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Perls’ Technique For The Demonstration of Haemosiderin – Method and Tips


Iron is absorbed in the duodenum by cells called enterocytes. It is then stored or combined with a transport protein molecule. This iron-protein complex is then taken to the bone marrow where the iron is incorporated into the substance known as haemoglobin which is involved in oxygen transportation.

Iron can be stored in the bone marrow and spleen in its ferric state (Fe3+) as haemosiderin when combined with protein. When haemoglobin is broken down by tissues this results in the formation of haemosiderin.

When there is excess iron in the body haemosiderin can be found deposited in organs that are involved with iron storage such as the spleen, bone marrow and liver. This condition is known as haemosiderosis.

A condition called haemochromatosis exists where the body indiscriminately absorbs iron resulting in the deposition of copious amounts of haemosiderin in many tissues.

Haemosiderin founds in histology sections is usually derived from the breakdown of damaged erythrocytes and can also be found absorbed by macrophages (siderophages).

The method used by the wide majority of histology laboratories for the demonstration of haemosiderin is the Perls’ technique. This method works by the hydrochloric acid (HCL) splitting off the bound protein which then allows the potassium ferrocyanide to bind with the Fe3+ and form ferric ferrocyanide (Prussian blue).

Below is the author’s favoured method.

SOLUTIONS

2% hydrochloric acid (HCL)

2% aqueous potassium ferrocyanide

1% aqueous neutral red

METHOD

1. Take sections to water

2. Mix equals parts of HCL and potassium ferrocyanide and filter onto sections. Leave for 15 minutes.

3. Wash for 5 minutes in running tap water.

4. Counterstain with neutral red for 1 minute.

5. Dehydrate, clear and mount.

TIPS

– Always include a positive control.

– Stronger staining results can be found by carrying out step 2 at higher temperatures (e.g. 37-56°C). This can result in false positive results. This author has found room temperature to suffice.

– The washing step (step 3) should not be decreased below 5 minutes as thorough washing is required to prevent a heavy dye precipitate resulting from the neutral red counterstain.

– The author has found neutral red to be the best counterstain. Do not use safranin as this can stain the Prussian blue granules a dark purple colour.

– Always mount in a DPX-type mountant as other mounting media results in fading of the stain.

– A common artefact is the presence of blue granules on and around the section. This can be due to expired HCL or potassium ferrocyanide. It can also be due to iron contaminants in the tap water. This can be fixed by replacing all steps from the cutting of the sections to the mounting of the stained slide that involve tap water with distilled water.

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