Containing Cells in Blood Smears

Principle. Methemoglobin combines with KCN to form cyanmethemoglobin, while oxyhemoglobin does not react with cyanides. Oxyhemoglobin functions as a peroxidase, whereas cyanmethemoglobin has very low perixodase activity.

Method. Add 1/50 vol of a 0.4 M KCN solution to blood anticoagulated with heparin or sodium citrate. Prepare thin smears from this mixture, dry, and immerse in the following mixture at room temperature: 80 mL of 96 % ethyl alcohol + 16 mL of 0.2 M citric acid + 5 mL of 30 % H2O2. Move the smears rapidly in the solution for about 1 min, then leave them in the solution for 2 min. Wash the smears first in methyl alcohol, then in distilled water, and stain with hematoxylin and erythrosin (see stain for HbF). Examine at 40 x using dry or oil-immersion magnification.

Interpretation. Oxy-Hb-containing cells stain a bright red. Cells that contain met-Hb (converted to cyanmet-Hb) are eluted and appear as shadows.

The same staining procedure can be used to differentiate erythrocytes with a glucose-6-phos-phate dehydrogenase (G-6-PDH) deficiency by combining it with the Brewer test (method of Betke, Kleihauer and Knotek). This test is based on the principle that hemoglobin converted to met-Hb by the addition of nitrite reduces to oxy-Hb in the presence of methylene blue and glucose. Red blood cells with a G-6-PDH deficit cannot undergo this reduction. Even after several hours, when all the methemoglobin in normal erythrocytes has converted back to oxy-Hb, cells with a G-6-PDH deficiency retain all or most of their met-Hb. This causes the deficient cells to appear "blank" with appropriate staining (see top of this section).

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