Dr. Muaz Osman Fagere

In biology, pigments are defined as substances occurring in

living matter that absorb visible light.

Pigments are differ in origin, chemical constitution, and

biological significance.

All pigments are able to absorb elecromagnatic energy

within a narrow band that lies approximately between 400

and 800 nm.

Pigments can be either organic or inorganic compounds

that remain insoluble in most solvents.

Pigments can be classified under the following headings:

1. Endogenous pigments:

These substances are produced either within tissues and

serve physiological functions or are by-products of normal

metabolic processes. They can be further subdivided into:

a) Haematogenous (blood derived pigments).

b) Non-Haematogenous pigments.

c) Endogenous minerals.

2. Artefact pigments:

These are deposits of artfactually produced materials caused

by the interaction between the certain tissue components and

some chemical substances such as fixatives.

3. Exogenous pigments and minerals:

These substances gain access to the body accidentally and

serve no physiological function. Entry is gained either by

inhalation into the lungs or implantation into the skin. Most

exogenous pigments are minerals few of which are actually

pigmented.

Haematogenous :

These group contain the following blood derived pigments:

I. Hemosiderins.

II. Haemoglobin.

III. Bile pigments.

IV. Porphyrins.

I. Hemosiderins

Hemosiderins pigments are seen as yellow to brown granules

and normally appear intracellularly. They contain iron in the

form of ferric hydroxide that is bound to a protein framework

and easily unmasked by various chemicals.

FIXATION

In unfixed tissue, hemosiderin is insoluble in alkalis but

freely soluble in strong acid solutions.

Fixative that contain acids but no formalin can alter it in a

such a way that reaction for iron are negative.

Certain type of iron found in tissues are not demonstrable

using traditional techniques. This is because the iron is

tightly bound within a protein complex ex; (haemoglobin &

myoglobin).

METHODS

1) Perls‘ Prussian Blue Reaction For Ferric Iron:

This method is considered by many to be the first classical histochemical reaction.

Treatment with an acid ferro-cyanide solution will result in the unmasking of ferric iron in the form of the hydroxide, Fe(OH)3 by dilute hydrochloric acid. The ferric iron then reacts with a dilute potassium ferro-cyanide solution to produce an insoluble blue compound , ferric ferro-cyanide (Prussian blue).

In this method avoid to use acid fixatives.

2) Hukill & Putt‘s for ferrous and ferric iron:

This method was claimed to be a more sensitive demonstration for the detection of both ferric and ferrous salts.

The method uses bathophenanthroline. and the resultant colour of any iron present in tissues is bright red.

Its important that bathophenanthroline is completely dissolved prior to use.

Dehydration with alcohol remove the resultant red colour.

II. Hemoglobin

Hemoglobin is basic conjugated protein that is responsible for the transportation for oxygen and carbon dioxide within the blood stream.

Its composed of a colourless protein (globin), and a red component (heam) which composed of protoporphyrin, a substance built up from pyrrol rings and combined with ferrous iron.

Histohemical demonstration of the ferrous iron is only possible if the close binding in the heam molecules is cleaved, this can be achieved by treatment with hydrogen peroxide.

Demonstration of hemoglbin is important in certain

pathological conditions such as casts in the lumen of renal

tubules in caeses of hemoglobinuria or active

glomerulonephritis.

Two types of demonstration methods can be used to stain

hemoglobin in tissue sections.

1. Benzidine-nitroprusside method

This method demonstrate the enzyme, hemoglobin

peroxidase, which is reasonably stable and withstand short

fixation and paraffin processing.

The method is not recommended due to the carcinogenicity

of benzidine.

2. Leuco Patent blue V

Fixation is by formalin or formal mercury, fixation in excess

of 36 hours may give unreliable results.

This method demonstrates peroxidase activity including the

peroxidase in other blood cells particularly in the lysosomes

of polymorphonuclear leucocytes and tissue peroxidase.

Heomglobin stained more greenish-blue colour by this

method.

III. Bile pigments

Bile pigments are bilirubin, biliveridin and hematoidin all

are results from the breakdown of red blood cells.

Demonstration of bile pigments arises mainly in the

histological examination of the liver where distinguishing

bile pigments from lipofuscin may be of significant

important.

1. Modified fouchet's technique for liver bile pigments.

The most commonly used routine method for bile pigments.

The pigment is converted to the green colour of biliveridin

and blue cholecyanin by the oxidative action of the ferric

chloride in the presences of Trichloroacetic acid.

its quick and simple to carry out.

2. Gmelin technique

The section treated with nitric acid and a changing colour

spectrum produced.

This method gives impermanent results.

IV. Porphyrin

These substances normally only occur in the tissues in

small amount.

They are considered to be precursors of the heme portion

of hemoglobin.

The porphyrias are rare pathological conditions that are

disorders of the biosynthesis of porphyrin and heme.

The pigment when seen in paraffin section and viewed

using polarized light shows as bright red in colour with a

centrally located drack Maltese cross.

The end