Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 19. Anemias: Red blood cell morphology and approach to diagnosis

Naveen Manchanda*

OUTLINE

Definition of Anemia

Patient History and Clinical Findings

Physiologic Adaptations

Mechanisms of Anemia

Ineffective and Insufficient Erythropoiesis

Blood Loss and Hemolysis

Laboratory Diagnosis of Anemia

Complete Blood Count with Red Blood Cell Indices

Reticulocyte Count

Peripheral Blood Film Examination and Red Blood Cell Morphology

Bone Marrow Examination

Other Laboratory Tests

Approach to Evaluating Anemias

Morphologic Classification of Anemia Based on Mean Cell Volume

Pathophysiologic Classification of Anemia and the Reticulocyte Count

Objectives

After completion of this chapter, the reader will be able to:

1. Define anemia and recognize laboratory results consistent with anemia.

2. Describe clinical findings in anemia.

3. Discuss the importance of the history and the physical examination in the diagnosis of anemia.

4. Explain how the body adapts to anemia and the symptoms experienced by the patient.

5. Distinguish among effective, ineffective, and insufficient erythropoiesis when given examples.

6. List laboratory procedures that are initially performed for the diagnosis of anemia.

7. Discuss the importance of the reticulocyte count in the evaluation of anemia.

8. Explain the importance of examining the peripheral blood film when investigating the cause of an anemia and distinguish the important findings.

9. Describe variations in red blood cell morphology such as inclusions and changes in shape, volume, or color.

10. Use an algorithm incorporating the absolute reticulocyte count to specify three groups of anemias involving decreased or ineffective red blood cell production and give one example of each.

11. Use an algorithm incorporating the mean cell volume to narrow the differential diagnosis of anemia.

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 “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. A point-of-care hemoglobin determination performed in the office was 9.0 g/dL. The physician then requested additional laboratory tests, including a CBC with a peripheral blood film examination and a reticulocyte count.

1. Why did the physician want the patient to come to the office before she prescribed therapy?

2. How do the mean cell volume and reticulocyte count help determine the classification of the anemia?

3. Why is the examination of the peripheral blood film important in the investigation of an anemia?

Red blood cells (RBCs) perform the vital physiologic function of oxygen delivery to tissues. Hemoglobin within the RBCs 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 hemoglobin concentration or number of RBCs results in decreased oxygen delivery to tissue, resulting in tissue hypoxia. Anemia is a common 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 an underlying disease or deficiency.45 Therefore, causes of anemia should be thoroughly investigated. This chapter provides an overview of the mechanisms, diagnosis, 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 has impaired function. The former is the more frequent cause.

Anemia is defined operationally as a reduction in the hemoglobin content of blood that can be caused by a decrease in RBCs, hemoglobin, and hematocrit below the reference interval for healthy individuals of similar age, sex, and race, under similar environmental conditions.4-8 The reference intervals are derived from large pools of “healthy” individuals; however, the definition of healthy is different for each of these groups. Thus these pools of “healthy” individuals may lack the heterogeneity required to be universally applied to any one of these populations of individuals.6 This fact has led to the development of different reference intervals for individuals of different sex, age, and race.

Examples of hematologic reference intervals 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 intervals 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.

Patient history and clinical findings

The history and physical examination are important components in making a clinical diagnosis of anemia. A decrease in oxygen delivery to tissues decreases the energy available to individuals to perform day-to-day activities. This gives rise to the classic symptoms associated with anemia, fatigue and shortness of breath. To elucidate the reason for a patient’s anemia, one starts by obtaining a good history that requires carefully questioning the patient, particularly with regard to diet, drug ingestion, exposure to chemicals, occupation, hobbies, travel, bleeding history, race or ethnic group, family history of disease, neurologic symptoms, previous medication, previous episodes of jaundice, and various underlying disease processes that result in anemia.47-9 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 petechiae), eyes (for pallor, jaundice, and hemorrhage), and mouth (for mucosal bleeding). The examination should also search for sternal tenderness, lymphadenopathy, cardiac murmurs, splenomegaly, and hepatomegaly.411 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 (discussed below).

