Case Study

After studying the material in this chapter, the reader should be able to respond to the following case study:

A 45-year-old female phoned her physician and complained of fatigue, shortness of breath on exertion, and general malaise. She requested some “B₁₂ shots” to make her feel better. The physician asked the patient to schedule an appointment so that she could determine the cause of the symptoms before offering treatment. The hematocrit performed in the office was 20%. The physician then requested additional laboratory tests, including a CBC with a peripheral blood film examination and a reticulocyte count.

Red blood cells (RBCs) perform the vital physiologic function of oxygen delivery to the tissues. The hemoglobin within the erythrocyte has the remarkable capacity to bind oxygen in the lungs and then release it appropriately in the tissues.¹ The term anemia is derived from the Greek word anaimia, meaning “without blood.”² A decrease in the number of RBCs, or the amount of hemoglobin in the RBCs, results in decreased oxygen delivery and subsequent tissue hypoxia. Anemia is a commonly encountered condition affecting an estimated 1.62 billion people worldwide.³ Anemia should not be thought of as a disease, but rather as a manifestation of other underlying disease processes.^4,5 Therefore, the cause of all anemias should be thoroughly investigated. This chapter provides an overview of the diagnosis, mechanisms, and classification of anemia. In the following chapters, each anemia is discussed in detail.

Definition of Anemia

A functional definition of anemia is a decrease in the oxygen-carrying capacity of the blood. It can arise if there is insufficient hemoglobin or the hemoglobin is nonfunctional. The former is the more frequent cause.

Anemia is defined operationally as a reduction, from the baseline value, in the total number of RBCs, amount of circulating hemoglobin, and RBC mass for a particular patient. In practice, this definition is not applicable, because a patient’s baseline value is rarely known.5,6 A more conventional definition is a decrease in RBCs, hemoglobin, and hematocrit below the reference range for healthy individuals of the same age, sex, and race, under similar environmental conditions.^4–8 Problems with this conventional definition may occur for several reasons. The reference ranges are derived from large pools of “normal” individuals; however, the definition of normal is different for each of these data sets. This has led to the development of different reference ranges, depending on which pool of individuals was used. Furthermore, these pools of individuals lack the heterogeneity required to be universally applied to all the different populations.⁶

Examples of hematologic reference ranges for the adult and pediatric populations are included on the inside cover of this text. They are listed according to age and sex, but race, environmental, and laboratory factors can also influence the values. Each laboratory must determine its own reference ranges based on its particular instrumentation, the methods used, and the demographics and environment of its patient population. For the purpose of the discussion in this chapter, a patient is considered anemic if the hemoglobin value falls below those listed in these tables.

Clinical Findings

The history and physical examination are important components in making a clinical diagnosis of anemia. The classic symptoms associated with anemia are fatigue and shortness of breath. If oxygen delivery is decreased, then patients will not have enough energy to perform their daily functions. Obtaining a good history requires carefully questioning the patient, particularly with regard to diet, drug ingestion, exposure to chemicals, occupation, hobbies, travel, bleeding history, ethnic group, family history of disease, neurologic symptoms, previous medication, jaundice, and various underlying diseases that produce anemia.4,7–9 Although inquiry in these areas can reveal common conditions that can lead to anemia, there are numerous other possibilities as well. Therefore, a thorough discussion is required to elicit any potential cause of the anemia. For example, iron deficiency can lead to an interesting symptom called pica.¹⁰ Patients with pica have cravings for unusual substances such as ice (pagophagia), cornstarch, or clay. Alternatively, individuals with anemia may be asymptomatic, as can be seen in mild or slowly progressive anemias.

Certain features should be evaluated closely during the physical examination to provide clues to hematologic disorders, such as skin (for pallor, jaundice, petechiae), eyes (for hemorrhage), and mouth (for mucosal bleeding). The examination should also look for sternal tenderness, lymphadenopathy, cardiac murmurs, splenomegaly, and hepatomegaly.4,7–9 Jaundice is important for the assessment of anemia, because it may be due to increased RBC destruction, which suggests a hemolytic component to the anemia. Measuring vital signs is also a crucial component of the physical evaluation. Patients experiencing a rapid fall in hemoglobin concentration typically have tachycardia (fast heart rate), whereas if the anemia is long-standing, the heart rate may be normal due to the body’s ability to compensate for the anemia.

