Perl's stain

An important topic and extremely important for practical purpose.

1. PERLS STAIN DR. MANOJ PANT

2. LAYOUT Introduction History Principle Staining protocols Quality controlling Clinical use

3. PIGMENTS Pigments are the substances occur­ring in living matter which absorb visible light and impart specific colour. Classified as: Endogenous pigments- produced either within tissues and serve a physiological function, or are by­ products of normal metabolic processes. Hematogenous pigments: Hemosiderin, Haemoglobin, Bile pigments. Non­-hematogenous pigments: Melanin, lipofuscins, chromaffin, Dubin-Jhonson pigments. Endogenous minerals: Calcium, copper, uric acid.

4. PIGMENTS 2. Artifact pigments- artifactually produced mate­rial caused by the interactions between certain tissue components and some chemical substances e.g. formalin and malaria pig­ments, mercury, starch. 3. Exogenous pigments and minerals- gain access to the body accidentally through a variety of methods e.g. tattoo pigments, asbestos, lead, silica, silver.

5. HEMOSIDERIN Yellowish-brown, iron-containing, granular pigment that is found within reticuloendothelial cells of bone marrow, spleen and liver. Hemosiderin is formed by partial degradation of aggregates of ferritin by lysosome. It is a conglomeration of iron, ferritin proteins and other subcellular constituents. It contains Iron in the form of ferric hydroxide which is bound to a protein framework. Hemosiderosis is a condition of iron overload where iron does not interfere with organ function; but hemochromatosis refers to a condition of iron overload associated with organ failure.

6. HISTORY Prussian blue was discovered in 1706 and was first used as synthetic colour in paints by Diesbach in Berlin. In 18th century, Prussian blue was uniform coat colour worn by infantry and artillery regiments of Prussian army and later by German soldiers. In 1867, German Pathologist, Max Perls described it as histochemical stain and hence Known as “ Perls Prussian Blue” or Berlin blue.

7. PRINCIPLE Tissue sections when treated with Hydrochloric acid, denatures the protein binding to hemosiderin molecules ,there by releasing Ferric (3+) ions. The Ferric ions combine with Potassium Ferrocyanide to form Ferric Ferrocyanide which is an insoluble bright blue pigment (Prussian blue)

8. STAINING PROTOCOL Fixation Formalin Avoid the use of acid fixatives. Sections Standard paraffin block sections. Reagents 2% Aqueous potassium ferrocyanide 2% Potassium ferrocyanide 1% Aqueous neutral red

9. STAINING PROTOCOL Method Take a test and control section to distilled water. Mix equal parts of 2% Hydrochloric acid and 2% Potassium ferrocyanide solutions and filter on to the sections. Leave for 30 min at room temperature, changing to fresh solution after 15 min. Wash in several changes of distilled water. Counter stain with the aqueous neutral red solution for 1 min. (stains other tissue components, nucleus & cytoplasm) Wash in distilled water. Dehydrate, clear with xylene and mount in DPX.

10. STAINING PROTOCOL Results Ferric iron - blue Nuclei - red Cytoplasm – Pale red

11. STAINING PROTOCOL Validation For assessment of Perls stain, section from spleen representing Gamma Gandy bodies in case of congestive splenomegaly is used as quality control. Section from control is stained along with test slide. The intensity and adequacy of staining is compared between control slide and test slide.

12. Other counter stains: 0.5% Aqueous neutral red 0.1% Safranine 0.5% Aqueous Eosin 0.1% Nuclear Fast red in 5% aluminium sulphate 0.5% Phloxine 0.5% Tartrazine Other controls: Bone marrow Postmortem lung tissue: iron positive macrophages (heart failure cells)

13. QUALITY CONTROLLING Avoid using outdated reagents or improperly stored reagents. Iron contamination of glassware must be prevented. Avoid washing with tap water as rust in the tap or iron in tap water may cause false positive staining . Use distilled water. Drain slides after each step to avoid solution carry over. Wash well after staining with neutral red, as traces of it can form a granular red deposit.

14. CLINICAL USE To demonstrate iron stores in bone marrow. Interpretation of Perls stain on BMA: GRADE 0 : Iron deficiency (a minimum of 7 BM particles must be available before concluding that hemosiderin is absent. GRADE 1,2,3 : Normal iron stores. GRADE 4 to 6: Increased iron stores. Grading of iron stores on BMA(after gale et al) Grade 2 Grade 5

15. CLINICAL USE Interpretation of iron stores on BM Bx : GRADE 0 : Iron deficiency. GRADE 1-2 : Normal stores. GRADE 3-4 : Increased stores. GRADING OF IRON STORES ON BMB

16. CLINICAL USE Demonstrate iron deposits in various organs: Liver (hemochromatosis) Lungs (Congestive heart failure) S pleen (Congestive splenomegaly) Cardiomyopathies Conjunctiva Placenta Intra- Alevolar deposition Portal macrophages Iron deposition in spleen

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