[Histonet] Wright giemsa on bone marrow core biopsies...

[lpwenk <@t> sbcglobal.net]

Smears can be stained with Wright Giemsa or with Leishmann Giemsa.

Fixed tissue require a different formulation, such as Wolbach Giemsa or
Jenner Giemsa. I've included our version of Jenner Giemsa.


JENNER GIEMSA


REAGENTS:

STOCK JENNER SOLUTION
Jenner dye  1.0 g
Absolute methanol (CH3OH)  400.0 mL
Dissolve together. Store at room temperature. Stable for months.

WORKING JENNER SOLUTION
Stock Jenner solution  25.0 mL
Distilled water  25.0 mL
JUST BEFORE USE, mix together. Good for 1 day only.

STOCK GIEMSA SOLUTION
Giemsa dye  1.0 g
Glycerin (C3H8O3)  66.0 mL
Absolute methanol (CH3OH)  66.0 mL
Mix together glycerin and Giemsa dye. Place in 60o C. oven for 30 minutes to
2 hours, until dissolved. Add methanol and mix. Store at room temperature in
a closed, dark brown bottle. Keep away from heat and sunlight, which will
shorten the life of the solution. Stable for about 1 year.

WORKING GIEMSA SOLUTIONS
Stock Giemsa solution  1.5 mL
Distilled water  50.0 mL
JUST BEFORE USE, mix together. Good for one day only.

0.5% ACETIC ACID WATER
Acetic acid, conc. (CH3COOH)  0.5 mL
Distilled water  99.5 mL
If this stain is done infrequently, make JUST BEFORE USE, mix together.
Stable for one day only.

PROCEDURE - Jenner-Giemsa:

1. Deparaffinize and hydrate slides through graded alcohol to distilled
water.

2. Place in 2 changes of absolute methanol 3 minutes each

3. Stain in WORKING Jenner solution  6 minutes

4. Transfer directly into WORKING Giemsa solution 45 minutes

THE FOLLOWING STEPS WILL DONE ONE SLIDE AT A TIME:

5. Rinse quickly in distilled water 3 seconds

6. Differentiate in 0.5% acetic acid, until can begin to see
chromatin pattern in the nuclei. Check with microscope 1-3 seconds

7. Rinse in distilled water 2-3 seconds

8. Complete differentiation of chromatin pattern until crisp and clear
in 2 changes of 95% ethanol. Check with microscope 1-10 seconds each

9.  Dehydrate in absolute ethanol, 2 changes 5-10 seconds each

10. Clear in xylene 10 seconds each

11.  Coverslip using a synthetic mounting media.


RESULTS:
Nuclei - blue
Cytoplasm, collagen, muscle, bone spicules - pink/blue/gray
Eosinophilic granules - pink
Basophilic granules - blue
Bacteria, parasitic protozoa cytoplasm - blue
Red blood cells - yellowish-pink


PROCEDURAL NOTES:

1.  Chromatin material of nuclei should be readily distinguished for good
differentiation.
2. If overdifferentiated, slides may be placed back into the Giemsa solution
and restained.
3. When differentiating, either in acetic acid or 95% ethanol, proceed one
slide at a time.



----- Original Message -----
From: "Goodwin, Diana" <GoodwinD <@t> pahosp.com>
To: "Histonet (E-mail)" <HistoNet <@t> Pathology.swmed.edu>
Sent: Wednesday, March 23, 2005 1:04 PM
Subject: [Histonet] Wright giemsa on bone marrow core biopsies...


> ...looking for a good method for tissue as opposed to smears.
>
> Thanks,
> Diana Goodwin
> Anatomic Pathology
> Pennsylvania Hospital
> 215-829-6532
> e-mail:  goodwind <@t> pahosp.com
>
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