Moderate anemias (hemoglobin concentration of 7 to 10 g/dL) may cause pallor of conjunctivae and nail beds but may not produce clinical symptoms if the onset of anemia is slow.4 However, depending on the patient’s age and cardiovascular state, symptoms such as dyspnea, vertigo, headache, muscle weakness, and lethargy can occur.48 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. Thus, severity of the anemia is gauged by the degree of reduction in hemoglobin, cardiopulmonary adaptation, and the rapidity of progression of the anemia.4

Physiologic adaptations

Anemia resulting from acute blood loss, such as with severe hemorrhage, can lead to profound changes in physiological processes that ensure adequate perfusion of vital organs and maintenance of homeostasis. In cases of severe blood loss, such as in trauma, blood volume decreases and hypotension develops, resulting in decreased blood supply to the brain and heart. As an immediate adaptation, there is sympathetic overdrive that results in increasing heart rate, respiratory rate, and cardiac output.478 In severe anemia, blood is preferentially shunted to organs that are key to survival, including the brain, muscle, and heart.478This results in oxygen being preferentially supplied to vital organs even in the presence of reduced oxygen-carrying capacity. In addition, 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 (Chapter 10).812 This is also a significant mechanism in chronic anemias that enables patients with low levels of hemoglobin to remain relatively asymptomatic. Thus with persistent anemia, the body develops physiologic adaptations to increase the oxygen-carrying capacity of a reduced amount of hemoglobin, which improves oxygen delivery to tissue. With persistent and severe anemia, however, the strain on the heart can ultimately lead to cardiac failure.

Reduced delivery of oxygen to tissues caused by reduced hemoglobin concentration elicits 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 circulation78 (Chapter 8).

It should be noted that with rapid blood loss, the hemoglobin and hematocrit may be initially unchanged because there is balanced loss of plasma and cells. However, as the drop in blood volume is compensated for by movement of fluid from the extravascular to the intravascular compartment or by administration of resuscitation fluid, there will be a dilution of RBCs and anemia. summarizes the body’s physiologic adaptations to anemia. Box 19-1

BOX 19-1

Human Body Adaptations to Anemia

Anemia caused by sudden loss of blood volume

The following adaptations occur in minutes to hours:

• Increase in heart rate, respiratory rate, and cardiac output

• Redistribution of blood flow from skin and viscera to heart, brain, and muscle

Anemia caused by slow loss of blood

The following adaptations occur over days to weeks:

• Decrease in hemoglobin-oxygen affinity by increasing the production of 2,3-bisphosphoglycerate

• Increase in erythropoietin production by kidneys

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 differentiate into erythroid (RBC) precursor cells, and the bone marrow releases reticulocytes (immature anucleated RBCs) that mature into RBCs in the peripheral circulation. Adequate RBC production requires several nutritional factors such as iron, vitamin B12, and folate. Globin (polypeptide chain) synthesis must also 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

Ineffective and insufficient erythropoiesis

Erythropoiesis is the term used for marrow erythroid proliferative activity. Normal erythropoiesis occurs in the bone marrow and is under the control of the hormone erythropoietin (produced by the kidney) and other growth factors and cytokines (Chapters 7 and 8).78 When erythropoiesis is effective, the 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 (deficient DNA synthesis due to 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, the peripheral blood hemoglobin is low, which triggers an increase in erythropoietin leading to increased erythropoietic activity. Although the RBC production rate is high, it is ineffective in that many of the defective RBC precursors undergo destruction in the bone marrow. The end result is a decreased number of circulating RBCs resulting in anemia.411

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 due to an autoimmune reaction (aplastic anemia, acquired pure red cell aplasia) or infection (parvovirus B19). Infiltration of the bone marrow with granulomas (sarcoidosis) or malignant cells (acute leukemia) can also suppress erythropoiesis.47

