Anemias: Red blood cell morphology and approach to diagnosis

Erythropoiesis is the term used for marrow erythroid proliferative activity. Normal erythropoiesis occurs in bone marrow and is under the control of the hormone erythropoietin (produced by the kidney) and other growth factors and cytokines (Chapters 4 and 5).^7,8 When erythropoiesis is effective, bone marrow is able to produce functional RBCs that replace the daily loss of RBCs.

Ineffective erythropoiesis refers to the production of erythroid precursor cells that are defective. These defective precursors often undergo apoptosis (programmed cell death) in the bone marrow before they have a chance to mature to the reticulocyte stage and be released into the peripheral circulation. Several conditions, such as megaloblastic anemia (impaired deoxyribonucleic acid [DNA] synthesis as a result of vitamin B₁₂ or folate deficiency), thalassemia (deficient globin chain synthesis), and sideroblastic anemia (deficient protoporphyrin synthesis), involve ineffective erythropoiesis as a mechanism of anemia. In these anemias, peripheral blood hemoglobin concentration is low, which triggers an increase in erythropoietin production leading to increased erythropoietic activity. Although the RBC production rate is high, it is ineffective in that many of the defective erythroid precursors undergo destruction in the bone marrow. The end result is a decreased number of circulating RBCs resulting in anemia.^4,11

Insufficient erythropoiesis refers to a decrease in the number of erythroid precursors in the bone marrow, resulting in decreased RBC production and anemia. Many factors can lead to the decreased RBC production, including a deficiency of iron (inadequate intake, malabsorption, excessive loss from chronic bleeding); a deficiency of erythropoietin (renal disease); or loss of the erythroid precursors as a result of an autoimmune reaction (aplastic anemia, acquired pure red cell aplasia) or infection (parvovirus B19). Erythropoiesis can also be suppressed by infiltration of the bone marrow space with leukemia cells or with nonhematopoietic cells (metastatic tumors, granulomas, or fibrosis), the latter called myelophthisic anemia with characteristic teardrop RBCs.^4,7,12

Anemia can also develop as a result of acute blood loss (such as a traumatic injury) or chronic blood loss (such as an intermittently bleeding colonic polyp). Increased hemolysis results in a shortened RBC life span, thus increasing the risk for anemia. Chronic blood loss induces iron deficiency as a cause of anemia. With acute blood loss and excessive hemolysis, the bone marrow takes a few days to increase production of RBCs.^4,7,8 This response may be inadequate to compensate for a sudden excessive RBC loss as in traumatic hemorrhage or in conditions with a high rate of hemolysis and shortened RBC survival. Numerous causes of hemolysis exist, including intrinsic defects in the RBC membrane, enzyme systems, or hemoglobin, or extrinsic causes such as antibody-mediated processes, mechanical fragmentation, or infection-related destruction.^4,7,8

To detect the presence of anemia, the medical laboratory professional performs a complete blood count (CBC) using an automated blood cell analyzer to determine the RBC count, hemoglobin concentration, hematocrit, RBC indices, white blood cell count, and platelet count (Chapter 12). The RBC indices include the mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) (Chapter 11).¹³ The most important of these indices is the MCV, a measure of the average RBC volume in femtoliters (fL). Reference intervals for these determinations are listed on the inside front cover and in Table 16.1. Automated blood cell analyzers also provide the red cell distribution width (RDW), an index of variation of cell volume in an RBC population (discussed later). A reticulocyte count should be performed for every patient with anemia. As with RBCs, automated analyzers provide accurate measurements of reticulocyte counts.

The RBC histogram provided by the automated analyzer is an RBC volume frequency distribution curve with the relative number of cells plotted on the ordinate and RBC volume (fL) on the abscissa. In healthy individuals the distribution is approximately Gaussian. Abnormalities include a shift in the curve to the left (smaller cell population or microcytosis) or to the right (larger cell population or macrocytosis). A widening of the curve occurs when a population of RBCs have different volumes causing a greater variation of RBC volume about the mean. The histogram complements the peripheral blood film examination in identifying abnormal RBC populations. A discussion of histograms with examples can be found in Chapters 12 and 13.

