U.S. patent application number 10/134575 was filed with the patent office on 2003-01-02 for method and composition of matter for enhancing staining of microorganisms.
This patent application is currently assigned to Ventana Medical Systems, Inc.. Invention is credited to Fischer, Timothy, Sherman, Walter.
Application Number | 20030003537 10/134575 |
Document ID | / |
Family ID | 23103893 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030003537 |
Kind Code |
A1 |
Fischer, Timothy ; et
al. |
January 2, 2003 |
Method and composition of matter for enhancing staining of
microorganisms
Abstract
The invention is directed to a method and composition of matter
for enhancing the staining of tissue or features of interest
therein by applying an effective amount of a bile salt in
combination with a special stain to a tissue suspected of harboring
a microorganism or other feature of diagnostic interest. The
particular invention herein is directed to detecting H. pylori, the
microorganism responsible for gastrointestinal ulcers. The
composition of matter is a sensitizer for Alcian Yellow stain.
Inventors: |
Fischer, Timothy; (Raleigh,
NC) ; Sherman, Walter; (Tucson, AZ) |
Correspondence
Address: |
VENTANA MEDICAL SYSTEMS, INC.
1910 INNOVATION PARK DRIVE
TUCSON
AZ
85737
US
|
Assignee: |
Ventana Medical Systems,
Inc.
|
Family ID: |
23103893 |
Appl. No.: |
10/134575 |
Filed: |
April 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60287679 |
Apr 30, 2001 |
|
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Current U.S.
Class: |
435/69.1 |
Current CPC
Class: |
G01N 2001/302 20130101;
G01N 1/30 20130101 |
Class at
Publication: |
435/69.1 |
International
Class: |
C12P 021/06 |
Claims
We claim:
1. A method of enhancing the staining of tissue or features of
diagnostic interest therein by applying an effective amount of a
bile salt in combination with a special stain to a tissue suspected
of harboring a microorganism or other feature of diagnostic
interest.
2. The method of claim 1 wherein said bile salt is sodium
desoxycholate.
3. The method of claim 1 wherein said special stain is Alcian
Yellow.
4. The method of claim 1 wherein said microorganism is H.
pylori.
5. A composition of matter comprising a bile salt in combination
with a special stain.
6. The composition of claim 5 wherein said bile salt is sodium
desoxycholate.
7. The composition of claim 5 wherein said special stain is Alcian
Yellow
8. The composition of claim 6 wherein said sodium desoxycholate has
at least a concentration of 0.32%.
9. The composition of claim 6 wherein said sodium desoxycholate has
at most a concentration of 0.64%.
10. The composition of claim 6 wherein additionally present is
sodium borate decahydrate.
Description
BACKGROUND
[0001] 1. Field of the Invention
[0002] This invention is in the general field of histology, in
particular the invention is an improved composition and method for
staining microorganisms.
[0003] 2. Description of Related Art
[0004] Histology is the practice of examination and classification
of tissue to aid in diagnosis of a disease or condition expressed
in the morphology of the affected tissue. The practice of staining
tissue or features of tissues with a visualizing agent to visualize
or detect them, and thus provide a scientific basis for diagnosing
a patient, is a very old art, going back over one hundred years.
Histology can be divided into three basic practice areas:
immunohistochemistry (use of antibodies to visualize tissue
structures), in situ hybridization (use of DNA/RNA probes to
detect) or special stains (chemical staining of tissue). Special
stains is the term given to a collection of chemically-based stains
that have been developed in response to difficult to stain tissue
types, unusual diseases, infectious diseases or other non-typical
situations affecting the tissue.
[0005] The staining of histologic sections of tissue for the
identification of H. pylori using a combination of Alcian Yellow
and Toluidine Blue was published by Leung et al. (Leung, J K,
Gibbob, K J, Vartanian, R K, "Rapid staining method for Heliobacter
pylori in gastric biopsies," J. Histol. 19:131-132 (1996).
SUMMARY OF THE INVENTION
[0006] The invention is directed to a method and composition of
matter for enhancing the staining of tissue or features of interest
therein by applying an effective amount of a bile salt in
combination with a special stain to a tissue suspected of harboring
a microorganism or other feature of diagnostic interest. The
particular invention herein is directed to detecting H. pylori, the
microorganism responsible for gastrointestinal ulcers.
[0007] The composition of matter is a sensitizer for Alcian Yellow
stain.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 includes two color pictures of a section of tissue on
a slide stained using the recipe and Alcian Yellow sensitizer of
the present invention. The top picture is a tissue section not
stained using the sensitizer, and the bottom picture is of a tissue
section wherein the recipe included in addition the step of using
the sensitizer.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0009] The staining of histologic sections for the identification
of Heliobactor pylori using a combination of Alcian Yellow and
Toluidine Blue was published by Leung et al (Leung, J K, Gibbon, K
J , Vartanian, R K: Rapid staining method for Heliobacter pylori in
gastric biopsies. J. Histol 19:131-132, 1996). The combination
normally results in H. pylori being stained blue, the mucin yellow
and the background blue. We were able to replicate the results of
this procedure using manual methods, but were unable to achieve
good staining of the H. pylori organisms when the procedure was
automated on the NexES.RTM. Special Stains instrument (Ventana
Medical Systems, Inc., Tucson, Ariz.).