Blood loss and hemolysis

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.478 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.478

Laboratory diagnosis of anemia

Complete blood count with red blood cell indices

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 count, and platelet count. The RBC indices include the mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) (Chapter 14).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 of the text. Automated hematology analyzers also provide the red cell distribution width (RDW), an index of variation of cell volume in a red blood cell population (discussed below). 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 (population of smaller cells or microcytosis) or to the right (larger cell population or macrocytosis). A widening of the curve caused by a greater variation of RBC volume about the mean can occur due to a population of RBCs with different volumes (anisocytosis). The histogram complements the peripheral blood film examination in identifying variable RBC populations. A discussion of histograms with examples can be found in Chapters 15 and 16.

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 an increased RDW 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.

Reticulocyte count

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. The reference interval for the absolute reticulocyte count is 20 to 115 × 109/L, based on an adult RBC count within the reference interval.47 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 is above the reference interval, but the absolute reticulocyte count is within the reference interval. For the degree of anemia, however, both of 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) (). The RPI is a better indication of the rate of RBC production than is the corrected reticulocyte count.Table 19-14 The reticulocyte count and derivation of RPI is discussed in Chapter 14.

TABLE 19-1

Formulas for Reticulocyte Counts and Red Blood Cell Indices

Test

Formula

Adult Reference Interval

Absolute reticulocyte count (× 109/L)

= [reticulocytes (%)/100] × RBC count (× 1012/L)

20–115 × 109/L

Corrected reticulocyte count (%)

= reticulocytes (%) × patient’s HCT (%)/45

Reticulocyte production index (RPI)

= corrected reticulocyte count/maturation time

In anemic patients, RPI should be > 3

Mean cell volume (MCV) (fL)

= HCT (%) × 10/RBC count (× 1012/L)

80–100 fL

Mean cell hemoglobin (MCH) (pg)

= HGB (g/dL) × 10/RBC count (× 1012/L)

26–32 pg

Mean cell hemoglobin concentration (MCHC) (g/dL)

= HGB (g/dL) × 100/HCT (%)

32–36 g/dL

HGB, Hemoglobin; HCT, hematocrit; RBC, red blood cell.

In addition, state-of-the-art automated hematology 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 15.

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 over time in acute blood loss.478 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, due to either insufficient or ineffective erythropoiesis.

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 ( and Tables 19-219-3). Examples of abnormal shapes and inclusions are provided in Figure 19-1.

Image 

FIGURE 19-1 Red blood cells (RBCs): varied RBC shapes and inclusions. Hb, Hemoglobin. Source: (Modified from Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St Louis, 2013, Elsevier, Saunders.)

TABLE 19-2

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/ 

Hb E 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 slit-like 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 quartz-like 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) 

Macroangiopathic hemolytic anemia** 

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

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

** Such as traumatic cardiac hemolysis.

† 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.

Hb, Hemoglobin; MCV, mean cell volume.

TABLE 19-3

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

Dark blue granules and filaments in cytoplasm (seen in reticulocytes)

Bluish tinge throughout cytoplasm; also called polychromasia (seen in polychromatic erythrocytes)

RNA

Hemolytic anemia 

After treatment for iron, vitamin B12, or folate deficiency

Basophilic stippling

Dark blue-purple, fine or coarse punctate granules distributed throughout cytoplasm

Dark blue-purple, fine or coarse punctate granules distributed throughout cytoplasm

Precipitated RNA

Lead poisoning 

Thalassemia 

Hemoglobinopathies 

Megaloblastic anemia 

Myelodysplastic syndrome

Howell-Jolly body

Dark blue-purple dense, round granule; usually one per cell; occasionally multiple

Dark blue-purple dense, round granule; usually one per cell; occasionally multiple

DNA (nuclear fragment)

