Endogenous Minerals

0 This group include the following :

1. Calcium.

2. Copper.

3. Uric acid and urates.

1. Calcium

0 Insoluble inorganic calcium salts are a normal constituent of bones and teeth.

0 From histochemical point of view, the free ionic calcium found in the blood can not be demonstrated.

0 Abnormal depositions of calcium can be found in necrotic areas of tissues associated with tuberculosis, infections (Gandy-Gamna bodies), atheroma in blood vessels and malakoplakia of the bladder.

0 The most common forms of calcium salts occurring in these conditions are phosphates and carbonates.

Calcium Demonstration

0 The use of dyes which act by forming chelate complexes with calcium has been practiced by many workers, these dyes includes:

0 Alizarin red S.

0 Purpurin.

0 Naphthochrome green B.

0 Nuclear fast red.

Alizarin red S.

0 Fixation by buffered neutral formalin, formal alcohol and alcohol is recommended.

0 Section to be used either paraffin or frozen section.

0 The staining solution of Alizarian must adjusted to pH4.2 so as the staining will be more specific for calcium.

0 This method is particularly useful in the identification and detection of small amounts of calcium like those seen in heterotopic calcification in the kidney (hypercalcinosis).

Copper

0 Many enzymes in the body would fail to function without the presence of copper.

0 Copper deficiency is extremely rare, copper accumulation is associated with Wilson's disease, the most important disorder of copper metabolism

0 This disease is a rare inherited autosomal recessive condition witch gives rise to copper deposition in the liver, basal ganglia of the brain and eye.

0 Copper deposition in the liver is also associated with the biliary cirrhosis and certain other hepatic disorder.

Copper Demonstration

0 Two methods are available fro copper demonstration:

1. Rubeanic acid method.

2. Modified rhodanine technique.

0 Excellent result can be obtained from the rubeanic acid method by using formalin fixed tissues.

0 Rhodanine technique demonstrate copper and copper associated protein (CAP), its considered to be the method of choice

Uric acid & Urates

0 Uric acid is the breakdown product of the body's protein.

0 The uric acid circulating in the blood is in the form of momosodium urates, which in the patients with gout may be high, forming a super saturated solution.

0 These high level may result in urate depostions which are water soluble in tissues causing:

0 Subcutaneous nodular deposits of urate crystal (tophi).

0 Synovitis and arthritis.

0 Renal disease and calculi.

Uric acid Demonstration

0 Lithium carbonate extraction-hexamine silver technique is a method of choice.

0 Fixation in alcohol is recommended and it will give more specific reaction.

0 Paraffin, frozen or celloidin section all can be used.

0 The urates will reduce the silver solution to give black colour.

0 More accurate control of incubation temperature is achievable using a thermostatically controlled water bath.

Artefact Pigments

0 This group of pigments comprises:

1. Formalin Pigment.

2. Malaria Pigments.

3. Schistosome Pigment.

4. Mercury Pigment.

5. Chromic Oxide Pigment.

1. Formalin Pigment

0 This pigment is seen as a brown or brownish-black deposit in tissue that have been fixed in acidic formalin.

0 The deposit is usually present in blood-rich tissues such as spleen.

0 The morphology of the pigment can vary but is commonly seen as a micro-crystalline.

0 It can be removed by treating unstained tissue section with saturated alcoholic picric acid.

0 Alcoholic solution of both sodium and potassium hydroxide will also remove the pigment, but it may affect the staining result.

0 The use of buffered neutral formalin will help to minimize the problem of formalin pigment deposition.

0 Changing the fixative solution on a regular basis is recommended for large blood-rich organs.

2. Malarial Pigment

0 This pigment is morphologically similar to formalin pigment and occasionally may be identical.

0 Its formed within, or in the region of red blood cells that contain the malarial parasite.

0 In case of cerebral malaria pigments can be seen in or over the red blood cells within the tiny blood capillaries of the brain.

0 Malarial pigment may also be present within phagocytic cells which have ingested infected red cell.

Extraction Method For Formalin & Malarial Pigment

0 Solution containing saturated alcoholic picric acid and absolute ethyl alcohol is used for the extraction.

0 The time necessary for the removal of formalin pigment will vary, depending on the amount of pigment present.

0 Malarial pigment usually requires treatment for at least 12 hours before removal is complete.

3. Schistosome Pigment

0 This pigment is occasionally seen in tissue sections where infestation with schistosoma can be seen.

0 The pigment which tend to be chunky, shows similar properties to those of both formalin and malarial pigment.

4. Mercury Pigment

0 This pigment is seen in tissues that have been fixed in mercury-containing fixatives.

0 They varies in its appearance but its usually seen as a brownish-black, extracellular crystal.

0 Prolonged storage of stained sections that containing mercury can bring about a change in the structure of the pigment it changes from crystalline form to a globular one.

0 Treatment of sections with iodine solutions such as Lugol's iodine is the classical method of removing the pigment, subsequent bleaching with a weak sodium thiosulfate solution completes the treatment.

5. Chromic Oxide Pigment

0 This pigment is very rare seen in tissue sections and is extremely difficult to produce intentionally.

0 When seen it present as a fine yellow-brown particulate deposit in the tissue, as a result of not washing in water, tissues that have been fixed in chromic acid or dichromate containing fixatives.

0 Its extracellular and can be removed from sections by treatment with 1% acid alcohol.

Exogenous pigments & Minerals

0 The majoring of the following substances are colourless, some of these substances are inert and unreactive, while other materials can be visualized in tissue sections using various histochemical methods.

0 Certain type of minerals gain access to the body by inhalation, ingestion or skin implantation, commonly as a result of industrial exposure.

0 Some minerals, in the form of dye complexes can be seen in the skin and adjacent lymph nodes as a result of tattooing.

0 Occasionally mineral deposition may occur due to medication or wound dressing.

0 The most common minerals seen in the tissue sections are: 1. Carbon.

2. Silica.

3. Asbestos.

0 Less common minerals seen in the tissue are: 1. Lead

2. Beryllium.

3. Aluminum.

4. Mercury.

5. Silver.

6. Bismuth.

1. Tattoo pigment : This is associated with skin and any adjacent lymphoid areas.

2. Carbon: this exogenous substance is the most commonly seen minerals in tissue and is easily recognized in stained tissue section, found in the lung and adjacent lymph nodes, the main sources are car exhausts and smoking.

Black pigmentation of the lung (anthracosis) is seen as massive deposition of carbon. Carbon is extremely inert and unreactive and fail to be demonstrated histochemically.

3. Silica: silica in the form of silicate is associated with the majority of all mined ores because are found in or near rocks that contain silica.

silica deposition lead to disease known as silicosis which present as a progressive pulmonary fibrotic condition which gives rise to impaired lung capacity and in some cases extremely disability.

Silica is unreactive substance and hence can not be demonstrated histochemicaaly.

4. Asbestos: its special form of silica, has been used for many years as a fire resistant and insulating material and break lining.

there are several types of asbestos and the fiber that cause pulmonary disease in man are called (amphiboles), the fibres are 5 – 100 um long and only 0.25 – 0.5 um in diameter, and can collect in the alveoli at the periphery of the lung.

tHE eND &

gOOD lUCK