Staining methods for histology and cytology specimens

• US 5,318,795 A
• Filed: 04/05/1993
• Issued: 06/07/1994
• Est. Priority Date: 05/07/1991
• Status: Expired due to Fees

• Current Assignee: Wesco Incorporated (HF HOLDINGS INC)
• Original Assignee: Wesco Incorporated (HF HOLDINGS INC)
• Inventors: Gibson, John F., Stokes, Barry O.
• Primary Examiner(s): Beck, Shrive
• Assistant Examiner(s): Dudash, Diana L.
• Application Number:
  US08/042,455
• Time in Patent Office: 428 Days
• Field of Search: 427/2, 427/4, 424/3
• US Class Current: 427/4
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 2001/317 spraying liquids onto surfaces

Abstract

In the field of staining methods for histology and cytology specimens, the improvements of the invention are three-fold. First included is a drying-clearing step whereby a specimen is cleared by drying. Thus, the use of hazardous chemical-clearing agents to remove washing-dehydrating solution prior to cover-slipping is eliminated. Second, in a staining method using spray staining equipment, the use of a low molarity, combination, bluing-washing solution which serves the dual purpose of a washing solution and a bluing reagent, eliminates the use of water in a staining procedure. Third, and also in a staining method using spray staining equipment, the use of a series of sprays of fresh, essentially anhydrous, washing-dehydrating solution to dehydrate a specimen replaces immersion in a series of washing-dehydrating solutions and thereby decreases the amount of chemical consumed and also avoids contamination of the reagent with water and specimen debris.

Claims

1. In a method for carrying out the staining of histology or cytology specimens adhered to a slide free of paraffin mounting, which method includes the following steps:

an optional step of hydrating the specimen adhered to a slide by applying water thereto;

applying excess stain to the specimen;

removing excess stain and/or water by exposing the specimen to a washing-dehydrating reagent;

clearing the specimen of residual washing-dehydrating reagent remaining from a previous washing-dehydrating step and thereby readying the specimen and slide for the application of a cover-slip mountant and a cover-slip;

the improvement comprising;

clearing the specimen by performing a drying-clearing step wherein the specimen is at least partially dried of the washing-dehydrating reagent thereby readying it for the application of a cover-slip mountant and a cover-slip without a chemical-clearing step.

2. A method according to claim 1 wherein the drying-clearing step is accomplished by applying forced air.

3. A method according to claim 1 wherein the drying-clearing step is accomplished by applying mild heat.

4. A method according to claim 1 wherein the drying-clearing step is accomplished by exposing the specimen to ambient evaporator hood or room conditions.

5. A method according to claim 1 wherein the drying-clearing step is performed in staining equipment including means for applying centrifugal force and the drying-clearing step is achieved by centrifugation.

6. A method according to claim 5, the improvement additionally comprising:

exposing a specimen to washing-dehydrating reagent to remove water and/or excess stain after said specimen has had water and/or excess stain applied to it by spraying the slide with an essentially anhydrous solution of washing-dehydrating reagent.

7. In a method according to claim 6, wherein there is additionally included the step of staining the specimen with hematoxylin after a hydration step and prior to any additional staining steps wherein an excess of hematoxylin is provided, the improvements additionally comprising:

removing excess hematoxylin after said hematoxylin has been applied to said specimen by spraying the slide with a combination bluing-washing solution having a concentration of a basic salt solute of about 0.01 molar yielding a solution with above about pH 5.0; and

raising the pH of the slide for the application of any subsequent reagent.