RDW is the coefficient of variation of RBC volume expressed as a percentage.¹³ It indicates the variation in RBC volume within the population measured and an increased RDW correlates with anisocytosis (variation in RBC diameter) on the peripheral blood film. Automated analyzers calculate the RDW by dividing the standard deviation of the RBC volume by the MCV and then multiplying by 100 to convert to a percentage. Clinical usefulness of the RDW is discussed later.

The reticulocyte count serves as an important tool to assess the bone marrow’s ability to increase RBC production in response to an anemia. Reticulocytes are young RBCs that lack a nucleus but still contain residual ribonucleic acid (RNA) to complete the production of hemoglobin. Normally they circulate peripherally for only 1 day while completing their development. The adult reference interval for the reticulocyte count is 0.5% to 2.5% expressed as a percentage of the total number of RBCs.¹³ The newborn reference interval is 1.5% to 6.0%, but these values change to approximately those of an adult within a few weeks after birth.¹³ An absolute reticulocyte count is determined by multiplying the percent reticulocytes by the RBC count (Table 16.1). The reference interval for the absolute reticulocyte count is 20 to 115 × 10⁹/L, based on an adult RBC count within the reference interval (inside front cover).^4,7 A patient with a severe anemia may seem to be producing increased numbers of reticulocytes if only the percentage is considered. For example, an adult patient with 1.5 × 10¹²/L RBCs and 3% reticulocytes has an absolute reticulocyte count of 45 × 10⁹/L. The percentage of reticulocytes exceeds the reference interval, but the absolute reticulocyte count is within the reference interval. For the degree of anemia, however, both these results are inappropriately low. In other words, production of reticulocytes within the reference interval is inadequate to compensate for an RBC count that is approximately one-third of normal.

Two successive corrections are made to the reticulocyte count to obtain a better representation of RBC production. First, to obtain a corrected reticulocyte count, one corrects for the degree of anemia by multiplying the reticulocyte percentage by the patient’s hematocrit and dividing the result by 45 (the average normal hematocrit). If the reticulocytes are released prematurely from the bone marrow and remain in the circulation 2 to 3 days (instead of 1 day), the corrected reticulocyte count must be divided by maturation time to determine the reticulocyte production index (RPI) (Table 16.1). The RPI is a better indication of the rate of RBC production than is the corrected reticulocyte count.⁴ The reticulocyte count and derivation of RPI is discussed in Chapter 11.

In addition, state-of-the-art automated blood cell analyzers determine the fraction of immature reticulocytes among the total circulating reticulocytes, called the immature reticulocyte fraction (IRF). The IRF is helpful in assessing early bone marrow response after treatment for anemia and is covered in Chapter 12.

Analysis of the reticulocyte count plays a crucial role in determining whether an anemia is due to an RBC production defect or to premature hemolysis and shortened survival defect. If there is shortened RBC survival, as in the hemolytic anemias, the bone marrow tries to compensate by increasing RBC production to release more reticulocytes into the peripheral circulation. Although an increased reticulocyte count is a hallmark of the hemolytic anemias, it can also be observed after acute blood loss (Chapter 20).^4,7,8 Chronic blood loss, on the other hand, does not lead to an appropriate increase in the reticulocyte count, but rather leads to iron deficiency and a subsequent low reticulocyte count. Thus an inappropriately low reticulocyte count results from decreased production of normal RBCs, as a result of either insufficient or ineffective erythropoiesis.

An important component in the evaluation of an anemia is examination of the peripheral blood film, with particular attention to RBC diameter, shape, color, and inclusions. The peripheral blood film also serves to verify the results produced by automated analyzers. Normal RBCs on a Wright-stained blood film are nearly uniform, ranging from 7 to 8 μm in diameter. Small or microcytic cells are less than 6 μm in diameter, and large or macrocytic RBCs are greater than 8 μm in diameter. Certain shape abnormalities of diagnostic value (such as sickle cells, spherocytes, schistocytes, and oval macrocytes) and RBC inclusions (such as malarial parasites, basophilic stippling, and Howell-Jolly bodies) can be detected only by studying the RBCs on a peripheral blood film (Tables 16.2 and 16.3). Examples of abnormal shapes and inclusions are provided in Figure 16.1.

