OBJECTIVES

After completing this chapter, the reader should be able to

  • Describe the physiology of blood cell development and bone marrow function

  • Discuss the interpretation and alterations of hemoglobin, hematocrit, and various red blood cell indices in the evaluation of macrocytic, microcytic, and normocytic anemias

  • Describe the significance of abnormal erythrocyte morphology, including sickling, anisocytosis, and nucleated erythrocytes

  • Name the different types of leukocytes and describe their primary functions

  • Calculate the absolute number of various types of leukocytes from the white blood cell count and differential

  • Interpret alterations in the white blood cell count, differential, and CD4 lymphocyte count in acute bacterial infections, parasitic infections, and human immunodeficiency virus infection

  • Identify potential causes of neutropenia and neutrophilia

This chapter reviews the basic functions and expected laboratory values of erythrocytes (red blood cells [RBCs]) and leukocytes (white blood cells [WBCs]). It also discusses, in an introductory manner, selected disorders of these two cellular components of blood. It must be remembered that the ability of laboratory medicine to discriminate between leukocytes is increasing, and many methods considered investigational in this edition may become routine components of blood examination in the future.

PHYSIOLOGY OF BLOOD CELLS AND BONE MARROW

The cellular components of blood are derived from pluripotential stem cells located in the bone marrow that can differentiate into RBCs, WBCs, and platelets (Figure 16-1). Bone marrow is a highly structured and metabolically active organ that normally produces 2.5 billion RBCs, 1 billion granulocytes, and 2.5 billion platelets/kilogram of body weight daily.1 Production can vary greatly from nearly 0 to 5 to 10 times normal. Usually, however, levels of circulating cells remain in a relatively narrow range (Table 16-1).2

TABLE 16-1.

Reference Ranges and Interpretative Comments for Common Hematologic Tests (Typical CBC)

TEST NAME

RANGEa REFERENCE

SI UNITS

COMMENTS

RBC

Men: 4.5–5.9 × 106 cells/μL

Women: 4.1–5.1 × 106 cells/μL

4.5–5.9 × 1012 cells/L

4.1–5.1 × 1012 cells/L

Hgb

Men: 14–17.5 g/dL

Women: 12.3–15.3 g/dL

140–175 g/L or 8.68–10.85 mmol/L

123–153 g/L or 7.63–9.49 mmol/L

Amount of Hgb in given volume of whole blood; indication of oxygen-transport capacity of blood; may be falsely elevated in hyperlipidemia

Hct

Men: 42% to 50%

Women: 36% to 45%

0.42–0.5

0.36–0.45

Percentage volume of blood comprised of RBCs; usually approximately three times Hgb

RBC indices MCV

80–96 fL/cell

80–96 fL/cell

Hct/RBC: average size of RBCs in a specimen; decreased in iron deficiency; increased in vitamin B12 and folate deficiency, cold agglutinins, reticulocytosis, hyperglycemia, and leukemias

MCH

27–33 pg/cell

27–33 pg/cell

Hgb/RBC: average amount of Hgb in RBCs in a specimen; decreased in iron deficiency; increased in vitamin B12 and folate deficiency

MCHC

33.4–35.5 g/dL

334–355 g/L

Hgb/Hct: average concentration of Hgb in RBCs in a specimen; decreased in iron deficiency, increased in hyperlipidemia and cold agglutinins

Reticulocyte count

0.5% to 2.5% of RBCs

0.005–0.025

Immature RBCs; increased in acute blood loss and hemolysis; decreased in untreated iron, vitamin B12, and folate deficiency

RDW

11.5% to 14.5%

0.115–0.145

Measure of variation in RBC volumes (anisocytosis): the larger the width percent, the greater the variation in size of RBCs; increased in early iron deficiency anemia and mixed anemias

WBC count

4.4–11.3 × 103 cells/μL

4.4–11.3 × 109 cells/L

Elevated by neutrophil demargination with exercise, glucocorticoids, epinephrine; decreased with cold agglutinins

Platelet count

150,000–450,000 cells/μL

150–450 × 109 cells/L

Elevated in presence of RBC fragments and microcytic erythrocytes; decreased in presence of large numbers of giant platelets and platelet clumps

MPV

6.8–10 fL

6.8–10 fL

SI = International System of Units.

Source: Adapted with permission from references 7 and 8.
FIGURE 16-1.
FIGURE 16-1.