8. A method of carrying out a Papanicolaou stain according to claim 7 wherein the spraying is performed by automated spray staining equipment including a centrifuge carousel for holding the slides and rotating them past a spraying apparatus at a selectable rotational speed, said equipment being capable of administering alternating cycles of 1) spraying a reagent on a specimen and 2) waiting for the applied reagent to diffuse through and interact with the specimen, the number of cycles and total length of time of each of said cycles of spraying and waiting comprising a total exposure of a specimen to a reagent, said method comprising the sequential steps of:

(a) exposing the specimen to washing-dehydrating reagent, the total exposure being sufficient to wash from the specimen any unwanted chemical residues that may be present in the specimen;

(b) exposing the specimen to a combination bluing-washing solution, i.e., a hydrating step, the total exposure being sufficient to remove the washing-dehydrating reagent and hydrate the specimen;

(c) staining the specimen with hematoxylin stain by exposing it to an excess of said stain, the total exposure being sufficient to stain susceptible elements of the cells of the specimen with hematoxylin;

followed by resetting to a higher rotational speed and spinning for a sufficient period of time to remove excess reagent;

followed by waiting for a sufficient length of time to stabilize the carousel so that the rotational speed can be reset to a speed suitable for spray application;

followed by exposing the specimen to a combination bluing-washing solution, the total exposure being sufficient to wash off remaining excess hematoxylin and raise the pH of the specimen to above 5.0 thereby turning the hematoxylin blue;

(d) staining the specimen with orange G stain by exposing said specimen to said stain, the total exposure being sufficient to stain keratinized cells of the specimen yellow to orange;

followed by resetting to a higher rotational speed and spinning for a sufficient period of time to partially remove excess stain;

followed by waiting for a sufficient length of time to stabilize the carousel so that the rotational speed can be reset to a speed suitable for spray application;

(e) exposing the specimen to washing-dehydrating reagent, the total exposure being sufficient to wash away excess orange G stain;

(f) staining the specimen with Papanicolaou EA stain by exposing the specimen to Papanicolaou EA stain, the total exposure being sufficient to stain differing elements of said specimen susceptible to said stain either red or blue;

followed by resetting to a higher rotational speed and spinning for a sufficient period of time to partially remove excess stain;

followed by waiting for a sufficient length of time to stabilize the carousel so that the rotational speed can be reset to a speed suitable for spray application;

(g) exposing the specimen to washing-dehydrating reagent the total exposure being sufficient to wash away excess EA stain;

(h) performing a drying-clearing step by spinning for a sufficient period of time to clear excess reagent and at least partially dry the specimen of washing-dehydrating reagent thereby readying the slide for the application of a cover-slip mountant and cover-slip.

9. A method for carrying out a hematoxylin and eosin stain according to claim 7 wherein the spraying is performed by automated spray staining equipment including a centrifuge carousel for holding the slides and rotating them past a spraying apparatus at a selectable rotational speed, said equipment being adapted to administer alternating cycles of 1) spraying a reagent on a specimen and 2) waiting for the applied reagent to diffuse through and interact with the specimen, the number of cycles and the total length of time of each of said cycles of spraying and waiting comprising a total exposure of a specimen to a reagent, said method comprising the steps of:

(a) exposing the specimen to washing-dehydrating reagent, the total exposure being sufficient to wash from the specimen any unwanted residues;

(b) exposing the specimen to bluing-washing solution, the total exposure being sufficient to remove the washing-dehydrating reagent and hydrate the specimen;

(c) staining the specimen with hematoxylin by exposing it to said stain, the total exposure being sufficient to stain the susceptible elements of the cells of the specimen with hematoxylin;

followed by resetting to a higher rotational speed and spinning for a sufficient period of time to remove excess reagent;

followed by waiting for a sufficient length of time to stabilize the carousel so that the rotational speed can be reset to a speed suitable for spray application, followed by exposing the specimen to bluing-washing solution, the total exposure being sufficient to wash off remaining excess hematoxylin and raise the pH of the specimen to above 5.0 And thereby turn the hematoxylin blue;

(d) staining the specimen with eosin Y stain by exposing the specimen to said stain, the total exposure being sufficient to stain the element susceptible to said stain red;

followed by resetting to a higher rotational speed and spinning for a sufficient period of time to remove excess reagent;

followed by waiting for a sufficient length of time to stabilize the carousel so that the rotational speed can be reset to a speed suitable for spray application;

(e) exposing the specimen to washing-dehydrating reagent, the total exposure being sufficient to wash away excess eosin Y stain;

(f) performing a drying-clearing step by spinning for a sufficient period of time to clear excess reagent and at least partially dry the specimen of the washing-dehydrating reagent thus readying the slide for the application of a cover-slip mountant and cover-slip.

