BETHESDA WITH HEMATOXILIN AND EOSIN (H&E) STAIN

PAPANICOLAOU - CYTOLOGY VARIANT STAIN

ECOLOGY EFFICIENT STAIN PROCEDURE USING H&E PREPARED WITH WATER

Humberto García-Alonso, M.D. FCAP; FASCP.(1), Patricia García-Alonso, M.D.(2) and Teresa Vela, M.D.(3)

(1) Pathologist. Hospital de Oncología I.M.S.S. México. Professor of Pathology. Facultad de Medicina. Universidad Nacional Autónoma de México. México. (Retired)

(2) Instituto de Perinatología, S.S. México.

(3) Department of Pathology. Instituto Nacional de Cancerología. S.S. México.

INTRODUCTION

In the routine Hematoxilin and Eosin (H&E) stain there is an alternative method that consists in the preparation of H&E stain without the use of solvents like alcohol and xilene using instead plain tap water to prepare the H&E solutions. This method gives an excellent staining quality for the routine use of the H&E stain in cytologic material. Papanicolaou1 mentions in his Atlas the use of aquous mixture of Hematoxilin and Eosin as a method he used in his earlier works. Mallory2 also refers the use of aquous solutions. It is of interest to review this stain method in view of its new cytological applications and its variants like the study of material to be classified using the Bethesda System 20013. Also to review this stain method for use in the fluid or liquid based thin preparation.

PREPARATION

A. FORMULA FOR THE PREPARATION OF 1000 ml. OF HEMATOXILIN SOLUTION

STEP 1

Hematoxilin * (dark crystals) . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g.

Hot tap water 70-80 °C . . . . . . . . . . . . . . . . . . . .  . . . . . . . 500 ml.

Mix and dilute completely

STEP 2

Prepare in a separate vase or flask.

Alum** (ammonium or potassium alum) . . . . . . . . . .  . . . . . . . 20 g.

Hot tap water 70-80 °C . . . . . . . . . . . . . . . . . . . . . . . . .  . . 500 ml.

Mix and dilute completely

STEP 3

Bring mixture of steps 1 and 2 together and mix well.

STEP 4

Add thymol***crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   1 g.

This is done to prevent the growth of fungal elements. The alum is a mordant so the hematoxilin solution can stain the nucleai.

STEP 5

Keep in a translucent flask or vase for a week at room temperature (not direct sunlight). Cover with a paper towel so that air can circulate. (Early "maturation" of the hematoxilin). After a week, change the solution to a dark flask or bottle tightly stopped. Keep it in a dark place at room temperature for 3 weeks. The solution can last up to 6 months (late "maturation" of hematoxilin). When you use the previous stock solution, dissolve all the crystals that may be formed in the flask (they are formed mainly from thymol).

B. FORMULA FOR THE PREPARATION OF 1000 ml. OF EOSIN STAIN

STEP 1

Eosin Y crystals**** . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10 g.

Hot tap water 70-80 °C . . . . . . . . . . . . . . . . .  . . . . . . . . . 1000 ml.

Mix and dilute well. No need to add thymol. Keep it in a dark flask in a stopped bottle (eosin doesn't have to "mature"). The eosin solution can be used immediately.

C. PREPARATION OF 10% ALUM WATER SOLUTION

Alum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 10 g.

Hot tap water 70-80 °C . . . . . . . . . . . . . . . . . . . . . . .  . . . . . . . . . . . . . 1000 ml.

Mix and dissolve well

You can keep adding this solution to replace the evaporation or consumption of the hematoxilin, as long as it retains the staining quality of nuclear elements. It can be checked with the one test slide at the microscope as a wet preparation. This solution is not to be added to the eosin stain dish.

D. GENERAL PROCEDURE

Prepare your slides properly identified and place them in a stain dish.

I.

  1. Place slide carrier in the hematoxilin dish for 3 minutes. It can vary from 3 to 6 minutes depending on the tissue origin of the cytological material and with the "maturation" of the hematoxilin solution. The intensity should be checked with a test slide for the desired and satisfactory color details of the nucleus. The hematoxilin solution is used only as a nuclear stain.
    Do not use recently prepared hematoxilin solution because it won't stain the nucleai. It has to "mature", ripen (3 to 4 weeks) before using it.
  2. Remove the slide carrier to the first tap water bath dish 3 dips (dip slowly).
  3. Change to a second water bath dish and leave it for 30 to 60 seconds so that the hematoxilin stain can "turn" in the tap water.