Schematic diagram of hematopoiesis.

In a fetus and child, blood cell formation or hematopoiesis occurs in the marrow of virtually all bones as well as in liver, spleen, and other visceral organs. With maturation, the task of hematopoiesis ceases in the liver and shifts to flat bones of the axial skeleton, such as the skull, ribs, pelvis, and vertebrae. The long bones, such as the femur and humerus, do not produce a large amount of blood cells in adulthood because the marrow in these bones is gradually replaced by fatty tissue. Radiation directed to large portions of hematopoietic bones, such as in patients treated for cancerous lesions such as bony metastases, can lead to deficient hematopoiesis. Similarly, preparation for a bone marrow transplant may include total body irradiation to destroy the hematopoietic cells of the recipient so that the grafted cells are not destroyed by residual host defenses.

Although most hematopoiesis occurs in the marrow, modern methods of identifying cellular characteristics have demonstrated that pluripotential cells—identified by a cellular expression of the surface marker CD34—also normally circulate in the blood.3

Although most of this chapter discusses laboratory analysis of blood obtained from the vein (peripheral venipuncture), an analysis of the bone marrow itself may be needed to diagnose or monitor various disease states, most commonly leukemias. Bone marrow specimens are usually obtained from the posterior iliac crest of the pelvis or, less commonly, from the sternum. Bone marrow sampling can involve an aspirate, a core biopsy, or both. The biopsy provides the advantage of examining the structure of the marrow stroma as well as the spatial relationship of the various hematopoietic cells.4

Blood pluripotential (stem) cells become increasingly differentiated in the bone marrow until they are committed to develop further into erythrocytes, platelets, or various leukocytes (Figure 16-1). Many regulatory proteins, including colony-stimulating factors, are involved in the differentiation and proliferation phases of hematopoiesis, but their functions and interrelationships are not yet fully understood. In addition to the colony-stimulating factors mentioned previously, proteins that stimulate hematopoiesis include erythropoietin, thrombopoietin, and various interleukins. Inhibitors of hematopoiesis are not as well defined but include interferons and lymphotoxins. When considering the response of neutrophils or erythrocytes to exogenously administered hematopoietic stimulants (eg, filgrastim, epoetin alfa), it is important to recall that normal physiologic hematopoietic regulation is more complex than the effect of one therapeutic protein would suggest. WBC formation involves local production of a combination of signaling proteins by cells of the hematopoietic microenvironment (eg, macrophages, T lymphocytes, osteoblasts, fibroblasts, and endothelial cells). Leukocyte-stimulating proteins, such as granulocyte-colony stimulating factor and granulocyte-macrophage colony-stimulating factor, are normally directed toward adjacent or closely approximated differentiating hematopoietic cells.3 In contrast, renal synthesis of the hormone erythropoietin is increased when oxygen tension in one or both kidneys decreases. Once it is released into the systemic circulation, erythropoietin stimulates erythrocyte precursors in the blood-forming areas of bone marrow.

Committed blood precursor cells undergo further differentiation in the bone marrow until they develop into mature cells. These developmental stages can be identified by differing morphologic or immunochemical staining characteristics. These same imaging techniques are used to identify the developmental phenotype of the cancerous WBCs of leukemia and lymphoma. Generally, only mature cellular forms are found in the circulating blood, and it is from this blood that clinical specimens are usually taken. As discussed later, the presence of immature forms of WBCs or RBCs in the blood typically indicates the presence of a pathologic process.

COMPLETE BLOOD COUNT

The complete blood count (CBC) is a frequently ordered laboratory test. It supplies useful information regarding the concentration of the different cellular and noncellular elements of blood and applies to multiple disorders. CBC is a misnomer because concentrations of cells/microliter, not counts, are measured and reported, and many hematologic tests are not included. Functionally, the CBC can be thought of as a routine or screening blood analysis given several tests are performed.