Moderate anemias (hemoglobin concentration of 7 to 10 g/dL) may not produce clinical signs or symptoms if the onset of anemia is slow.⁴ Depending on the patient’s age and cardiovascular state, however, moderate anemias may be associated with pallor of conjunctivae and nail beds, dyspnea, vertigo, headache, muscle weakness, lethargy, and other symptoms.^4,7–9 Severe anemias (hemoglobin concentration of less than 7 g/dL) usually produce tachycardia, hypotension, and other symptoms of volume loss, in addition to the symptoms listed earlier. The severity of the anemia is gauged by the degree of reduction in RBC mass, cardiopulmonary adaptation, and the rapidity of progression of the anemia.⁴

Physiologic Adaptations

Reduced delivery of oxygen to tissues caused by reduced hemoglobin causes an increase in erythropoietin secretion by the kidneys. Erythropoietin stimulates the RBC precursors in the bone marrow, which leads to the release of more RBCs into the circulation (see Chapter 8). With persistent anemia, the body implements physiologic adaptations to increase the oxygen-carrying capacity of a reduced amount of hemoglobin. Heart rate, respiratory rate, and cardiac output are increased for a more rapid delivery of oxygenated blood to tissues. In addition, the tissue hypoxia triggers an increase in RBC 2,3-bisphosphoglycerate that shifts the oxygen dissociation curve to the right (decreased oxygen affinity of hemoglobin) and results in increased delivery of oxygen to tissues (see Chapter 10).¹¹ This is a significant mechanism in chronic anemias that enables patients with low levels of hemoglobin to remain relatively asymptomatic. With persistent and severe anemia, however, the strain on the heart can ultimately lead to cardiac failure.

Mechanisms of Anemia

The life span of the RBC in the circulation is about 120 days. In a healthy individual with no anemia, each day approximately 1% of the RBCs are removed from circulation due to senescence, but the bone marrow continuously produces RBCs to replace those lost. Hematopoietic stem cells develop into erythroid precursor cells, and the bone marrow appropriately releases reticulocytes that mature into RBCs in the peripheral circulation. Adequate RBC production requires several nutritional factors, such as iron, vitamin B₁₂, and folate. Globin synthesis also must function normally. In conditions with excessive bleeding or hemolysis, the bone marrow must increase RBC production to compensate for the increased RBC loss. Therefore, the maintenance of a stable hemoglobin concentration requires the production of functionally normal RBCs in sufficient numbers to replace the amount lost.4,7,8

To detect the presence of anemia, the medical laboratory professional performs a complete blood count (CBC) using an automated hematology analyzer to determine the RBC count, hemoglobin concentration, hematocrit, RBC indices, white blood cell (WBC) count, and platelet count. The RBC indices include the mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) (see Chapter 14).¹³ The most important of these indices is the MCV, a measure of the average RBC volume in femtoliters (fL). Reference ranges for these determinations are listed on the inside front cover of the text. Automated hematology analyzers also provide an RBC histogram and the red blood cell distribution width (RDW). A relative and absolute reticulocyte count, described subsequently, should be performed for every patient when anemia is found. Automated analyzers are available to perform reticulocyte counts with greater accuracy and precision than manual counting methods.

The RBC histogram is an RBC volume frequency distribution curve with the relative number of cells plotted on the ordinate and RBC volume in femtoliters on the abscissa. With a normal population of RBCs, the distribution is approximately gaussian. Abnormalities include a shift in the curve to the left (microcytosis) or to the right (macrocytosis), and a widening of the curve caused by a greater variation of RBC volume about the mean or by the presence of two populations of RBCs with different volumes (anisocytosis). The histogram complements the peripheral blood film examination in identifying variant RBC populations.¹² (A discussion of histograms with examples can be found in Chapter 39.)

The 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 correlates with anisocytosis 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. The usefulness of the RDW is discussed later.

Peripheral Blood Film Examination

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 as a quality control to verify the results produced by automated analyzers. Normal RBCs on a Wright-stained blood film are nearly uniform, ranging from 6 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 18-2 and 18-3). Examples of abnormal shapes and inclusions are provided in Figure 18-1.

