Wright Information and Courses from MediaLab, Inc.

Pappenheimer bodies, while visible on a Wright's stained smear, should be Perls' Prussian blue stain, which is specific for iron. Pappenheimer bodies are seen in certain types of anemia characterized by an increase in the storage of iron, such as sideroblastic anemia and thallassemia. These inclusions are also seen in the peripheral blood following a splenectomy. In a healthy person with a normal spleen, Pappenheimer bodies are destroyed before the erythrocytes enter the peripheral circulation.

To verify that red cell inclusions contain iron, it is necessary to use an iron stain, such as Perl's Prussian blue. The iron-containing granules are called siderotic granules. A mature erythrocyte containing siderotic granules is referred to as a siderocyte, while an immature (nucleated) erythrocyte containing siderotic granules is known as a sideroblast. A Pappenheimer body is a siderotic granule which is visible on Wright stain. All Pappenheimer bodies are Prussian blue positive, but not all siderotic granules are visible on Wright's stain as Pappenheimer bodies.

Although the nucleus has been extruded, the reticulocyte is still considered immature because it retains numerous organelles needed for hemoglobin production, such as ribosomes, mitochondria, and fragments of the Golgi apparatus. The reticulocyte is slightly larger (10 microns) than the mature erythrocyte. A reticulocyte normally remains in the bone marrow for one or two days before entering the circulation and its final 24 hours of maturation. The red cell is mature when hemoglobin production is complete and the organelles have disintegrated. Reticulocytes normally make up 0.5 - 1.5% of the peripheral blood red cells. They appear blue/gray on the Wright's stained smear. The residual RNA in the cytoplasm causes the blue/gray color. The terms, polychromasia or polychromatophilic, are used to describe these cells on a Wright's stained preparation. A supravital stain such as new methylene blue N or brilliant cresyl blue is used to stain reticulocytes for an actual count.

The first group is composed of erythrocytes or red blood cells (RBC's). The main function of the erythrocytes is the transport of oxygen from the lungs to the body tissues. Most of the cells in this Wright's stained peripheral blood smear are red cells. On is shown at the arrowhead.

All of these peripheral blood cells have different characteristics. In order to accurately identify each of them, a peripheral blood film must be made, preferably from capillary blood or blood anticoagulated with EDTA (Ethylenediaminotetracetic Acid). EDTA, in contrast to many other anticoagulants, preserves cellular morphology. The individual characteristics of each cell type are made visible by staining the blood films with the Wright stain, and observing them under the microscope.

Normal red cells are seen in this field. Mature erythrocytes can be described as round, elastic, non-nucleated, bi-concave discs which appear buff colored on Wright's stained smears. Notice that many of the cells have an area of central pallor which covers about one-third of the cell. The pallor occurs as a result of the disc-shaped cells being spread on the slide. Normal mature red blood cells have an average diameter of 7.2 microns with a range of 6-9 microns. This is approximately the same size as the nucleus of a small lymphocyte, which is often used as a guideline when determining the size of the red cells on a slide. The average thickness of a normal mature red blood cell is 2.1 microns with a mean corpuscular volume (MCV) of 87 cubic mircons/femtoliters.

In addition to the amount of hemoglobin present, the color of the cell must also be considered. Completely mature red cells appear buff-colored, while slightly immature non-nucleated red cells (reticulocyte stage) appear blue/gray on Wright's stained smears due to the presence of residual ribonucleic acid (RNA).The terms used to describe these cells are polychromasia or polychromatophilia. Polychromatophilic cells are frequently larger in size than mature red cells and can be distinguished from both types of macrocytes by this distinctive color.

Vacuoles are areas of the cytoplasm which do not stain with Wright's stain and appear as holes in the cytoplasm. Their composition may vary; some will contain remnants of bacterial digestion, autodigestion in an aging cell, while still others may contain fat. It is not possible to differentiate the various types of vacuoles on Wright's stained smears under light microscopy. They may be seen occasionally in an aging granulocyte, but are seen more frequently and are significant in cases of bacterial infection and septicemia.

Illustrated in the photograph is a normal bone marrow smear stained with Wright/Giemsa stain. Note the evenly distributed cells with normal maturation in both the myeloid and erythroid maturation sequences.An estimation of the percentage composition of cells can be made by experienced observers from scanning of multiple fields. In some instances a detailed differential count of 300 or more cells must be made.In normal bone marrows, the myeloid to erythroid ratio (M:E ratio)ranges from 1.2:1 to 5:1.A ratio of less than 1.2:1 indicates depressed leukopoiesis or erythroid hyperplasia. Ratios of 6:1 or greater usually indicates infection, erythroid hypoplasia, or chronic myelogenous leukemia.An assessment of the overall cellularity is also useful. In general, cellularity of less than 25% indicates hypoplasia; greater than 75% indicates hyperplasia.