Dr. Muaz Osman Fagere

Transcription

1 Dr. Muaz Osman Fagere

2 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.

3 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

4 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.

5 Haematogenous : These group contain the following blood derived pigments: I. Hemosiderins. II. III. IV. Haemoglobin. Bile pigments. 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.

6 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).

7 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.

8 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.

9 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.

10 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.

11 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.

12 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.

13 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.

14 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.

15 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.

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