TABLE 18-2

Description of Red Blood Cell (RBC) Abnormalities and Commonly Associated Disease States

RBC Abnormality	Cell Description	Commonly Associated Disease States
Anisocytosis	Abnormal variation in RBC volume or diameter	Hemolytic, megaloblastic, iron deficiency anemia
Macrocyte	Large RBC (>8 µm in diameter), MCV >100 fL	Megaloblastic anemia Myelodysplastic syndrome Chronic liver disease Bone marrow failure Reticulocytosis
Oval macrocyte	Large oval RBC	Megaloblastic anemia
Microcyte	Small RBC (<6 µm in diameter), MCV <80 fL	Iron deficiency anemia Anemia of chronic inflammation Sideroblastic anemia Thalassemia HbE disease and trait
Poikilocytosis	Abnormal variation in RBC shape	Severe anemia Certain shapes helpful diagnostically
Spherocyte	Small, round, dense RBC with no central pallor	Hereditary spherocytosis Immune hemolytic anemia Extensive burns (along with schistocytes)
Elliptocyte, ovalocyte	Elliptical (cigar-shaped), oval (egg-shaped), RBC	Hereditary elliptocytosis or ovalocytosis Iron deficiency anemia Thalassemia major Myelophthisic anemias
Stomatocyte	RBC with slitlike area of central pallor	Hereditary stomatocytosis Rh deficiency syndrome Acquired stomatocytosis (liver disease, alcoholism) Artifact
Sickle cell	Thin, dense, elongated RBC pointed at each end; may be curved	Sickle cell anemia Sickle cell–β-thalassemia
Hb C crystal	Hexagonal crystal of dense hemoglobin formed within the RBC membrane	Hb C disease
Hb SC crystal	Fingerlike or quartzlike crystal of dense hemoglobin protruding from the RBC membrane	Hb SC disease
Target cell (codocyte)	RBC with hemoglobin concentrated in the center and around the periphery resembling a target	Liver disease Hemoglobinopathies Thalassemia
Schistocyte (schizocyte)	Fragmented RBC due to rupture in the peripheral circulation	Microangiopathic hemolytic anemia* (along with microspherocytes) Traumatic cardiac hemolysis Extensive burns (along with microspherocytes)
Helmet cell (keratocyte)	RBC fragment in shape of a helmet	Same as schistocyte
Folded cell	RBC with membrane folded over	Hb C disease Hb SC disease
Acanthocyte (spur cell)	Small, dense RBC with few irregularly spaced projections of varying length	Severe liver disease (spur cell anemia) Neuroacanthocytosis (abetalipoproteinemia, McLeod syndrome)
Burr cell (echinocyte)	RBC with blunt or pointed, short projections that are usually evenly spaced over the surface of cell; present in all fields of blood film but in variable numbers per field†	Uremia Pyruvate kinase deficiency
Teardrop cell (dacryocyte)	RBC with a single pointed extension resembling a teardrop or pear	Primary myelofibrosis Myelophthisic anemia Thalassemia Megaloblastic anemia

Hb, Hemoglobin; MCV, mean cell volume.

^*Such as thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, disseminated intravascular coagulation.

^†Cells with similar morphology that are unevenly distributed in a blood film (not present in all fields) are likely due to a drying artifact in blood film preparation; these artifacts are sometimes called crenated RBCs.

TABLE 18-3

Erythrocyte Inclusions: Description, Composition, and Some Commonly Associated Disease States

Inclusion	Appearance in Supravital Stain	Appearance in Wright Stain	Inclusion Composed of	Associated Diseases/Conditions
Diffuse basophilia	Granules and filaments	Bluish tinge throughout cytoplasm; also called polychromasia	RNA	Hemolytic anemia After treatment for iron, vitamin B12, or folate deficiency
Basophilic stippling (punctate basophilia)	Granules and filaments	Blue-purple granules distributed throughout cytoplasm	Precipitated RNA	Lead poisoning Thalassemia Hemoglobinopathies Abnormal heme synthesis
Howell-Jolly body	Dense, round, granule	Dense, round, blue or purple granule; usually one per cell; occasionally multiple	DNA (nuclear fragment)	Hyposplenism After splenectomy Megaloblastic anemia Hemolytic anemia
Heinz body	Round granule attached to inner membrane	Not visible	Denatured hemoglobin	Glucose-6-phosphate dehydrogenase deficiency Unstable hemoglobins Oxidant drugs/chemicals
Pappenheimer bodies*	Clusters of small granules	Clusters of small, light blue granules, often near periphery of cell	Iron	Sideroblastic anemia Hemoglobinopathies Hyposplenism Megaloblastic anemia
Cabot ring	Rings or figure-eights	Blue rings or figure-eights	Remnant of mitotic spindle	Megaloblastic anemia Myelodysplastic syndromes
Hb H	Fine, evenly dispersed granules	Not visible	Precipitate of β chains of hemoglobin	Hb H disease

Hb, Hemoglobin.