Most clinical laboratories use an automated method to determine the CBC. Results are usually accurate, reproducible, and rapidly obtained. Numerous measured and calculated values are included in a CBC (Table 16-1). These results traditionally include the following values:

  • Erythrocyte count or RBC

  • Leukocyte count or WBC

  • Hemoglobin (Hgb)

  • Hematocrit (Hct)

  • RBC (Wintrobe) indices: mean cell (corpuscular) volume (MCV), mean Hgb content, mean cell Hgb concentration (MCHC), and RBC distribution width (RDW)

  • Platelet estimate or count and mean platelet volume

  • Reticulocyte count

  • Erythrocyte sedimentation rate (ESR)

When a “CBC with differential” is ordered, the various types of WBCs are also analyzed (see White Blood Cell Count and Differential section). The reliability of the results can be doubtful if (1) the integrity of the specimen is questionable (inappropriate handling or storage) or (2) the specimen contains substances that interfere with the automated analysis. Grossly erroneous results are usually flagged for verification by another method. Manual microscopic review of the blood smear may be used to resolve unusual automated results (and if the counts are lower in specific pathologic or spurious myeloid conditions).1

Note that laboratory value reference ranges vary slightly among laboratories.

Red Blood Cell Count

Normal adult range: men, 4.5 to 5.9 × 106 cells/μL (4.5 to 5.9 × 1012 cells/L); women, 4.1 to 5.1 × 106 cells/μL (4.1 to 5.1 × 1012 cells/L)

The red blood cell (RBC) count is the number of red cells in a given volume of blood. The international unit for reporting blood cells is for a 1-L volume, but it is still common to see values reported in cells/microliter (μL), or less commonly in cells/cubic millimeter (mm3). After puberty, women have slightly lower counts (and Hgb/Hct) than men, partly because of their menstrual blood loss and because of higher concentrations of androgens (an erythropoietic stimulant) in men. The RBC count in all anemias is by definition below the normal range, and this decrease causes a proportionate decrease in Hct and Hgb. In clinical practice, the Hgb and Hct are more commonly used to define the presence or absence of anemia. The reticulocyte is the cell form that precedes the mature RBC or erythrocyte. During the entire maturation process, Hgb is produced, gradually filling the cytoplasm. The reticulocyte does not contain a nucleus but possesses remnants of the nucleus or endoplasmic reticulum. The mature erythrocyte contains neither an organized nucleus nor nucleic acids. Reticulocytes persist in the circulation for 1 to 2 days before maturing into erythrocytes.2,5,6

Mature erythrocytes have a median lifespan of 120 days under normal conditions. They are removed from the circulation by macrophages in the liver, spleen, bone marrow, and other reticuloendothelial organs. The erythrocytes are tested for flexibility, size, and integrity in these organs as the cells pass through areas of osmotic, pH, or hypoxic stress.5

Variability in the size of RBCs is termed anisocytosis, and variation in the normal biconcave disc shape is termed poikilocytosis. Such abnormalities are seen with iron deficiency or periods of increased erythrocyte production and RBC damage.6

White Blood Cell Count

Normal range: 4.4 to 11.3 × 103 cells/μL (4.4 to 11.3 × 109 cells/L)

The white blood cell count is an actual count of the number of leukocytes in a given volume of blood. Unlike RBCs, leukocytes have a nucleus and normally represent five different mature cell types. The various percentages of the five mature and WBC types comprise the WBC differential, which is discussed later in this chapter.

Hemoglobin

Normal range: men 14 to 17.5 g/dL (140 to 175 g/L); women 12.3 to 15.3 g/dL (123 to 153 g/L)

The hemoglobin (Hgb) value is the amount of this metalloporphyrin-protein contained in a given volume (100 mL or 1 L) of whole blood. The Hgb concentration provides a direct indication of the oxygen-transport capacity of the blood. As the major content of the RBCs, Hgb is proportionately low in patients with anemia. Fluid volume must be taken into consideration because Hgb and Hct are sensitive to the volume status of a patient, making the setting of the evaluation paramount in its interpretation (ambulatory versus acute care versus critical care).

Hematocrit

Normal range: men 42% to 50% (0.42 to 0.5); women 36% to 45% (0.36 to 0.45)

The hematocrit (Hct), also known as the packed cell volume, is the percentage volume of blood that is composed of erythrocytes. To manually perform the Hct test, a blood-filled capillary tube is centrifuged to settle the erythrocytes. Then, the percentage volume of the tube that is composed of erythrocytes is calculated.7 The Hct is usually about three times the value of the Hgb, but disproportion can occur when cells are substantially abnormal in size or shape. Like Hgb, Hct is usually low in patients with anemia and is useful in evaluation for surgical procedures and reversal of coagulopathies.