Finally, a review of the white blood cells and platelets may help show that a more generalized bone marrow problem is leading to the anemia. For example, hypersegmented neutrophils can be seen in vitamin B₁₂ or folate deficiency, whereas blast cells and decreased platelets may be an indication of acute leukemia. Chapter 13 contains a complete discussion of the peripheral blood film evaluation. Information from the blood film examination always complements the data from the automated blood cell analyzer.

The cause of many anemias can be determined from the history, physical examination, and results of laboratory tests on peripheral blood. When the cause cannot be determined, however, or the differential diagnosis remains broad, bone marrow aspiration and biopsy may help in establishing the cause of anemia.^4,8 Bone marrow examination is indicated for a patient with an unexplained anemia associated with or without other cytopenias, fever of unknown origin, or suspected hematologic neoplasm. Bone marrow examination evaluates hematopoiesis and can determine whether there is an infiltration of abnormal cells into the bone marrow. Important findings in bone marrow that can point to the underlying cause of the anemia include abnormal cellularity (e.g., hypocellularity in aplastic anemia); evidence of ineffective erythropoiesis and megaloblastic changes (e.g., folate/vitamin B₁₂ deficiency or myelodysplastic syndromes); lack of iron on iron stains of bone marrow (the gold standard for diagnosis of iron deficiency); and the presence of granulomata, fibrosis, infectious agents, and tumor cells that may be inhibiting normal erythropoiesis. Chapter 14 discusses bone marrow procedures and bone marrow examination in detail.

Other tests that can assist in the diagnosis of anemia can be performed on a bone marrow specimen as well, including immunophenotyping of membrane antigens by flow cytometry (Chapter 28), cytogenetic studies (Chapter 30), and molecular analysis to detect specific genetic mutations and chromosome abnormalities in leukemia cells (Chapter 29).

Other laboratory tests that can assist in establishing the cause of anemia include routine urinalysis (to detect hemoglobinuria or an increase in urobilinogen) with a microscopic examination (to detect hematuria or hemosiderin) and analysis of stool (to detect occult blood or intestinal parasites). Also, certain chemistry studies are very useful, such as serum haptoglobin, lactate dehydrogenase, and unconjugated bilirubin (to detect excessive hemolysis) and renal and hepatic function tests. With more patients having undergone gastric bypass surgery for obesity, certain rare deficiencies such as insufficient copper have become more common as another nutritional deficiency that can cause anemia.¹⁴

After the hematologic laboratory studies are completed, the anemia may be classified based on reticulocyte count, MCV, and peripheral blood film findings. Iron studies (including serum iron, total iron-binding capacity, transferrin saturation, and serum ferritin) are valuable if an inappropriately low reticulocyte count and a microcytic anemia are present. Serum vitamin B₁₂ and serum folate assays are helpful in investigating a macrocytic anemia with a low reticulocyte count, whereas a direct antiglobulin test can differentiate autoimmune hemolytic anemias from other hemolytic anemias. Because of the numerous potential etiologies of anemia, the specific cause needs to be determined to initiate appropriate therapy.¹¹

The MCV is an extremely important tool and is key in the morphologic classification of anemia. Microcytic anemias are characterized by an MCV of less than 80 fL with small RBCs (less than 6 μm in diameter). Microcytosis is often associated with hypochromia (increased central pallor in RBCs) and an MCHC of less than 32 g/dL. Microcytic anemias are caused by conditions that result in reduced hemoglobin synthesis. Heme synthesis is diminished in iron deficiency, iron sequestration (chronic inflammatory states), and defective protoporphyrin synthesis (sideroblastic anemia, lead poisoning). Globin chain synthesis is insufficient or defective in thalassemia and in Hb E disease. Iron deficiency is the most common cause of microcytic anemia; the low iron level is insufficient for maintaining normal erythropoiesis. Although iron deficiency anemia is characterized by abnormal iron studies, the early stages of iron deficiency do not result in microcytosis or anemia and are manifested only by reduced iron stores. The causes of iron deficiency vary in infants, children, adolescents, and adults, and it is imperative to find the cause before beginning treatment (Chapter 17).