^*Blue (siderotic) granules observed in Prussian blue stain.

Finally, a review of the WBCs 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. (See Chapter 15 for a complete discussion of the peripheral blood film evaluation.) Additional information from the blood film examination always complements the data from the automated hematology analyzer.

Approach to Evaluating Anemias

The approach to the patient with anemia begins with taking a complete history and performing a physical examination.4,5,7–9 For example, new-onset fatigue and shortness of breath suggest an acute drop in the hemoglobin concentration, whereas a lack of symptoms suggests a long-standing or congenital condition. A strict vegetarian may not be getting enough vitamin B₁₂ in the diet, whereas an individual with alcoholism may not be getting enough folate. A large spleen may be an indication of hereditary spherocytosis, whereas a stool positive for occult blood may indicate iron deficiency. The complete history and physical examination can yield information to narrow the possible cause or causes of the anemia and thus lead to a more rational approach to ordering the appropriate diagnostic tests.

The first step in the laboratory diagnosis of anemia is detecting its presence by the accurate measurement of the hemoglobin, hematocrit, and RBC count and comparison of these values with the reference range for healthy individuals of the same age, sex, race, and environment. Knowledge of previous hematologic results is often extremely valuable. A reduction of 10% or more in hematologic values may be the first clue that an abnormal condition may be present.4,6,15

There are numerous causes of anemia, so a rational algorithm to evaluate this condition is required. Several of the tests already discussed help guide the approach to evaluating patients with anemia, including the CBC, reticulocyte count, RBC indices (particularly the MCV), and examination of the peripheral blood film.

Reticulocyte Count and Anemia Classification

The absolute reticulocyte count is useful in initially classifying anemias into the categories of decreased RBC production (decreased reticulocyte count) and shortened RBC survival (increased reticulocyte count). When the reticulocyte count is decreased, the MCV can further classify the anemia into three subgroups: (1) normocytic anemias, (2) microcytic anemias, and (3) macrocytic anemias. Figure 18-2 presents an algorithm that illustrates how anemias can be evaluated based on the reticulocyte count and MCV.

Mean Cell Volume and Anemia Classification

Microcytic anemia is characterized by an MCV of less than 80 fL with small RBCs (less than 6 µm in diameter). Hypochromia, characterized by an MCHC of less than 32 g/dL and increased central pallor in the RBCs, may accompany the microcytosis. Microcytic anemias are caused by conditions that result in reduced hemoglobin synthesis: iron deficiency, inability to utilize iron (chronic inflammatory states), globin synthesis defect (thalassemia), and heme synthesis defect (sideroblastic anemia, lead poisoning). The most common microcytic anemia results from an iron level that is insufficient for maintaining normal erythropoiesis; it is characterized by abnormal results on iron studies. Early iron deficiency is manifested only by reduced iron stores without anemia or microcytosis. The causes of iron deficiency vary in infants, children, adolescents, and adults, and it is imperative to find the cause before beginning treatment (see Chapter 19).

Macrocytic anemia is characterized by an MCV greater than 100 fL with large RBCs (greater than 8 µm in diameter). Macrocytic anemias may be megaloblastic or nonmegaloblastic. Megaloblastic anemias are caused by conditions that impair synthesis of deoxyribonucleic acid (DNA), such as vitamin B₁₂ and folate deficiency or myelodysplasia. Nuclear maturation lags behind cytoplasmic development as a result of 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 (see Chapter 20). Pernicious anemia is one cause of vitamin B₁₂ deficiency, whereas malabsorption secondary to inflammatory bowel disease is one cause of folate deficiency. A megaloblastic anemia is characterized by oval macrocytes and hypersegmented neutrophils in the peripheral blood and by megaloblasts or large nucleated RBC 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) occur as well.

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

Normocytic anemia is 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 CBC instrument (see Chapter 39). Some normocytic anemias develop due to the premature destruction and shortened survival of RBCs (hemolytic anemias), and they are characterized by an elevated reticulocyte count. Other normocytic anemias develop due to a decreased production of RBCs and are characterized by a decreased reticulocyte count. Figure 18-3 presents an algorithm for initial evaluation of anemia based on the MCV.

Summary

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review Questions