10. In a method for carrying out the staining of histology or cytology specimens whereby the following steps are included:

exposing a slide having a specimen fixed to it to a washing-dehydrating reagent;

removing washing-dehydrating reagent and hydrating the specimen by exposing the slide to an aqueous reagent;

staining susceptible elements of the cells of the specimen with hematoxylin by exposing the specimen to an excess of said stain, removing excess stain by exposing the specimen to an aqueous reagent, exposing the specimen to a bluing solution having a pH above 5.0 to turn the hematoxylin blue and readying the specimen for the application of subsequent reagents;

removing the excess stain and/or water by exposing the specimen to a washing-dehydrating reagent remaining from a previous washing-dehydrating step and thereby readying the specimen and slide for the application of a cover-slip mountant and a cover-slip; and

clearing the specimen of residual washing-dehydrating reagent remaining from a previous washing-dehydrating step and thereby readying the slide for the application of a cover-slip mountant and a cover-slip;

the improvement comprising;

exposing the slide to a combination bluing-washing solution having a concentration of a basic salt solute of about 0.01 molar yielding a solution with above about pH 5.0, said exposure raising the pH of the specimen above pH 5.0 thereby turning the hematoxylin blue, washing away excess hematoxylin, and readying the specimen for application of a subsequent reagent.

11. A method according to claim 10 wherein the solute is sodium bicarbonate.

12. A method according to claim 10 wherein the improvement additionally comprises:

clearing residual washing-dehydrating solution remaining from the washing step from the specimen by at least partially drying residual washing-dehydrating from the specimen and thereby readying the slide for the application of a cover-slip mountant and a cover-slip.

13. A method according to claim 10, the improvements additionally comprising:

exposing the slide to washing-dehydrating reagent by spraying said slide with an essentially anhydrous washing-dehydrating reagent solution.

14. In a method for carrying out the staining of a histology or cytology specimen including the step of applying to a slide having a specimen affixed thereto, a reagent, other than a washing-dehydrating reagent, the improvement comprising:

removing said reagent from the specimen by repeatedly spraying the slide with an essentially anhydrous washing-dehydrating reagent.

15. A method according to claim 14, wherein the washing-dehydrating reagent is selected from the group consisting of glycols, ketones, esters and ethers of 2 to 5 carbon atoms, alcohols of 1 to 3 carbon atoms, and mixtures thereof.

16. A method according to claim 14, wherein the washing-dehydrating reagent consists of essentially anhydrous ethanol, other essentially anhydrous alcohols of from 1 to 3 carbon atoms, and mixtures thereof.

17. A method of readying for the application of a cover-slip mountant and a cover-slip a histology or cytology specimen that has been affixed to a slide and to which stain and/or water has been applied and excess stain and/or water removed by application of a washing-dehydrating reagent, comprising the step of at least partially drying the washing-dehydrating reagent applied to the specimen thereby readying the specimen and slide for application of a cover-slip mountant and cover-slip without a chemical clearing step.

18. In a method for carrying out the staining of histology or cytology specimens wherein is included the step of staining the susceptible elements of the specimen with hematoxylin wherein an excess of hematoxylin is provided, the improvements comprising:

removing excess hematoxylin by spraying the slide with a combination bluing-washing solution having a concentration of a basic salt solute of no more than about 0.01 molar yielding a solution with above about pH 5.0 and raising the pH of the slide to above pH 5.0 thereby turning the hematoxylin blue and readying the slide for the application of a subsequent reagent.

19. A method according to claim 18 wherein the basic salt solute is sodium bicarbonate.