Red Blood Cell Indices

Because the following laboratory tests specifically assess RBC characteristics, they are called RBC indices. These indices, which assess the size and Hgb content of the RBC, may be useful in the evaluation of anemias, polycythemia, and nutritional disorders. The MCV is measured directly, whereas the MCHC and MCH are calculated from the Hgb, MCV, and RBC count using predetermined formulas. Because of its dependence on cell size, MCH is rarely used in clinical practice, whereas the MCHC is sometimes used to assess RBCs for their Hgb concentration and color.

Mean Corpuscular Volume

Normal range: 80 to 96 fL/cell (80 to 96 f L/L SI)

The mean cell (corpuscular) volume (MCV) is an estimate of the average volume of RBCs and is the most clinically useful of the RBC indices. It can be calculated by dividing the Hct by the RBC count, but it is now determined by averaging the directly measured size of thousands of RBCs with modern hemocytometry instruments.

Abnormally large cells have an increased MCV and are called macrocytic. Vitamin B12 and folate deficiency cause the formation of macrocytic erythrocytes, which corresponds to a true increase in MCV. In contrast, a false increase in MCV may be observed when a patient has reticulocytosis, an increase in the number of reticulocytes in the peripheral blood, because reticulocytes are larger than mature erythrocytes.7,8 The MCV may also be falsely increased in hyperglycemia due to osmotic expansion of the erythrocyte. When erythrocytes are mixed with diluting fluid to perform the test, the cells swell because the diluent is relatively hypotonic compared with the patient’s hyperglycemic blood. Abnormally small cells with a decreased MCV are called microcytic. A decrease in the MCV implies some abnormality in Hgb synthesis. The most common cause of microcytosis is iron deficiency.9 Some patients have simultaneous microcytic and macrocytic anemias (eg, iron and folic acid deficiencies), and in those patients, the MCV may not be predictive of the patient’s overall status.

Mean Corpuscular Hemoglobin

Normal range: 27 to 33 pg/cell

The mean cell (corpuscular) hemoglobin (MCH) is a measure of the oxygen-carrying capacity (ie, Hgb) of each cell. It is calculated as the quotient of Hgb/RBC. The presence of Hgb adds color to the erythrocyte and picks up the dyes of RBC stains for microscopic viewing. Cells that have decreased amounts of Hgb are referred to as being hypochromic, such as in iron deficiency.

Mean Corpuscular Hemoglobin Concentration

Normal range: 33.4 to 35.5 g/dL (334 to 355 g/L)

The mean cell (corpuscular) hemoglobin concentration (MCHC) is the Hgb divided by the Hct, and this calculation is usually around 33 g/dL (330 g/L) because the Hct is usually three times the Hgb. Some laboratories do not report the MCH, as the MCHC reports the Hgb per volume of blood rather than per erythrocyte and, therefore, provides a more direct index of the oxygen carrying capacity of the blood. Iron deficiency is the only anemia in which the MCHC is routinely low although it can also be decreased in other disorders of Hgb synthesis.7,8 In this case, RBCs are described as hypochromic (pale). MCHC can be falsely elevated in hyperlipidemia. The Wintrobe indices are averages for the patient’s blood, and normal values may be reported by automated methods, even in the presence of a mixed (normal + abnormal) erythrocyte population.

Red Blood Cell Distribution Width

Normal range: 11.5% to 14.5% (0.115 to 0.145)

The RBC distribution width (RDW) is an indication of the variation in RBC size, termed anisocytosis.8 The RDW is reported as the coefficient of variation of the MCV (standard deviation/mean value). This value is used primarily with other tests to differentiate iron deficiency anemia from thalassemias and to identify the presence of a mixed anemia. The RDW increases in macrocytic anemias and in early iron deficiency, often before other tests show signs of this kind of anemia. However, it is not specific for iron deficiency anemia. Mild forms of thalassemia often are microcytic but have a normal or only slightly elevated RDW.

Platelet Count and Mean Platelet Volume

Normal range: 150,000 to 450,000 cells/μL (150 to 450 × 109 cells/L)

The platelet estimate or count, often included routinely in the CBC with differential, and mean platelet volume are discussed with other coagulation tests in Chapter 17.