Hyposplenism 

Postsplenectomy 

Megaloblastic anemia 

Hemolytic anemia 

Thalassemia 

Myelodysplastic syndrome

Heinz body

Round, dark blue-purple granule attached to inner RBC membrane

Not visible

Denatured hemoglobin

Glucose-6-phosphate dehydrogenase deficiency 

Unstable hemoglobins 

Oxidant drugs/chemicals

Pappenheimer bodies***

Irregular clusters of small, light to dark blue granules, often near periphery of cell

Irregular clusters of small, light to dark blue granules, often near periphery of cell

Iron

Sideroblastic anemia 

Hemoglobinopathies 

Thalassemias 

Megaloblastic anemia 

Myelodysplastic syndrome 

Hyposplenism 

Post-splenectomy

Cabot ring

Rings or figure-eights

Blue rings or figure-eights

Remnant of mitotic spindle

Megaloblastic anemia 

Myelodysplastic syndromes

Hb H

Fine, evenly dispersed, dark blue granules; imparts “golf ball” appearance to RBCs

Not visible

Precipitate of β-globin chains of hemoglobin

Hb H disease

* Inclusions of hemoglobin crystals (Hb S, Hb C, Hb SC) are covered in Table 19-2.

** Such as new methylene blue.

*** Stain dark blue and are called siderotic granules when observed in Prussian blue stain.

Hb, Hemoglobin.

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 B12or folate deficiency, whereas blast cells and decreased platelets may be an indication of acute leukemia. Chapter 16 contains a complete discussion of the peripheral blood film evaluation. Information from the blood film examination always complements the data from the automated hematology analyzer.

Bone marrow examination

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, a bone marrow aspiration and biopsy may help in establishing the cause of anemia.48 A 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 malignancy. A bone marrow examination evaluates hematopoiesis and can determine if there is an infiltration of abnormal cells into the bone marrow. Important findings in the 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 syndrome); lack of iron on iron stains of the 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 17 discusses bone marrow procedures and bone marrow examination in detail.

Other tests that can assist in the diagnosis of anemia can be performed on the bone marrow sample as well, including immunophenotyping of membrane antigens by flow cytometry (Chapter 32), cytogenetic studies (Chapter 30), and molecular analysis to detect specific genetic mutations and chromosome abnormalities in leukemia cells (Chapter 31).

Other laboratory tests

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 hemolytic anemias due to other causes. Because of the numerous potential etiologies of anemia, the specific cause needs to be determined to initiate appropriate therapy.11

Approach to evaluating anemias

The approach to the patient with anemia begins with taking a complete history and performing a physical examination.47 For example, new-onset fatigue and shortness of breath suggest an acute drop in the hemoglobin concentration, whereas minimal or lack of symptoms suggests a long-standing condition where adaptive mechanisms have compensated for the drop in hemoglobin. 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. Thus a 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 and cost-effective 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, MCV and RBC count and comparison of these values with the reference interval for healthy individuals of the same age, sex, race, and environment. Knowledge of previous hematologic values is valuable as a reduction of 10% or more in these values may be the first clue that an abnormal condition may be present.4615

There are numerous causes of anemia, so a rational algorithm to initially evaluate this condition utilizing the above-mentioned tests is required. A reticulocyte count and a peripheral blood film examination are of paramount importance in evaluating anemia.

The remainder of this chapter discusses the importance of individual RBC measurements, the MCV, reticulocyte count, and RDW, and how they assist in classifying anemias so as to arrive at a specific diagnosis. Two widely used classification schemes for anemias relate to the morphology of red cells and the pathophysiological condition responsible for the patient’s anemia.

Morphologic classification of anemia based on mean cell volume

The MCV is an extremely important tool and is key in the morphologic classification of anemia. Microcytic anemia is characterized by an MCV of less than 80 fL with small RBCs (less than 6 μm in diameter). Microcytosis is often associated with hypochromia, characterized by an increased central pallor in the 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 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 20).

