Staining techniques: Perl's Prussian Blue/Safranin in Cats (Felis) | Vetlexicon
felis - Articles

Staining techniques: Perl's Prussian Blue/Safranin

ISSN 2398-2950


Synonym(s): Perls, Prussian blue

Introduction

  • Hemosiderin combines with potassium ferrocyanide to form a colored pigment, potassium ferric ferrocyanide.
  • Iron-positive material stains blue, rest of cell and background stain dark pink (contrast).

Uses

  • Bone marrow - evaluate iron stores, helps to classify cause of anemia Anemia: overview.

Anemia due to iron deficiency, has decreased bone marrow storage of iron. Anemia due to hemolysis or decreased production has increased bone marrow iron stores.

  • Siderocytes/sideroblasts RBC with iron that appear as focal areas of basophilic stippling.
  • Sideroleukocyte - leukocytes containing iron-positive inclusions.
  • Hemosiderin - in macrophages in TW/BAL or body cavity fluids or other specimens.

Advantages

  • 1% acidic potassium ferrocyanide solution only has a shelf life of a few hours, therefore must always be made fresh.
  • Biohazard since need to store strong acids in clinic/laboratory.
  • 2% Potassium ferrocyanide must be kept in a brown bottle, stored in a dark cupboard to keep it stable.

Time required

Preparation

  • 5 minutes.
  • 2% HCL = 500 ml distilled H2O + 28 ml concentrated HCL.
  • 0.1 % Safranin solution (1% acidified potassium ferrocyanide) = 2% HCL +  2% K ferrocyanide.

Requirements

Materials required

Minimum equipment

  • Coplin Jars; 100 x oil immersion objective.

Minimum consumables

  • 28% HCL + 2% Potassium ferrocyanide (keep in brown bottle in the dark) to make 1.0% acidified potassium ferrocyanide solution (0.1% Safranin solution).

Preparation

Technique

Approach

Step 1 -

  • Fix smeared slides in a coplin jar filled with methyl alcohol for a minimum of 10 minutes (overnight preferable).

Step 2 -

  • Then place in fresh 1% acidified potassium ferrocyanide solution for 10 mins.

Step 3 -

  • Gently wash in running tap water for 10 minutes.
  • Shake off excess water.

Step 4 -

  • Dip smears into 0.1% safranin counter stain solution for 10 seconds.
  • Rinse smears in tap water.

Step 5 -

  • Dry.
  • Examine under 100 x oil immersion objective.

Aftercare

Immediate Aftercare

Monitoring

Pathological

  • Indicates previous hemorrhage if iron is identified in macrophages.

Outcomes

Further Reading

Publications

Refereed papers

Other sources of information

  • Harvey J W (2001) Atlas of Veterinary Hematology. Blood and Bone Marrow of Domestic Animals. W B Saunders. pp 14-15.
  • Lewis  H B & Rebar A H (1979) Bone marrow Evaluation in Veterinary Practice. Ralston Purina Company, ISB 78-68556,  pp 64-65.