Reticulocyte Count

Normal range: 0.5% to 2.5% of RBCs (0.005 to 0.025)

Reticulocytes are almost-mature RBC that contain nuclear fragments. Normally, only a small number of reticulocytes are in the peripheral circulation. When the bone marrow increases the production of RBC, more reticulocytes are released into the peripheral circulation. In anemia, the reticulocyte count or reticulocyte index (RI) reflects not only the level of bone marrow production but also a decline in the total number of mature erythrocytes that normally dilute the reticulocytes. Therefore, the reticulocyte count would double in a person whose bone marrow production is unchanged but whose Hct has fallen from 46% to 23%. The RI corrects for the transient increase in reticulocyte release that may be seen even in hypoproliferative anemias. It is calculated as follows:
RI=measured%reticulocytes×patients Hct/normal Hct

where a normal RI is <3%.7

In persons with anemia secondary to acute blood loss or hemolysis, even the corrected reticulocyte count is increased.5,7 This increase reflects an attempt by the bone marrow to compensate for the lack of circulating erythrocytes by speeding bone marrow production and release of RBCs. In contrast, persons with untreated anemia secondary to iron, folate, or vitamin B12 deficiency are unable to increase their reticulocyte count appropriate to the degree of their anemia. Appropriate treatment of an anemia should be accompanied by an increase in the reticulocyte count, typically in 5 to 7 days.

The reticulocyte count can be useful in identifying drug-induced bone marrow suppression in which the percentage of circulating reticulocytes may be close to zero.

ERYTHROCYTE SEDIMENTATION RATE

Normal range: men 1 to 15 mm/hr; women 1 to 20 mm/hr (increases with age)

Numerous physiologic and disease states are associated with the rate at which erythrocytes settle from blood, termed the erythrocyte sedimentation rate (ESR). Erythrocytes normally settle slowly in plasma but settle rapidly when they aggregate because of electrostatic forces. Each cell normally has a net negative charge and repels other erythrocytes because like charges repel each other. Many plasma proteins are positively charged and are attracted to the surface charge of one or more erythrocytes, thereby promoting erythrocyte aggregation.10 Nonmicrocytic anemia, pregnancy, multiple myeloma, and various inflammatory diseases (including infections) can increase the ESR. Sickle cell disease, high doses of corticosteroids, liver disease, microcytosis, carcinomas, and congestive heart failure can decrease the ESR.7

Although the ESR may be used to confirm a diagnosis supported by other tests, it is rarely used alone for a specific diagnosis. Rather, the ESR is sometimes useful as a nonspecific biomarker for monitoring the activity of inflammatory conditions (eg, temporal [giant cell] arteritis, polymyalgia rheumatica, rheumatoid arthritis, and osteomyelitis).10 The ESR is often higher when the disease is active due to increased amounts of circulating proteins, termed acute phase reactants (eg, fibrinogen), and falls when the intensity of the disease decreases.

The ESR is usually measured using either the Wintrobe or the Westergren method. Anticoagulated blood is diluted and placed in a vertical glass tube of standard size. After 1 hour, the distance from the plasma meniscus down to the top of the erythrocyte column is recorded as the ESR in millimeters per hour.10,11

LABORATORY ASSESSMENT OF ANEMIA

The functions of the erythrocyte are to transport and protect Hgb, the molecule used for oxygen and carbon dioxide transport. Anemia is practically defined by a decrease in either the Hct or the Hgb concentration below the normal range for age and gender. Anemia is not a disease in itself but a manifestation of an underlying disease process. Appropriate treatment of the patient with anemia must include identification and treatment of the underlying cause of the condition. Signs and symptoms of anemia depend on its severity (how low is the Hgb/Hct or H/H) and the rapidity with which it has developed. Severe, acute blood loss results in more dramatic symptoms than an anemia that took months to develop because with chronic loss some compensatory adaptation may occur. Patients with mild anemia are often asymptomatic (ie, absence of pallor, weakness, and fatigue), but severely symptomatic patients may manifest shortness of breath, tachycardia, and palpitations even at rest. The presence of patient signs and symptoms must always be considered when interpreting test results.

Anemia can be caused by decreased production, increased destruction, or loss of RBC.12,13 The first two situations can often be differentiated by the reticulocyte count, which is decreased in the former and increased in the latter. The MCV is commonly used to characterize the possible etiology of anemia. This method is useful because different causes of anemia lead to different erythrocyte morphology. Figure 16-2 outlines this approach. Only the more common causes of anemia are included, but others can be fit into this outline. Other laboratory tests that are useful in differentiating the anemias are described later. Usual laboratory findings are also included in each section (Table 16-2).

