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Lesson 7-4
 Special Stains


The members of the granulocytic series contain an enzyme, peroxidase, which liberates the oxygen from hydrogen peroxide. This enzyme is more prominent in mature forms. A benzidine derivative is used as an indicator of peroxidase activity. The indicator is oxidized and precipitates in the form of brown to blue granules. This stain is used to help differentiate leukemias.

NOTE: Follow manufacture's instructions for all special stains.

Peroxidase positive cells are identified by yellow-green to blue and brown-green granules. Cells of the granulocyte series from the promyelocyte through the segmented neutrophil are peroxidase positive. The degree of peroxidase activity increases as the granulocytes mature. Monocytes may show a weak reaction. All other cells are negative.

The oxidizing enzyme in the granules of the granulocytic leukocytes deteriorates rapidly in vitro. It is, therefore, necessary to use fresh blood in making the preparation.

  • Smears should be prepared within one hour of obtaining the specimen and stained within three hours after they are prepared.
  • The monocyte is thought to be slightly peroxidase positive through the phagocytization of peroxidase positive granules of ruptured cells.
  • Myeloblasts can show weak peroxidase activity using this method.
  • Addition of 4.9 mg of sodium cyanide to the stain inhibits peroxidase activity in all granulocytes except eosinophils.
  • If greater nuclear detail is required, counter stain with laqueous cresyl violet acetate for one minute or in freshly prepared Giemsa stain for 10 minutes.
  • Giemsa stain is prepared as follows: Mix 3.8 g Giemsa stain powder and 200 ml glycerin. Incubate at 60șC for two hours. Add 312 ml absolute methanol; dilute the staining solution 1:10 with 5 sodium carbonate before use.


Blood smears are fixed and stained for alkaline phosphatase activity.


Count two slides (100 cells per slide) on each patient, rating the segmented neutrophils according to how much black staining of the granules is observed. If no staining is noted, the rating is 0; if slight black staining is noted, the rating is 1+, if a medium amount of black staining is noted, the rating is 2+, if a heavy amount of dark black staining is observed, the rating is 3+, and if there is heavy black staining covering all the cytoplasm, the rating is 4+.

 After 100 cells per slide are rated, figure the score-giving cells counted as 0--no score; cells rated as 1+ get a score of 1 each; cells rated as 2+ get a score of 2 each, etc.

Report the total number of cells, giving their ratings and score. Report the total score for each individual slide. Average the two total scores and report the average. Also, always report the normal score range.

Patients with infections, polycythemia, and myeloproliferative disorders demonstrate increased alkaline phosphatase activity. In patients with acute or chronic granulocytic leukemia, alkaline phosphatase activity is decreased.

Normal Values

Scores of 13 to 130 have been obtained in healthy adults. However, the attending physician should interpret whether values are normal or abnormal.


Blood is mixed with methyl violet solution and a smear is prepared. Heinz-bodies stained with methyl violet are purple, round or oval granules, one-two microns in diameter within the erythrocytes.

Iron granules present in erythrocytes stain blue.

  • Heinz-bodies are invisible in Wright-stained preparation.
  • They can be observed in reticulocyte preparations and by the use of phase microscopy.
  • Heinz-bodies are thought to be denatured hemoglobin. They are usually demonstrated in hemolytic anemias caused by toxic agents, including vegetable and animal poisons.


Siderocytes are erythrocytes containing iron granules. The granules are blue when stained with Prussian blue.

Iron granules present in erythrocytes stain blue.

  • Siderocytes occur in several anemias, lead poisoning, and after splenectomy.
  • On Wright-stained preparation, the granules are bluish-purple and are called Pappenheimer bodies.


David L. Heiserman, Editor

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Revised: June 06, 2015