Figure 1. Maturation of erythroid cells in the bone marrow
Pleuripotent stem cells give rise to erythrocytes by the process of erythropoiesis. The stem cell looks like a small lymphocyte and lacks the functional capabilities of the erythrocyte. The stem cells have the capacity of infinite division something the mature cells lack. Some of the daughter cells arising from the stem cell acquire erythroid characters over generations and time. Most of the erythroid cells in the bone marrow have a distinct morphology but commitment to erythroid maturation is seen even in cells that have not acquired morphological features distinctive of the erythroid lineage. These cells are recognized by the type of colonies they form in vitro. Two such cells are recognized. Burst-forming unit erythroid (BFU-E) arise from the stem cell and gives rise to colony-forming unit erythroid (CFU-E). CFU-E gives rise to pronormoblast, the most immature of erythroid cells with a distinct morphology (figure 1). BFU-E and CFU-E form a very small fraction of bone marrow cells and are not important in diagnosis. Examination of Romanovsky stained (Giemsa, Wright’s) bone marrow smears is central to the haematological diagnosis. Morphologically five erythroid precursors are identifiable in the bone marrow stained with Romanovsky stains. The five stages from the most immature to the most mature are the proerythroblast, the basophilic normoblast (early erythroblast), polychromatophilic normoblast (intermediate erythroblast), orthochromatophilic normoblast (late erythroblast) and reticulocyte (figure 1). As the cell matures the following morphological changes take place
- Cell becomes smaller
- Nucleus becomes smaller, chromatin more clumped and the nucleoli disappear
- Cytoplasm shrinks
- The cytoplasmic basophilia decreases: Haemoglobin is a major constituent of the red cell takes a pink to red colour on staining with Romanovsky stains. The machinery to synthesize haemoglobin (ribosomes) must appear before haemoglobin. Ribosomes make the cytoplasm basophilic (blue) because of their RNA content. As the haemoglobin content approaches the desired levels the number of ribosomes decreases. The cytoplasm of the maturing erythroid cell captures these changes and changes from deep blue (mainly ribosomes) in basophilic normoblast to polychromatophilic (ribosomes and haemoglobin) in polychromatophilic normoblast and resembling that of a erythrocyte (mainly haemoglobin) in orthochromatophilic normoblast.
- The earliest nucleated stages are least numerous and the later stages the most numerous
Proerythroblast: Proerythroblast (figure 2) is a large cell (12-20μm in size or about 1.5-3 times a normal erythrocyte) with a large nucleus that occupies almost 80% of the cell and a blue cytoplasm that forms a thin rim around the nucleus. The chromatin is finely granular and stripped. The nucleus shows multiple nucleoli (the multiple pale staining areas in the nucleus). The cytoplasm may show a small pale area that corresponds to the Golgi apparatus and may have a pale perinuclear halo. While it is usually possible to tell a proerythroblast from other blasts (myeloblasts, lymphoblasts and monoblasts) by it’s more homgenous of cytoplasm, larger size ,a chromatin that coarser and the perinuclear halo in very immature cells this distinction may be impossible.
Basophilic Normoblast: The basophilic normoblast (figure 3, 4 and 5) is a smaller (12-17μm) cell. The nucleus is round like that of a proerythroblast but lacks nucleoli. Condensation of chromatin with the appearance of heterochromatin begins at this stage giving the nucleus a coarse and granular appearance. The number of ribosomes peak at in basophilic normoblasts and this reflects in the cytoplasmic colour. The basophilic normoblast has one of the bluest cytoplasm amongst the bone marrow cells. It may have a perinuclear halo. The nucleus may assume a wheel spoke arrangement like a plasma cell. The spoke wheel arrangement, the blue cytoplasm and similar size makes the basophilic normoblast resemble a plasma cell (figure 4). The plasma cell is elliptical with an eccentric nucleus while the basophilic normoblast is round with a central nucleus.
Polychoromatophilic Normoblast: The polychromatophilic normoblast (figure 4) is a smaller (12-15μm). The distinguishing feature of this stage is the appearance of haemoglobin which reduces the basophilia of the cytoplasm. The chromatin shows a greater degree of clumping and irregular dense areas of staining are seen in the nucleus. Nucleoli are nor seen. Figure 4 shows adjacent basophilic and polychromatophilic normoblasts to contrast the size, the clumping of the chromatin and cytoplasmic staining (see normoblast maturation for more images of maturing polychromatophilic normoblasts).
Orthochromaphilic Normoblast: The process of haemoglobinization is almost complete by the stage of orthochromatophilic normoblast (figure 6). “Ortho-” in Greek means straight, upright or correct. Though orthochromatophilic suggests that the colour of the cytoplasm is the same as mature erythrocyte, this is not the case. The orthochromatophilic normoblast is the nucleated erythroid precursor that is closest to a mature erythrocyte in terms of size (8-12μm) and cytoplasmic staining. The cytoplasm however retains a blue tinge much like a reticulocyte. The nucleus is greatly condensed, shrunk and assumes a variety of bizarre shapes (buds, clover leaves, double spheres). The chromatin is greatly condensed and almost completely homogenous.
Reticulocyte: The Orthochromatophilic normoblasts finally extrudes the nucleus and reticulocyte (figure 7)is formed. The reticulocyte is about 20% larger (7-9μm) than the mature erythrocyte. The lifespan of the reticulocyte is about 3 days. It spends 2 of these in the bone marrow. A day after appearing in the peripheral blood the reticulocyte looses the blue colour and becomes a erythrocyte.
|BFU-E, CFU-E||The two cells are indistingushible from blasts of other series. They posses no morphological characters that indicates their erythroid origin|
|Pronormoblast||12-20 μ||Fine chromatin, many nucleoli||Blue, a perinuclear halo and a small pale area (Golgi apparatus) may be seen|
|Basophilic Normoblast||12-17 μ||Granular chromatin, no nucleoli||Very deep blue, perinuclear halo may be seen.|
|Polychromatophilic Normoblast||12-15 μ||Cromatin is visibly clumped with dark staining areas||Basophilia reduced but still not as pink as an erythrocyte|
|Orthochromatophilic normoblast||8-12 μ||A featureless nucleus with dense chromatin||Almost the colour of a reticulocyte|
|Reticulocyte||7-9μ||No Nucleus||Slightly blue compared to an erythrocyte|
|The nucleus of the small lymphocyte and the normoblast are about 7.5μ|
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