FIGURE 16-2.
FIGURE 16-2.

Use of erythrocyte morphology in differential diagnosis of anemia.

TABLE 16-2.

Qualitative Laboratory Findings for Various Types of Anemiaa

VITAMIN B12 DEFICIENCY

FOLATE DEFICIENCY

IRON DEFICIENCY

ACUTE BLOOD LOSS

HEMOLYTIC ANEMIA

ANEMIA OF CHRONIC DISEASE

RBC

Hgb

Hct

MCV

↔↓

MCH

↔↓

MCHC

↔↓

RDW

Reticulocyte count

↔↓

Serum vitamin B12

Serum folate

Serum methylmalonate

Serum homocysteine

Ferritin

Serum iron

TIBC

Transferrin saturation

Serum haptoglobin

Plasma-free Hgb

Autoantibodies

+

aSome tests with no change (↔) are left empty for clarity. Autoantibodies positive for antibody-mediated immune hemolysis. Patients with multiple causes of anemia, such as iron deficiency and inflammation, may have a confusing laboratory picture.

Macrocytic Anemia

Macrocytic anemia is a lowered Hgb value characterized by abnormally enlarged erythrocytes. The two most common causes are vitamin B12 and folic acid deficiencies. Drugs that cause macrocytic anemia mainly interfere with proper use, absorption, and metabolism of these vitamins (Table 16-3) (Minicase 1).

TABLE 16-3.

Examples of Causes of Drug-Induced Macrocytic Anemia

ALTERED FOLATE ABSORPTION

Alcohol

Aminosalicylic acid

Antimalarials

Erythromycin

Estrogen

Oral contraceptives

Ampicillin and other penicillins

Phenytoin

Nitrofurantoin

Tetracyclines

VITAMIN B12 MALABSORPTION

Colchicine

Isoniazid

Metformin

Neomycin

Para-aminosalicylic acid

Proton pump inhibitors

ALTERED PURINE METABOLISM

Allopurinol

Azathioprine

Fludarabine

Cladribine

Mercaptopurine

Methotrexate

Mycophenolate mofetil

Pentostatin

Thioguanine

ALTERED PYRIMIDINE SYNTHESIS

Capecitabine

Cytosine arabinoside

Fluorouracil

Gadolinium

Gemcitabine

Hydroxyurea

Leflunomide

Methotrexate

Mercaptopurine

Nitrous oxide

Teriflunomide

Trimethoprim

VITAMIN B12 INACTIVATION

Nitrous oxide

UNCLEAR MECHANISM

Imatinib

Sunitinib

Source: Adapted with permission from references 1416,18.

Vitamin B12 Deficiency

Vitamin B12 is also known as cobalamin. The normal daily requirement of vitamin B12 is 2 to 5 mcg.14-16 It is stored primarily in the liver, which contains approximately 1 mcg of vitamin/g of liver tissue. Overall, the body has B12 stores of approximately 2,000 to 5,000 mcg. Therefore, if vitamin B12 absorption suddenly ceased in a patient with normal liver stores, several years would pass before any abnormalities occurred because of vitamin deficiency.

Anemia with Increased Mean Cell Volume

Anna B., a 45-year-old woman with alcoholism, is admitted to the hospital because of pneumonia. Her physical exam reveals an emaciated patient with ascites, dyspnea, fever, cough, and weakness. No cyanosis, jaundice, or peripheral edema is evident. Her peripheral neurologic exam is within normal limits, as are her serum electrolytes, urea nitrogen, creatinine, and glucose. The following CBC results are obtained:

TEST NAME

RESULT

REFERENCE RANGE

RBC

3 × 106 cells/μL

4.1–5.1 × 106 cells/μL for women

WBC

4.6 × 103 cells/μL

4.4–11.3 × 103 cells/μL

Hgb

10.3 g/dL

12.3–15.3 g/dL for women

Hct

30.9%

36% to 45% for women

MCV

110.8 fL/cell

80–96 fL/cell

RDW

15.4%

11.5% to 14.5%

Platelets

174,000 cells/μL

150,000–450,000 cells/μL

Neutrophils

68%

45% to 73%

Bands

6%

3% to 5%

Monocytes

11%

2% to 8%

Eosinophils

2%

0% to 4%

Basophils

2%

0% to 1%

Lymphocytes

11%

20% to 40%

QUESTION: What abnormalities are present? What is the likely cause?

