyond which no further spreading occurs. In the preferred embodiment, a sample of post coital cervical mucus 12 containing sperm 13 in FIG. 2 and 2-b is disposed on a glass slide 20. The slide 20 is sprayed with a dry aerosol application of microspheres or microfibers of uniform diameter 10 such that the application produces a selected density of microspheres or microfibers per square centimeter. The slide is then covered with a coverslip 25 applying pressure until no further spread of mucus is possible. A definite "stop point" of the expansion of the mucus layer is observed as the coverslip 25 engages the microsphere 10 beyond which no further spreading occurs. As an alternate embodiment, fiberglass or synthetic microfibers 11 of uniform diameter are disposed onto the sample 12 thereby acting as spacers between the microscope slide 20 and the coverslip 25. Those skilled in the art will recognize that any inert symmetrical microparticle that has uniform diameters through two or more separate planes will provide a suitable equivalent for microspheres or microfibers.

Table l gives a sample raw count of 15 samples for a given patient. The first two columns give total and motile sperm counts per field on a slide prepared in accordance with the present invention, and the next two columns give counts for 15 samples from the same patient on a slide prepared in the traditional manner:

TABLE 1

Studies of many patients have shown that interpatient variability is reduced by a factor of 81% for motility and 82% for total count by this invention. As can be seen the results of total and motile sperm recorded when the post coital test is practiced in accordance with the present invention in this case are greater than the results recorded when the traditional method of practicing the post coital test is utilized. False negative results are reduced by 30% by the post coital test practiced in accordance with the present invention.

A device for dispensing the microspheres or microfibers is illustrated generally at 30 in FIG. 4. The microspheres or microfibers are suspended 34 in a volatile fluid carrier propellant in the device's reservoir 32. The reservoir 32 is under pressure 36 such that when the mechanical discharge means 38 is depressed the microsphere suspension 34 is discharged in aerosol fashion 40. The port 39 in the mechanical discharge means 38 is selectively sized to prevent clogging when the microsphere suspension 34 is discharged onto a microscope slide.

As an alternative embodiment the microspheres 10 or microfibers 11 may be disposed into the sample 12 on the microscope slide 20 in a sprinkling fashion by a sprinkling device illustrated generally as 31 in FIG. 6. The shaking device 31 comprises a reservoir 32* which receives the microparticles 46. The microparticles 46 are shaken through one or more orifices 44. A diffusing means 42 disperses the microparticles 46 in order to randomize and diffuse the sprinkling pattern.

From the foregoing description, it will be recognized by those skilled in the art that a method and device using microspheres for microscopically examining gelatinous or liquid specimens, such as post coital cervical mucus or specimens in a liquid environment, and for quantifying the post coital test, offering advantages over the prior art has been provided. Specifically, the method and device of the present invention provide a method to accurately and reproducibly quantify the post coital test by controlling the thickness of the mucus layer in the post coital test by delivering a homogenous preparation of uniformly sized, inert and nontoxic plastic microspheres or microfibers which act as spacers to keep the microscope slide at a uniform distance from an overlying cover slip thereby insuring that a constant volume is inspected with each microscope field. The method of the present invention further provides a method for delivering a dry microsphere or similar microparticle into a specimen that is to be microscopically examined without resulting in the dilution of the specimen and without altering certain physical characteristics of the fluid to be observed, i.e. viscosity, pH, osmolarity, ionic strength, and concentrations of solutes and allows adequate mixing of the microspheres with the specimen.

While a preferred embodiment has been shown and described, it will be understood that it is not intended to limit the disclosure, but rather it is intended to cover all modifications and alternate methods falling within the spirit and the scope of the invention as defined in the appended claims.

Claims

Having thus described the aforementioned invention, I Claim:

1. A method for mounting a coverslip over a gelatinous specimen comprising: fixing a distance between a microscope slide and a coverslip whereby said fixed distance receives said specimen for microscopic analysis.

2. The method of claim 1 wherein said method further comprises: applying said specimen to a microscope slide; disposing a plurality of uniformly sized microparticles upon said slide such that said microparticles are interspersed in said specimen; and applying a cover slip over said specimen such that said coverslip is supported by said microparticles above said specimen.

3. A method for mounting a coverslip over a specimen contained within a liquid environment comprising: applying a' specimen to a microscope slide; disposing a plurality of uniformly sized microparticles upon said slide such that said microparticles are interspersed in said specimen; applying a cover slip over said specimen such that said coverslip is supported by said microparticles above said specimen;

4. The method of claim 3 wherein microspheres of uniform diameter are disposed upon said slide.

5. The method of claim 4 wherein said microspheres of uniform diameter are disposed in aerosol fashion upon said slide.

6. The method of claim 4 wherein said microspheres of uniform diameter are disposed in a sprinkling fashion upon said slide.

