This document discusses Perls stain, which is used to identify iron deposits in tissue samples. It provides background on pigments in living tissue, including endogenous pigments like hemosiderin and hematogenous pigments. The history of Prussian blue and its use as Perls stain is described. The principle of the stain is that hydrochloric acid releases ferric ions from hemosiderin, which then react with potassium ferrocyanide to form insoluble Prussian blue pigment. Staining protocols, quality control, and clinical applications for identifying iron deposits in organs are covered.

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:
1. Endogenous pigments- produced either within tissues and serve a
physiological function, or are by products of normal metabolic
processes.
a. Hematogenous pigments: Hemosiderin, Haemoglobin, Bile
pigments.
b. Non-hematogenous pigments: Melanin, lipofuscins, chromaffin,
Dubin-Jhonson pigments.
c. 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)
• Spleen (Congestive splenomegaly)
• Cardiomyopathies
• Conjunctiva
• Placenta
Intra-Alevolar deposition
Portal macrophages Iron deposition in spleen

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