PERL’S / PRUSSIAN BLUE STAINING
Synonyms: Siderocyte stain or Prussian blue reaction. A simple simple method method for staini staining ng non-hemo non-hemoglo globin bin iron iron in erythr erythrocy ocytes tes,, 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 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 method is particularly particularly useful in studying studying conditions conditions accompanied by 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 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 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) state) is see seen n as small small blue blue or blue-gr blue-green een siderocy siderocyte te granules found found in the cytoplasm of developing developing RBC’s. RBC’s. This is iron that has not been incorporated incorporated into hemoglobin. hemoglobin. If one or more of these free iron granules are observed, observed, the RBC is called a siderocyte. In the healthy individual, up to 1% of the RBC’s may be siderocytes. Increas Increased ed sideroc siderocyt ytes es are observe observed d in thalas thalassem semia ia major major,, lea lead d poison poisoning ing,, leukem leukemia, ia, alcoholism, alcoholism, hemolytic hemolytic anemias, anemias, megaloblastic megaloblastic anemias, anemias, and splenectomy. splenectomy. Increased Increased siderocytes are an indicator of abnormal hemoglobin synthesis. Siderocyte granules are obse observ rved ed in nucl nuclea eate ted d red red bloo blood d cell cellss and and may may be foun found d in the the bone bone marr marrow ow reticul reticulocy ocytes tes.. These These granule granuless are not normal normal to the mature mature erythr erythrocy ocytes tes 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 + K 4Fe(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 blood is placed on on one end of of a 3" by 1" glass glass slide slide and using using a spreader spreader o o slide at an angle of approximately 25 to 45 , a wedge type smear is made and allowed to dry. dry. Smears may be made using two two cover glasses. A drop of blood is placed placed in the center of one of the cover cover glasses. glasses. The other cover cover glass is is placed over the drop of blood so that the corners of each cover glass form an eight pointed star. star. The two cover glasses are pulled apart and allowed allowed to dry face up. 2. Blood slides slides are air dried, dried, then then fixed fixed in methanol methanol for for up to 10 minutes minutes.. 3. Pour Pour the working working solution solution into into a staini staining ng dish to be immerse immersed d in wat waterba erbath th of o o 50 C to 55 C. 4. The slides slides are stained stained in Prussi Prussian an blue reagent reagent (sometime (sometimess called called Perl’s Perl’s reagent) reagent) for up to 30 minutes. 5. Rinse with tap water water for for up to 20 minutes minutes and and next with with distille distilled d water water. 6. The slides slides are then then counters counterstai tained ned with with eosin or 1% safrani safranin n for a few second seconds. s. Rinse and dry.
7. To determine determine the percenta percentage ge of siderocyte siderocytes, s, do this: FIRST: determine the number of siderocytes per 1,000 RBC. RBC. SECOND: use the formula # siderocytes sideroc ytes counted / 1000 RBC’s (times) 100, THIRD: report the percent siderocytes. 8. Sampl Samplee probl problem em:: 65 sidero siderocy cyte tess divi divided ded (÷) by 1000 1000 RBC RBC then then the answe answerr 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 pancytopenic and the the abnormal, immature, or reactive cell densities are low, low, [2] examining a patient diagnosed with megaloblastic 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 buffy coat coat is prepared prepared by filling filling a Wintr Wintrobe obe tube or hematoc hematocrit rit tube tube with blood. 2. It is recom recomme mende nded d that that the Wintr Wintrob obee tube tube be ce cent ntri rifu fuged ged at 1000 rpm for six six minutes. 3. The hematocrit hematocrit tube for for a shorter shorter time in in the hemotocrit hemotocrit centrifuge, centrifuge, 2 minutes minutes is is recommended. These reduced centrifuge times will cause less less cellular distortion. distortion. 4. For the hematocr hematocrit it tube, tube, score the tube just below below the the red cell cell line and and break. 5. Touch ouch the tube with with the buffy buffy coat coat to a glass glass slide slide and allow allow a small amoun amountt of plasma to add to the it. 6. Mix Mix the the buf buffy coat coat in the plasm plasmaa and and then then make make the the buf buffy coat coat film film,, air air dry and stain. 7. The buf buffy coat in the Wintrobe may be aspirat rated with a pipett ette and transferred to a watch glass and mixed with plasma. 8. Make Make the buff buffy y coat coat film, film, the the air dry dry and stai stain. n. 9. The slides slides are stained stained in Prussi Prussian an blue reagent reagent (sometime (sometimess called called Perl’s Perl’s reagent) reagent) for up to 30 minutes. 10. The slides are then counterstained with eosin or safranin. For spleen and bone marrow.: marrow.:
Procedure: 1. 2.
Deparaffinize and hydrate sections to distilled water. Mix equal parts of hydrochloric acid and potassium ferrocyanide prepared immediately before use. Immerse slides in this solution for 20 minutes.
3. 4.
Wash in distilled water, 3 changes. 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. 6. 7. 8.
Rinse twice in distilled water. Dehydrate through 95% and 2 changes of 100% alcohol. Clear in xylene, 2 changes, 3 minutes each. Coverslip with resinous mounting medium.
Results:
Iron (ferric form) ------------------------- bright blue Nuclei --------------------------------------- red Cytoplasm -------------------------------- pink