7. The method of claim 4 wherein said microspheres are dry when disposed upon said slide.

8 . The method of claim 3 wherein fiberglass microfibers of uniform diameter are disposed upon said slide.

9 . The method of claim 8 wherein said fiberglass microfibers of uniform diameter are disposed in aerosol fashion upon said slide.

10 . The method of claim 8 wherein said fiberglass microfibers of uniform diameter are disposed in a sprinkling fashion upon said slide.

11 . The method of claim 3 wherein said fiberglass microfibers of uniform diameter are disposed in aerosol fashion upon said slide by said device such that a microfiber-specimen mixture with a density of about 40 to about 60 microfibers per square centimeters is achieved.

12. The method of claim 11 wherein said microfibers are dry when disposed upon said slide.

13. A method for quantifying the post coital test and preventing flow injury comprising: fixing a distance between a microscope slide and a coverslip whereby said fixed distance receives a sample of post coital cervical mucus for quantitative analysis facilitating determination of the concentration of sperm or other cells in said sample of post coital cervical mucus.

14. The method of claim 13 wherein said method further comprises: applying a sample of post coital cervical mucus to a microscope slide; disposing a plurality of uniformly sized microparticles upon said slide such that said microparticles are interspersed in said sample of post coital cervical mucus; and applying a cover slip over said sample of post coital cervical mucus such that said coverslip exerts pressure on said sample of post coital cervical mucus causing said sample to expand thus filling said fixed distance.

15. A method for quantifying the post coital test and preventing flow injury to sperm comprising: applying a sample of post coital cervical mucus to a microscope slide; disposing a plurality of uniformly sized microparticles upon said slide such that said microparticles are interspersed in said sample of post coital cervical mucus; applying a cover slip over said sample of post coital cervical mucus such that said coverslip exerts pressure on said sample of post coital cervical mucus causing said sample to expand thus filling said fixed distance whereby viable and dead sperm in a plurality of fields of view may be microscopically counted thus allowing the sperm concentration in terms of number of sperm per volume to be calculated.

16. The method of claim 15 wherein microspheres of uniform diameter are disposed upon said slide.

17. The method of claim 16 wherein microspheres of uniform diameter are disposed in aerosol fashion upon said slide.

18. The method of claim 16 wherein microspheres of uniform diameter are disposed in a sprinkling fashion upon said slide.

19. The method of claim 15 wherein fiberglass microfibers of uniform diameter are disposed upon said slide.

20. The method of claim 19 wherein fiberglass microfibers of uniform diameter are disposed in aerosol fashion upon said slide.

21. The method of claim 19 wherein fiberglass microfibers of uniform diameter are disposed in a sprinkling fashion upon said slide.

22. The method of claim 15 wherein fiberglass microfibers of uniform diameter are disposed in aerosol fashion upon said slide by said device such that a microfiber-mucus mixture with a density of about 40 to about 60 microfibers per square centimeter is achieved.

23. A device for dispensing microbeads into a specimen contained within a liquid environment prior to mounting a coverslip over said specimen said device comprising: a reservoir for containing a suspension under pressure; a fluid carrier contained in said reservoir said fluid carrier being volatile such that said fluid carrier acts as a dry propellant; microparticles of uniform diameter in suspension in said fluid carrier; mechanical means for discharging said microparticles; a discharge port in said device selectively sized such that said microparticles of uniform diameter can be discharged, without clogging, onto a microscope slide in an aerosol fashion.

24. The device of claim 23 wherein said fluid carrier suspends microspheres of uniform diameter.

25. The device of claim 23 wherein said fluid carrier suspends fiberglass microfibers of uniform diameter.

26. A sprinkling device for delivering a random distribution of microparticles of uniform diameter onto a microscope slide which comprises: a reservoir for containing the dry microparticles; and one or more perforated dispersion screens which disperse said microparticles in a random fashion onto said microscope slide.

27. The sprinkling device of claim 26 wherein said sprinkling device further comprises one or more orifices in said reservoir to allow the exit of a controlled amount of said microparticles onto said dispersion screens.

28. The sprinkling device of claim 26 wherein said sprinkling device delivers dry microspheres of uniform diameter.

29. The sprinkling device of claim 26 wherein said sprinkling device delivers dry fiberglass microfibers of uniform diameter.

30. The sprinkling device of claim 27 wherein said sprinkling device delivers dry microspheres of uniform diameter.

31. The sprinkling device of claim 27 wherein said sprinkling device delivers dry fiberglass microfibers of uniform diameter.