Macrocytic anemia is 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 the bone marrow. 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 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 21). 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 macrocytic anemias 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 megaloblastic maturation 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 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 hematology analyzer (Chapters 15and 16). 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. The hemolytic anemias can be further divided into those due to intrinsic causes (membrane defects, hemoglobinopathies, and enzyme deficiencies) and those due to extrinsic causes (immune and nonimmune RBC injury). A direct antiglobulin test helps differentiate immune-mediated destruction from the other causes. In the other hemolytic anemias, reviewing the peripheral blood film is vital for determining the cause of the hemolysis (Table 19-2Table 19-3, and Figure 19-1). Hemolytic anemias are discussed in Chapters 23 through 26 [Chapter 23 Chapter 24 Chapter 25 Chapter 26 ].

Other normocytic anemias develop due to a decreased production of RBCs and are characterized by a decreased reticulocyte count. presents an algorithm for initial morphologic classification of anemia based on the MCV.Figure 19-2

Image 

FIGURE 19-2 Algorithm for morphologic classification of anemia based on mean cell volume (MCV). Anemia of chronic liver disease is multifactorial and can be normocytic. ↑, Increased; ↓, decreased; Hb, hemoglobin; N, normal; retic, absolute reticulocyte count.

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 19-3 presents an algorithm that illustrates how anemias can be classified based on the absolute reticulocyte count and MCV.47

Image 

FIGURE 19-3 Algorithm for evaluating causes of anemia based on absolute reticulocyte count and mean cell volume (MCV). The list of anemias contains examples; there are numerous other causes not listed. Anemia of chronic liver disease is multifactorial and can be normocytic. DIC, Disseminated intravascular coagulation; Hb, hemoglobin; HUS, hemolytic uremic syndrome; RBC, red blood cell; TTP, thrombotic thrombocytopenic purpura.

Morphologic classification and the red blood cell distribution width

The RDW can help determine the cause of an anemia when used in conjunction with the MCV. Each of the three MCV categories mentioned previously (normocytic, microcytic, macrocytic) can also be subclassified by the RDW as homogeneous (normal RDW) or heterogeneous (increased or high RDW), according to Bessman and colleagues.716 For example, a decreased MCV with an increased RDW is suggestive of iron deficiency (Table 19-4). This classification is not absolute, however, because there can be an overlap of RDW values among some of the conditions in each MCV category.

TABLE 19-4

Morphologic Classification of Anemia Based on Red Blood Cell Mean Volume (MCV) and Red Blood Cell Distribution Width (RDW)*

 

MEAN CELL VOLUME

 

Decreased

Normal

Increased

RDW Normal

α- or β-thalassemia trait 

Anemia of chronic inflammation 

Hb E disease/trait

Anemia of chronic inflammation 

Anemia of renal disease 

Acute hemorrhage 

Hereditary spherocytosis

Aplastic anemia 

Chronic liver disease 

Alcoholism 

Chemotherapy

RDW Increased

Iron deficiency 

Sickle cell–β-thalassemia

Early iron, folate, or vitamin B12 deficiency 

Mixed deficiency of iron + vitamin B12 or folate 

Sickle cell anemia 

Hb SC disease 

Myelodysplastic syndrome

Folate or vitamin B12 deficiency 

Myelodysplastic syndrome 

Cold agglutinin disease 

Chronic liver disease 

Chemotherapy

* This classification scheme is not absolute because there can be overlap of RDW values among some of the conditions in each MCV category.

Modified from Bessman JD, Gilmer PR, Gardner FH: Improved classification of anemias by MCV and RDW, Am J Clin Pathol 80:324, 1983; and Marks PW: Approach to anemia in the adult and child. In Hoffman R, Benz EJ, Silberstein LE, et al, editors. Hematology: basic principles and practice, ed 6, Philadelphia, 2013, Elsevier, Saunders, p. 423.

Hb, hemoglobin; MCV, mean cell volume; RDW, red blood cell distribution width.

