Hemochromatosis, esp., liver. excess iron deposition is stained as blue granules

       Gastic ulver 2nd to iron overdose:

FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

2% Hydrochloric Acid Solution

Hydrochloric acid, concentrated -----------------------------------------------                2.0 ml

Distilled water -----------------------------------------------------------------                98.0 ml

10% Triton X-100

Triton X-100 ------------------------------------------------------------------                10.0 ml

Distilled water -----------------------------------------------------------------                90.0 ml

       Add a few grains of thymol to prevent the growth of fungi.

1% Potassium Ferrocyanide Solution

Potassium ferrocyanide ---------------------------------------------------------                1.0 gm

Distilled water ------------------------------------------------------------------                98.0 ml

10% Triton X-100 -------------------------------------------------------------                2.0 ml

Hydrochloric Acid-Potassium Ferrocyanide Solution

2% hydrochloric acid ----------------------------------------------------------                20.0 ml

1% potassium ferrocyanide ----------------------------------------------------                20.0 ml

       Prepare just before use and discard after use.

Nuclear Fast Red (Kernechtrot) Solution

       Dissolve 0.1 gm nuclear fast red in 100 ml of 5% solution of aluminum sulfate

with the aid of heat.  Cool, filter, add a few grains of thymol as a preservative.

• Use positive control slides.

1. Deparaffinize and hydrate to distilled water.
2. Place the slides in the hydrochloric acid-potassium ferrocyanide solution for 30 minutes at room temperature.
3. Rinse with five changes of distilled water.
4. Nuclear fast red solution for 3 minutes.
5. Rinse with three changes of distilled water.
6. Dehydrate in graded alcohols.
7. Clear in xylene, three or four changes.
8. Mount with synthetic resin.

Ferric iron pigment ------------------------------------------------        bright blue

Nuclei ------------------------------------------------------------        red

Cytoplasm --------------------------------------------------------        pink

Hemosiderin is a breakdown product of hemoglobin and is thought to be composed of ferric iron and protein.  It may be present in tissues in certain pathologic conditions such as hemochromatosis.  This yellow-brown pigment is insoluble in alkalis and water but is soluble in acid even after fixation.  Treating a tissue section with 10% sulfuric acid overnight will usually remove this pigment.

The principle of Pearl’s Prussian blue reaction is that potassium ferrocyanide will form ferric ferrocyanide (Prussian blue) with reactive ferric salts in an acid solution.  Dilute hydrochloric acid liberates loosely bound ferric iron from protein.

For many years we used Gomori’s method for demonstrating ferric iron.  The potassium ferrocyanide and hydrochloric acid solutions used in this method are much stronger that those used in Pearl’s method.  Because most texts on histologic technic indicate the use of Pearl’s method we decided to do a comparative study of the two methods.  The staining results on a variety of tissues, including plastic bone marrow sections, were almost identical in this study.  Therefore, we decided to use Pearl’s method routinely because it is more cost effective.

Triton X-100 is added to the potassium ferrocyanide solution because it helps to stabilize the hydrochloric acid-potassium ferrocyanide solution.  If Triton X-100 is omitted there is a greater tendency for the solution to break down resulting in a fine blue precipitate forming in the Coplin jar and sometimes on the slides and tissue sections.

Lillie, R.D.:  Histopathologic Technic and Practical Histochemistry, 4th ed., New York, McGraw-Hill, 1976, p. 507.