1

throughout the cytoplasm or may encircle the nucleus of erythroblasts like a ring. Normally the granules are very fine and can be identified in erythroblasts only by closely examining the smears with oil-immersion microscopy in a darkened room. Generally 1 to 4 fine granules will be seen, rarely more. When iron deficiency is present, the percentage of sideroblasts is reduced to less than 15 %. Sideroblasts containing coarse iron granules that form a partial or complete ring around the nucleus (ringed sideroblasts) are definitely abnormal. The detection of siderocytes has little practical relevance: they are increased in the same diseases as sideroblasts, and they are elevated in the peripheral blood following splenectomy, as the spleen normally removes iron from intact red blood cells.

Regarding (b): the content of stored iron is assessed by examining bone marrow fragments in smears or sections. Iron stored in macrophages may occur in a diffusely scattered form, a finely granular form, or in the form of larger granules or clumps that may cover part of the nucleus. Iron can also be demonstrated in plasma cells as a result of alcohol poisoning or sideroblastic anemia and hemochromatosis.

The differential diagnosis afforded by iron stain is summarized in Table 1.

1.10 Cytochemical Determination of Glycogen in Blood Cells by the Periodic Acid Schiff Reaction and Diastase Test (PAS Reaction)

Principle

This method is based on the oxidation of a-gly-cols in carbohydrates and carbohydrate-containing compounds. The resulting polyaldehydes are demonstrated with the Schiff reagent (leukofuch-sin).

Reagents

• Periodic acid solution, 1 %, in distilled water.

• Sulfite water: add tap water to 10 mL of a 10 % sodium metabilsulfite solution (Na2S2O5) and 10 mL of 1mol/L HCL to make a volume of 200 mL. The stock solutions can be stored in the refrigerator; the mixture should always be freshly prepared.

• Prepare Schiff reagent (commercially available) as follows: completely dissolve 0.5 g pararosa-niline in 15 mL of 1 mol/L HCl by shaking (no heating) and add a solution of 0.5 g potassium metabisulfite (K2S2O5) in 85 mL distilled water. The clear, bright red solution will gradually lighten to a yellowish color. After 24 h, shake with 300 mg activated charcoal (powdered) for 2 min and then filter. The colorless filtrate is ready to use and, when stored in a dark stoppered bottle in a cool place, will keep for several months. Schiff reagent that has turned red should no longer be used!

Method

• Fix the smears for 10 min in a mixture of 10 mL 40 % formalin and 90 mL ethanol (alternative: fix for 5 min in formalin vapor).

• Wash for 5 min in several changes of tap water.

• Place the smears in 1 % periodic acid for 10 min (prepared fresh for each use).

• Wash in at least two changes of distilled water and dry.

• Place in Schiff reagent for 30 min (in the dark at room temperature).

• Rinse in sulfite water (changed once) for 23 min.

• Wash in several changes of distilled water for 5 min.

• Nuclear stain with hemalum for approx. 10 min, then blue in tap water for approx. 15 - 20 min, and air dry.

Even older slides that have been stained with Giemsa or Pappenheim can be reused for the PAS reaction. Specimens that have been treated several times with oil or xylene should not be used for PAS staining. The smears can be placed unfixed in periodic acid after washing in distilled water (Step 3) to remove the color.

Interpretation

PAS-positive material in the cytoplasm may produce a diffuse red stain or may appear as pink to burgundy-red granules, flakes, or clumps of varying size that may occupy large areas of the cytoplasm. The distribution of PAS-positive material in normal leukocytes is summarized in the Table. Some plasma cells, macrophages, and osteoblasts may also show a positive PAS reaction, and megakaryocytes are strongly positive.

Table 1. Differential diagnosis by iron stain in the bone marrow

Sideroblasts

Iron-storing reticulum cells, sideromacrophages

Special features

finely granular, 1-4 granules

Isolated, mostly finely granular deposits

Siderocytes in peripheral blood 0 - 0.3 %0

Hypochromic anemias - Iron deficiency

< 15 % finely granular

None

Serum Fe Q

- Infection, tumor

< 15 % finely granular

Increased finely granular or (rarely) coarsely granular deposits

Serum Fe Q

- Sideroachrestic anemias (RARS)

> 90 % coarsely granular; ringed sideroblasts (> 15 %)

Greatly increased, many diffuse or coarsely granular deposits

Serum Fe q , siderocytes may be increased

- Lead poisoning

> 90 % coarsely granular; ringed sidero-blasts

Greatly increased, many diffuse or coarsely granular deposits

Serum Fe q , siderocytes may be increased

- Thalassemia

> 90 % coarsely granular; ringed sidero-blasts

Greatly increased, many diffuse or coarsely granular deposits

Serum Fe q , siderocytes may be increased

Hemolytic anemias

< 80 % finely granular

Increased finely granular or (rarely) coarsely granular deposits

Secondary sideroachrestic anemias

< 80 % finely granular

Increased finely granular or (rarely) coarsely granular deposits

Vitamin B6 deficiency

< 80 % finely granular

Increased finely granular or (rarely) coarsely granular deposits

Megaloblastic anemias

< 80 % finely granular

Increased finely granular or (rarely) coarsely granular deposits

Aplastic anemias

< 80 % finely granular

Increased finely granular or (rarely) coarsely granular deposits

Myeloproliferative disorders

< 80 % finely granular

Increased finely granular or (rarely) coarsely granular deposits

Hemochromatosis

< 80 % finely granular

Increased

Plasma cells contain iron

Bone marrow is not useful for diagnosis except positive plasma cells

Postsplenectomy state

< 80 % finely granular

Somewhat increased

Siderocytes greatly increased

PAS reaction in normal leukocytes

Cell type

PAS reaction

Myeloblast

0

Promyelocyte

(+)

Myelocyte

+

Metamyelocyte

++

Band and

+++

segmented cells

Eosinophils

+ (intergranular reaction)

Basophils

+ (granular!)

Monocytes

(+) to +

Lymphocytes

0 to + (granular)

+ = positive; ++ = markedly positive; +++ = strongly positive

+ = positive; ++ = markedly positive; +++ = strongly positive

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