PERL’S / PRUSSIAN BLUE STAINING

Synonyms: Siderocyte stain or Prussian blue reaction. 

A simple method for staining non-hemoglobin iron in erythrocytes, normoblasts, macrophages, and other cells containing particulate iron in new or old films of cellular fluids or imprints of tissues and organs is presented. This method consists in using the prussian blue reagent as a type of counterstain; no separate decolorization is necessary. The preparations obtained resemble the original preparations except that iron stands out as a vivid blue-green material.

The method is particularly useful in studying conditions accompanied by varying degrees of iron excess or hemosiderosis of the marrow. The stainable iron is all virtually the same color. The diffuse blue-green color of the cytoplasm of macrophages might be attributed to the more soluble form of iron, ferritin. Stainable iron is visible in normoblasts and erythrocytes as well as in macrophages in sections subjected to the prussian blue reaction.

A prussian blue method using formalin fixation on fresh films of marrow has also been shown to be useful in the demonstration of particulate iron in previously stained films of marrow and blood. The formalin fixation appears to bring about a higher percentage of siderocytes.

Free iron (Fe+3 state) is seen as small blue or blue-green siderocyte granules found in the cytoplasm of developing RBC’s.  This is iron that has not been incorporated into hemoglobin.  If one or more of these free iron granules are observed, the RBC is called a siderocyte.

In the healthy individual, up to 1% of the RBC’s may be siderocytes.  Increased siderocytes are observed in thalassemia major, lead poisoning, leukemia, alcoholism, hemolytic anemias, megaloblastic anemias, and splenectomy.  Increased siderocytes are an indicator of abnormal hemoglobin synthesis. Siderocyte granules are observed in nucleated red blood cells and may be found in the bone marrow reticulocytes.  These granules are not normal to the mature erythrocytes seen in peripheral blood.

PRINCIPLE Dilute mineral acid hydrolysis releases ferric ions from protein bound tissue deposits, which, in the presence of ferrocyanide ions, is precipitated as the highly coloured and highly water-insoluble complex, potassium ferric ferrocyanide, Prussian blue. FeCl3 + K4Fe(CN)6 = KFeFe(CN)6¯ + 3KCl 

Ferrous ions do not produce a coloured reaction product, thus are excluded from the visualisation. Tissue deposits containing ferric ions are invariably haemosiderin. The original method of Perls applied the ferrocyanide and acid as separate reagents. 

SOLUTIONS AND REAGENTS:   20% Aqueous Solution of Hydrochloric Acid: Hydrochloric acid, concentrated ------------ 20 ml Distilled water -------------------------------------- 80 ml Mix well.   10% Aqueous Solution of Potassium Ferrocyanide: Potassium ferrocyanide, Trihydrate -----10 g Distilled water ---------------------------------------------------------- 100 ml Mix to dissolve  Working Solution: Mix equal parts of 20% hydrochloric acid and 10% potassium ferrocyanide solution JUST before use.

METHODS:

Iron staining can be performed on bone marrow, touch preps, EDTA peripheral blood, biopsies and buffy coat preparations.

For blood slides:

Procedure:

1. A drop of blood is placed on one end of a 3" by 1" glass slide and using a spreader slide at an angle of approximately 25o to 45o, a wedge type smear is made and allowed to dry.  Smears may be made using two cover glasses.  A drop of blood is placed in the center of  one of the cover glasses.  The other cover glass is placed over the drop of blood so that the corners of each cover glass form an eight-pointed star.  The two cover glasses are pulled apart and allowed to dry face up. 

2. Blood slides are air dried, then fixed in methanol for up to 10 minutes.3. Pour the working solution into a staining dish to be immersed in waterbath of

50oC to 55oC.4. The slides are stained in Prussian blue reagent (sometimes called Perl’s reagent)

for up to 30 minutes.5. Rinse with tap water for up to 20 minutes and  next with distilled water.

6. The slides are then counterstained with eosin or 1% safranin for a few seconds.  Rinse and dry.

7. To determine the percentage of siderocytes, do this: FIRST:  determine the number of siderocytes per 1,000 RBC.SECOND: use the formula # siderocytes counted / 1000 RBC’s (times) 100, THIRD: report the percent siderocytes. 

8. Sample problem: 65 siderocytes divided (÷) by 1000 RBC then the answer is multiplied (×) 100 = 6.5%

For buffy coat preparations:

The buffy coat preparation may be required if the following are suspected: [1]   a blood specimen that is pancytopenic and the abnormal, immature, or reactive cell densities are low, [2]   examining a patient diagnosed with megaloblastic anemia for nucleated red         blood cells and/or hypersegmented neutrophils, [3]    looking for plasma cells, [4]    tumor cells in blood indicating metastasis, [5]    facilitate the search for bacteria and/or parasites (NOTE: erythrocytes containing malarial parasites tend to concentrate at the top of the red cell layer).

Procedure:

1. The buffy coat is prepared by filling a Wintrobe tube or hematocrit tube with blood. 

2. It is recommended that the Wintrobe tube be centrifuged at 1000 rpm for six minutes. 

3. The hematocrit tube for a shorter time in the hemotocrit centrifuge, 2 minutes is recommended.  These reduced centrifuge times will cause less cellular distortion. 

4. For the hematocrit tube, score the tube just below the red cell line and break.  5. Touch the tube with the buffy coat to a glass slide and allow a small amount of

plasma to add to the it. 6. Mix the buffy coat in the plasma and then make the buffy coat film, air

dry and stain. 7. The buffy coat in the Wintrobe may be aspirated with a pipette and

transferred to a watch glass and mixed with plasma.8. Make the buffy coat film, the air dry and stain.9. The slides are stained in Prussian blue reagent (sometimes called Perl’s reagent)

for up to 30 minutes. 10. The slides are then counterstained with eosin or safranin. 

For spleen and bone marrow.:

Procedure:   1.       Deparaffinize and hydrate sections to distilled water.

2.       Mix equal parts of hydrochloric acid and potassium ferrocyanide prepared immediately before use. Immerse slides in this solution for 20 minutes.

3.       Wash in distilled water, 3 changes. 4.       Counterstain with nuclear fast red for 5 minutes.

Nuclear Fast Red (Kernechtrot) Solution   Nuclear fast red ------------------------- 0.1 g Aluminum sulfate ------------------------ 5 g Distilled water ---------------------------100 ml Dissolve aluminum sulfate in water. Add nuclear fast red and slowly heat to boil and cool. Filter and add a grain of thymol as a preservative.

5.       Rinse twice in distilled water. 6.       Dehydrate through 95% and 2 changes of 100% alcohol. 7.       Clear in xylene, 2 changes, 3 minutes each. 8.       Coverslip with resinous mounting medium.

Results:         Iron (ferric form) ------------------------- bright blue       Nuclei --------------------------------------- red       Cytoplasm -------------------------------- pink