Pathophysiologic classification of anemias and the reticulocyte count

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 blood loss, which have increased reticulocyte counts. Some anemias have more than one pathophysiologic mechanism. Box 19-2 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.

BOX 19-2

Pathophysiologic Classification of Anemias

Anemia caused by decreased production of red blood cells

Hematopoietic stem cell failure: acquired and inherited aplastic anemia

Functional impairment of erythroid precursors:

• Disturbance of DNA synthesis: megaloblastic anemia

• Disturbance of hemoglobin synthesis: iron deficiency anemia, thalassemia, sideroblastic anemia, anemia of chronic inflammation

• Disturbance of proliferation and differentiation of erythroid precursors: anemia of renal failure, anemia associated with marrow infiltration

Anemia caused by increased red blood cell destruction or loss

Intrinsic abnormality

• Membrane defect: hereditary spherocytosis, hereditary elliptocytosis, pyropoikilocytosis, paroxysmal nocturnal hemoglobinuria

• Enzyme deficiency: glucose-6-phosphate dehydrogenase deficiency, pyruvate kinase deficiency

• Globin abnormality: sickle cell anemia, other hemoglobinopathies

Extrinsic abnormality

• Immune causes: warm-type autoimmune hemolytic anemia, cold agglutinin disease, paroxysmal cold hemoglobinuria, hemolytic transfusion reaction, hemolytic disease of the fetus and newborn

• Nonimmune red blood cell injury: microangiopathic hemolytic anemia (thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, HELLP syndrome, disseminated intravascular coagulation), macroangiopathic hemolytic anemia (traumatic cardiac hemolysis), infectious agents (malaria, babesiosis, bartonellosis, clostridial sepsis), other injury (chemicals, drugs, venoms, extensive burns)

• Blood loss: acute blood loss anemia

HELLP, Hemolysis, elevated liver enzymes, and low platelets syndrome.

The list of anemias is not all-inclusive; numerous other conditions are not listed.

Modified from Prchal JT: Clinical manifestations and classification of erythrocyte disorders. In Prchal JT, Kaushansky K, Lichtman MA, et al, editors: Williams hematology, ed 8, New York, 2010, McGraw-Hill. Available at: 6108487. Accessed September 28, 2013.

Summary

• Anemia is defined conventionally as a decrease in RBCs, hemoglobin, and hematocrit below the reference interval for healthy individuals of the same age, sex, and race, under similar environmental conditions.

• Diagnosis of anemia is based on history, physical examination, symptoms, and laboratory test results.

• Many anemias have common manifestations. Careful questioning of the patient may reveal contributing factors, such as diet, medications, occupational hazards, and bleeding history.

• A thorough physical examination is valuable in determining the cause of anemia. Some of the areas that should be evaluated are skin, nail beds, eyes, mucosa, lymph nodes, heart, and size of the spleen and liver.

• Moderate anemias (hemoglobin concentration between 7 and 10 g/dL) may not manifest clinical symptoms if the onset is slow. Severe anemias (hemoglobin concentration of less than 7 g/dL) usually produce pallor, dyspnea, vertigo, headache, weakness, lethargy, hypotension, and tachycardia.

• Laboratory procedures helpful in the initial diagnosis of anemia include the complete blood count (CBC) with RBC indices and the red blood cell distribution width (RDW), reticulocyte count, and examination of the peripheral blood film with emphasis on RBC morphology. Examination of a peripheral blood film is very important in the diagnosis of hemolytic anemias.

• Bone marrow examination is not usually required for diagnosis of anemia but is indicated in cases of unexplained anemia, fever of unknown origin, or suspected hematologic malignancy. Other tests are indicated based on the RBC indices, history, and physical examination, such as serum iron, total iron-binding capacity, and serum ferritin (for microcytic anemias), and serum folate and vitamin B12 (for macrocytic anemias).

• The reticulocyte count and mean cell volume (MCV) play crucial roles in investigation of the cause of an anemia.