II.

  1. Remove the slide carrier to the eosin dish for 5 to 10 seconds. 
  2. Remove the slide carrier to a tap water dish and then again to another clean tap water dish to wash the excess of colorant. No need to use alcohol or xilene after the last water dish wash.
III.
  1. To dry, place the slide carrier in an oven and then mount for microscopic examination.

IV.

  1. The stain should be perfectly differentiated, dark blue for the nucleus and different red tones for the counter stain of the cytoplasm. If this is not achieved, the stain that is not "working" should be checked and changed. Both stains can be filtered each day and if necessary additional stain or 10% alum water solution can be added. This last one only to the hematoxilin solution as previously mentioned.
  2. If large numbers of slides are processed, the solution may be preserved for a longer period of time if the slide carrier is rested on several paper towels for a few seconds to dry the excess solution between the different passes.

COMMENTARIES:

This procedure eliminates the use of toxic substances like alcohol and xilene in the preparation of H&E stains. Reduces the time and cost of the H&E preparation. There is an excellent quality of the stain for routine staining procedures for cytologic smears provided you acquire skills and experience in the preparation of the solutions and in the microscopic examination of the material with this procedure. To obtain the best quality results the stains*-**** should be obtained from any prestigious commercial company on the market, as well as the other substances**-***.

BIBLIOGRAPHY:

  1. Papanicolaou,G.N.: Atlas of Exfoliative Cytology. Cambrige. Harvard University Press. 1954; Published for the Commonwealth Fund by Harvard University Press, Cambrige, Mass. 1963. Pg. 6.
  1. Mallory F.B.; A.M,; M.D.; S.D. Pathological Technique.                                Philadelphia & London. W.B. Sunders Company. Copyright, 1938, by W.B. Sunders Company. Reprinted June, 1942 and September, 1944. Pgs. 70 and 91

  1. Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002; 287: 2114-2119

NOTES:

We do not have any affiliation with any commercial products or companies. The article is solely for academic and general information on a cytology variant stain.

Technical assistance can be arranged for nonprofit organizations.

Commentaries can be sent to humbertogarciaalonso@yahoo.com.mx

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BETHESDA WITH HEMATOXYLIN AND EOSIN (H&E) STAIN

PAPANICOLAOU - CYTOLOGY VARIANT STAIN

ECOLOGY EFFICIENT STAIN PROCEDURE USING H&E PREPARED WITH WATER

Humberto García-Alonso, M.D. FCAP; FASCP.(1), Patricia García-Alonso, M.D.(2) and Teresa Vela, M.D.(3)

(1) Pathologist. Hospital de Oncología I.M.S.S. México. Professor of Pathology. Facultad de Medicina. Universidad Nacional Autónoma de México. México. (Retired)

(2) Instituto de Perinatología, S.S. México.

(3) Department of Pathology. Instituto Nacional de Cancerología. S.S. México.

INTRODUCTION

In the routine Hematoxylin and Eosin (H&E) stain there is an alternative method that consists in the preparation of H&E stain without the use of solvents like alcohol and xylene using instead plain tap water to prepare the H&E solutions. This method gives an excellent staining quality for the routine use of the H&E stain in cytologic material. Papanicolaou1 mentions in his Atlas the use of aquous mixture of Hematoxylin and Eosin as a method he used in his earlier works. Mallory2 also refers the use of aquous solutions. This stain method can be used for the routine cytologic material to be classified using the Bethesda System 20013, and also can be used for the fluid or liquid based thin preparation method. We think that with this method the cost of the cytology routine stains will be significantly reduced, and will help, in screening programs for public health purposes, as well as for routine laboratory work

PREPARATION

A. FORMULA FOR THE PREPARATION OF 1000 ml. OF HEMATOXYLIN SOLUTION

STEP 1

Hematoxylin * (dark crystals) . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g.

Hot tap water 70-80 °C . . . . . . . . . . . . . . . . . . . .  . . . . . . . 500 ml.

Mix and dilute completely

STEP 2

Prepare in a separate vase or flask.

Alum** (ammonium or potassium alum) . . . . . . . . . .  . . . . . . . 20 g.

Hot tap water 70-80 °C . . . . . . . . . . . . . . . . . . . . . . . . .  . . 500 ml.

Mix and dilute completely

STEP 3

Bring mixture of steps 1 and 2 together and mix well.

STEP 4

Add thymol***crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   1 g.