Macrocytic anemias are characterized by an MCV greater than 100 fL with large RBCs (greater than 8 μm in diameter). Macrocytic anemias arise from conditions that result in megaloblastic or nonmegaloblastic red cell development in bone marrow. Megaloblastic anemias are caused by conditions that impair synthesis of DNA, such as vitamin B₁₂ and folate deficiency or myelodysplasia. Nuclear maturation lags behind cytoplasmic development as a result of the impaired DNA synthesis. This asynchrony between nuclear and cytoplasmic development results in larger cells. All cells of the body are ultimately affected by the defective production of DNA (Chapter 18). Pernicious anemia is one cause of vitamin B₁₂ deficiency, whereas pregnancy with increased requirements is a leading cause of folate deficiency. Megaloblastic anemia is characterized by oval macrocytes and hypersegmented neutrophils in the peripheral blood and by megaloblasts or large nucleated erythroid precursors in the bone marrow. The MCV in megaloblastic anemia can be markedly increased (up to 150 fL), but modest increases (100 to 115 fL) are most common.

Nonmegaloblastic forms of macrocytic anemias are also characterized by large RBCs, but in contrast to megaloblastic anemias, they are typically related to membrane changes caused by disruption of the cholesterol-to-phospholipid ratio. These macrocytic cells are mostly round, and the erythroid precursors in the bone marrow do not display megaloblastic changes. Macrocytic anemias are often seen in patients with chronic liver disease, alcohol abuse, and bone marrow failure. It is rare for the MCV to be greater than 115 fL in nonmegaloblastic anemias.

Normocytic anemias are characterized by an MCV in the range of 80 to 100 fL. The RBC morphology on the peripheral blood film must be examined to rule out a dimorphic population of microcytes and macrocytes that can yield a normal MCV. The presence of a dimorphic population can also be verified by observing a bimodal distribution on the RBC histogram produced by an automated blood cell analyzer (Chapters 12 and 13). Some normocytic anemias develop as a result of the premature destruction and shortened survival of RBCs (hemolytic anemias), and they are characterized by an elevated reticulocyte count. The hemolytic anemias can be further divided into those that result from intrinsic causes (membrane defects, hemoglobinopathies, and enzyme deficiencies) and those that result from extrinsic causes (immune and nonimmune RBC injury). A direct antiglobulin test helps differentiate immune-mediated RBC destruction from other causes of hemolysis. In the hemolytic anemias, reviewing the peripheral blood film provides vital information for determining the cause of the hemolysis (Tables 16.2 and 16.3 and Figure 16.1). Hemolytic anemias are discussed in Chapters 20 to 24.

Other normocytic anemias develop as a result of a decreased production of RBCs and are characterized by a decreased reticulocyte count (Chapter 19). Figure 16.2 presents an algorithm for initial morphologic classification of anemia based on the MCV.

Morphologic classification of anemias and the reticulocyte count

The absolute reticulocyte count is useful in initially classifying anemias into the categories of decreased or ineffective RBC production (decreased reticulocyte count) and excessive RBC loss (increased reticulocyte count). Using the morphologic classification in the first category, when the reticulocyte count is decreased, the MCV can further classify the anemia into three subgroups: normocytic anemias, microcytic anemias, and macrocytic anemias. The excessive RBC loss category includes acute hemorrhage and the hemolytic anemias with shortened RBC survival. Figure 16.3 presents an algorithm that illustrates how anemias can be classified based on the absolute reticulocyte count and MCV.^4,7

In a pathophysiologic classification of anemia, related conditions are grouped by the mechanism causing the anemia. In this classification scheme, the anemias caused by decreased RBC production have inappropriately low reticulocyte counts (e.g., disorders of DNA synthesis and aplastic anemia) and are distinguished from other anemias caused by increased RBC destruction (intrinsic and extrinsic abnormalities of RBCs) or acute blood loss, which have increased reticulocyte counts. Some anemias have more than one pathophysiologic mechanism. Box 16.2 presents a pathophysiologic classification of anemia based on the causes of the abnormality and gives one or more examples of an anemia in each classification.