• The morphologic classification of anemias is based on the MCV and includes normocytic, microcytic, and macrocytic anemias. The MCV, when combined with the reticulocyte count and the RDW, also can aid in classification of anemia.

• Major subgroups of the pathophysiologic classification include anemias caused by decreased RBC production and those caused by increased RBC destruction or loss. Anemias may have more than one pathophysiologic cause.

• The cause of anemia should be determined before treatment is initiated.

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

Answers can be found in the Appendix.

1. Which of the following patients would be considered anemic with a hemoglobin value of 14.5 g/dL? Refer to reference intervals inside the front cover of this text.

a. An adult man

b. An adult woman

c. A newborn boy

d. A 10-year-old girl

2. Common clinical symptoms of anemia include:

a. Splenomegaly

b. Shortness of breath and fatigue

c. Chills and fever

d. Jaundice and enlarged lymph nodes

3. Which of the following are important to consider in the in the patient’s history when investigating the cause of an anemia?

a. Diet and medications

b. Occupation, hobbies, and travel

c. Bleeding episodes in the patient or in his or her family members

d. All of the above

4. Which one of the following is reduced as an adaptation to long-standing anemia?

a. Heart rate

b. Respiratory rate

c. Oxygen affinity of hemoglobin

d. Volume of blood ejected from the heart with each contraction

5. An autoimmune reaction destroys the hematopoietic stem cells in the bone marrow of a young adult patient, and the amount of active bone marrow, including RBC precursors, is diminished. The RBC precursors that are present are normal in appearance, but there are too few to meet the demand for circulating red blood cells, and anemia develops. The reticulocyte count is low. The mechanism of the anemia would be described as:

a. Effective erythropoiesis

b. Ineffective erythropoiesis

c. Insufficient erythropoiesis

6. What are the initial laboratory tests that are performed for the diagnosis of anemia?

a. CBC, iron studies, and reticulocyte count

b. CBC, reticulocyte count, and peripheral blood film examination

c. Reticulocyte count and serum iron, vitamin B12, and folate assays

d. Bone marrow study, iron studies, and peripheral blood film examination

7. An increase in which one of the following suggests a shortened life span of RBCs and hemolytic anemia?

a. Hemoglobin

b. Hematocrit

c. Reticulocyte count

d. Red cell distribution width

8. Which of the following is detectable only by examination of a peripheral blood film?

a. Microcytosis

b. Anisocytosis

c. Hypochromia

d. Poikilocytosis

9. Schistocytes, ovalocytes, and acanthocytes are examples of abnormal changes in RBC:

a. Volume

b. Shape

c. Inclusions

d. Hemoglobin concentration

10. Refer to Figure 19-3 to determine which one of the following conditions would be included in the differential diagnosis of an anemic adult patient with an absolute reticulocyte count of 20 × 109/L and an MCV of 65 fL.

a. Aplastic anemia

b. Sickle cell anemia

c. Iron deficiency

d. Folate deficiency

11. Which one of the following conditions would be included in the differential diagnosis of an anemic adult patient with an MCV of 125 fL and an RDW of 20% (reference interval 11.5% to 14.5%)? Refer to Table 19-4.

a. Aplastic anemia

b. Sickle cell anemia

c. Iron deficiency

d. Vitamin B12 deficiency

References

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11.  Adamson J. W, Longo D. L. Anemia and polycythemia. Available at: http://www.accessmedicine.com/content.aspx?aID=9113377 In: Longo D. L, Fauci A. S, Kasper D. L, et al. Harrison’s principles of internal medicine. 18th ed. New York : McGraw-Hill 2012 Accessed 28.09.13.

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14.  Halfdanarson T. R, Kumarm N, Li C. Y, et al. Hematologic manifestations of copper deficiency a retrospective review. Eur J Haemotol; 2008; 80:523-531.

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*Ann Bell and Dr. Rakesh Mehta have contributed to the admirable framework and contents of this chapter.