This is done to prevent the growth of fungal elements. The alum is a mordant so the hematoxylin solution can stain the nucleai.

STEP 5

Keep in a translucent flask or vase for a week at room temperature (not direct sunlight). Cover with a paper towel so that air can circulate. (Early "maturation" of the hematoxylin). After a week, change the solution to a dark flask or bottle tightly stopped. Keep it in a dark place at room temperature for 3 weeks. The solution can last up to 6 months (late "maturation" of hematoxylin). When you use the previous stock solution, dissolve all the crystals that may be formed in the flask (they are formed mainly from thymol).

B. FORMULA FOR THE PREPARATION OF 1000 ml. OF EOSIN STAIN

STEP 1

Eosin Y crystals**** . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10 g.

Hot tap water 70-80 °C . . . . . . . . . . . . . . . . .  . . . . . . . . . 1000 ml.

Mix and dilute well. No need to add thymol. Keep it in a dark flask in a stopped bottle (eosin doesn't have to "mature"). The eosin solution can be used immediately.

C. PREPARATION OF 10% ALUM WATER SOLUTION

Alum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 10 g.

Hot tap water 70-80 °C . . . . . . . . . . . . . . . . . . . . . . .  . . . . . . . . . . . . . 1000 ml.

Mix and dissolve well

You can keep adding this solution to replace the evaporation or consumption of the hematoxylin, as long as it retains the staining quality of nuclear elements. It can be checked with the one test slide at the microscope as a wet preparation. This solution is not to be added to the eosin stain dish.

D. GENERAL PROCEDURE

Prepare your slides properly identified and place them in a stain dish.

I.

  1. Place slide carrier in the hematoxylin dish for 3 minutes. It can vary from 3 to 6 minutes depending on the tissue origin of the cytological material and with the "maturation" of the hematoxylin solution. The intensity should be checked with a test slide for the desired and satisfactory color details of the nucleus. The hematoxylin solution is used only as a nuclear stain.
    Do not use recently prepared hematoxylin solution because it won't stain the nucleai. It has to "mature", ripen (3 to 4 weeks) before using it.
  2. Remove the slide carrier to the first tap water bath dish 3 dips (dip slowly).
  3. Change to a second water bath dish and leave it for 30 to 60 seconds so that the hematoxylin stain can "turn" in the tap water.

II.

  1. Remove the slide carrier to the eosin dish for 5 to 10 seconds. 
  2. Remove the slide carrier to a tap water dish and then again to another clean tap water dish to wash the excess of colorant. No need to use alcohol or xylene after the last water dish wash.

III.

  1. To dry, place the slide carrier in an oven and then mount for microscopic examination.

IV.

  1. The stain should be perfectly differentiated, dark blue for the nucleus and different red tones for the counter stain of the cytoplasm. If this is not achieved, the stain that is not "working" should be checked and changed. Both stains can be filtered each day and if necessary additional stain or 10% alum water solution can be added. This last one only to the hematoxylin solution as previously mentioned.
  2. If large numbers of slides are processed, the solution may be preserved for a longer period of time if the slide carrier is rested on several paper towels for a few seconds to dry the excess solution between the different passes.

COMMENTARIES:

This procedure eliminates the use of toxic substances like alcohol and xylene in the preparation of H&E stains. Reduces the time and cost of the H&E preparation. There is an excellent quality of the stain for routine staining procedures for cytologic smears provided you acquire skills and experience in the preparation of the solutions and in the microscopic examination of the material with this procedure. To obtain the best quality results the stains*-**** should be obtained from any prestigious commercial company on the market, as well as the other substances**-***.

BIBLIOGRAPHY:

  1. Papanicolaou,G.N.: Atlas of Exfoliative Cytology. Cambrige. Harvard University Press. 1954; Published for the Commonwealth Fund by Harvard University Press, Cambrige, Mass. 1963. Pg. 6.

2.     Mallory F.B.; A.M,; M.D.; S.D. Pathological Technique.                                Philadelphia & London. W.B. Sunders Company. Copyright, 1938, by W.B. Sunders Company. Reprinted June, 1942 and September, 1944. Pgs. 70 and 91

  1. Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002; 287: 2114-2119

NOTES:

We do not have any affiliation with any commercial products or companies. The article is solely for academic and general information on a cytology variant stain.

Technical assistance can be arranged for nonprofit organizations.

Commentaries can be sent to humbertogarciaalonso@yahoo.com.mx

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