[0010] To achieve a stable Toluidine stain solution, the published
formulation was modified. Instead of adding a dilute solution of
sodium hydroxide to the diluted Toluidine Blue stock solution just
prior to use, we dispensed a solution of 0.025 M sodium borate, pH
9.30+/-0.05 to the tissue on the slide, followed by dispensing the
Toluidine Blue dye. We used this same technique previously with
Toluidine Blue solution applied as a counterstain in the automated
AFB II Staining kit (Ventana Medical Systems, Tucson, Ariz.). In an
analysis of this data, the inventors concluded that the nonionic
detergent (Tween 20) used in the Water Wash solution interfered
with binding of the Toluidine Blue dye to the microorganisms. We
modified the borate buffer solution to add a series of compounds
that we suspected might neutralize the effect of the nonionic
detergent. Surprisingly, only one compound, sodium desoxycholate
(Sigma-Aldrich, Cat. No. D6750), achieved the desired effect, and
the effect was shown to be optimum at a SDC concentration between
0.32%- 0.64%. We chose the near midpoint concentration of 0.05% SDC
for further experiments.
[0011] The temperature of the NexES Special Stains instrument was
reduced to ambient by leaving the heat turned off, and the
concentration of Alcian Yellow and Toluidine dyes was optimized for
the lower temperature (pp 16-23, AY notebook 3, attached). An
experiment was then performed to test the optimized reagents
against three (3) tissues cut at 3, 4 and 5 micron thicknesses. The
optimized reagent performed satisfactorily in comparison to manual
stains.
[0012] A "special stain" as defined herein is any chemically-based
stain useful for histological analysis that is not an
immunohistochemical stain, or an in situ hybridization stain. A
representative listing of such stains may be found in "Theory and
Practice of Histological Techniques," Bancroft, John D., Stevens,
Alan, eds., Fourth Ed. (1996), Churchill Livingstone Press, New
York.
[0013] The invention is directed to a method for enhancing the
staining of tissue or features of interest therein by applying an
effective amount of a bile salt in combination with a special stain
to a tissue suspected of harboring a microorganism or other featue
of diagnostic interest. The particular invention herein is directed
to detecting H. pylori, the microorganism responsible for
gastrointestinal ulcers. On the NexES Special Stains instrument,
the method is practiced according to the following general recipe,
all of which are carried out at room temperature:
[0014] 1) The slide with tissue adhered to it was rinsed using a
solution of Tween 20.
[0015] 2) 200 uL of Alcian Yello Oxidizer was added to the slide,
and it was incubated for four minutes.
[0016] 3) Liquid Coverslip.TM. ("LCS") was applied.
[0017] 4) Rinsed slide.
[0018] 5) Adjusted slide volume and applied LCS.
[0019] 6) Incubated for 4 more minutes.
[0020] 7) Rinsed.
[0021] 8) Applied Volume Adjust, and then 200 uL Alcian Yellow
Clarifier.
[0022] 9) Incubated for eight minutes, and the applied LCS.
[0023] 10) Rinsed.
[0024] 11) Adjusted slide volume, applied LCS
[0025] 12) Incubated four minutes.
[0026] 13) Rinsed slide.
[0027] 14) Applied volume adjust, then added 200 uL of Alcian
Yellow Stain.
[0028] 15) Incubated four minutes, applied LCS.
[0029] 16) Rinsed.
[0030] 17) Volume adjusted, and added 200 uL Alcian Yellow
Sensitizer.
[0031] 18) Incubated four minutes, applied LCS.
[0032] 19) Volume adjusted, and then added 200 uL Alcian Yellow
Toluidine Blue.
[0033] 20) Incubated four minutes, blowoff.
[0034] 21) Rinsed.
[0035] The invention described herein is called "Alcian Yellow
Sensitizer" and comprises 1.9% sodium borate decahydrate and 0.5%
sodium desoxycholate, in aqueous solution (DI water). The preferred
range of concentration for the sodium desoxycholate is 0.32% to
about 0.64%. 1 N NaOH is added to balance pH to between 9.25 and
9.35. All of the following are available from Ventana Medical
Systems, Tucson, Ariz.: Alcian Yellow Toluidine Blue, P/N 10551.
Alcian Yellow Stain, P/N 10549. Alcian Yellow Oxidizer, P/N 10514.
Alcian Yellow Clarifier, P/N 10012. Rinse and volume adjust
solutions are stock solutions, also available from Ventana.
[0036] Results are best seen in relation to FIG. 1. FIG. 1 includes
two color pictures of a section of tissue on a slide stained using
the recipe and Alcian Yellow sensitizer of the present invention.
The top picture is a tissue section stained without using the
sensitizer, and the bottom picture is of a tissue section wherein
the recipe included in addition the step of using the sensitizer.
The arrows point to areas where H. pylori are present. The bottom
picture shows a remarkable enhancement of staining in the small
blue dots, which is the H. pylori microorganism.
* * * * *