DISCUSSION: The patient has anemia, evidenced by the low RBC, Hgb, and Hct. The increased MCV identifies this as a macrocytic anemia. The RDW is elevated, indicating variability in the size of the erythrocytes. These findings are typical of folic acid deficiency, a common finding in persons with alcoholism due to poor nutrition. Folic acid deficiency is more common than vitamin B12 deficiency because body stores of folic acid are not as large. However, vitamin B12 deficiency must also be ruled out as it may arise with or without a concurrent folate deficiency. Vitamin B12 deficiency may arise from poor nutrition but is more commonly caused by disorders such as pernicious anemia. It is critical that both serum folate and vitamin B12 concentrations be measured in this patient to guide appropriate supplementation. Replenishment of folate in a patient with vitamin B12 deficiency may temporarily improve the values of the CBC, but failure to appropriately replenish vitamin B12 can lead to irreversible brain and nerve damage.

The absorption of vitamin B12 is complex, and the mechanisms responsible are still being defined. Vitamin B12 is ingested mostly in meats, eggs, and dairy products. Therefore, strict vegans may develop vitamin B12 deficiency over time if supplements are not ingested. Some supplements have forms of B12, such as those made by the blue-green algae Spirulina, which are active vitamins in bacterial assays but are not active vitamins for humans. Other cobamides structurally related to cobalamin are found in plasma after ingesting other animal and plant-based foods. Only the cobamide with an attached 5,6-dimethylbenzimidazole group is correctly termed cobalamin and is active in humans.14

Dietary B12 is usually bound nonspecifically to food proteins, and gastric acid and pepsin are required to hydrolyze the vitamin from the protein. Aging patients with decreasing stomach acid production may be less able to free vitamin B12 from meat protein. Freed B12 is bound with high affinity to protein R, which is a large protein secreted in saliva. The cobalamin–protein R complex moves to the duodenum, where proteases denature protein R and allow the freed vitamin to bind to intrinsic factor, which is secreted by the parietal cells of the stomach and is resistant to the intestinal proteases. Patients may develop autoantibodies to intrinsic factor and thereby develop vitamin B12 deficiency. The B12–intrinsic factor complex binds to cubulin at the ileal epithelium. The B12 translocates, dissociates, and then enters the circulation bound to transcobalamin, which is largely homologous with intrinsic factor.14 When vitamin B12 deficiency occurs, there are several steps in the absorption of vitamin B12 that may be responsible for the deficiency.

Vitamin B12 deficiency may arise from inadequate intake of the vitamin or from a deficiency of the intrinsic factor required for the effective ileal absorption of the vitamin. Inadequate dietary intake is a rare cause of vitamin B12 deficiency, usually occurring only in vegans who abstain from all animal food, including milk and eggs.15,16 Defective production of intrinsic factor is a common cause of the deficiency.1416 The gastric mucosa can fail to secrete intrinsic factor because of atrophy, especially in elderly persons, due to autoimmune diseases or due to surgical removal of the stomach. Disorders that affect the ileum, such as Crohn disease, also may impair B12 absorption.

Clinical and laboratory diagnosis

Vitamin B12 is necessary for deoxyribonucleic acid (DNA) synthesis in all cells, for the synthesis of neurotransmitters, and for metabolism of homocysteine. Therefore, B12 deficiency leads to signs and symptoms involving many organ systems.14 The most notable symptoms involve the following systems:

  • Gastrointestinal (GI) tract (eg, loss of appetite, smooth and sore tongue, and diarrhea or constipation)

  • Central nervous system (eg, paresthesias in fingers and toes, loss of coordination of legs and feet, tremors, irritability, somnolence, abnormalities of taste and smell and dementia)

  • Hematopoietic system (anemia)

Nuclear maturation retardation occurs in the developing cells in the bone marrow due to slowed DNA synthesis. The morphologic result—cells with an immature and enlarged nuclei (megaloblasts) but a cytoplasm that matures normally—causes mature cells to be larger than normal. The resulting anemia is called a macrocytic, megaloblastic anemia, which has both morphologic characteristics of nuclear maturation retardation.14 Visual inspection of smears of both peripheral blood and bone marrow reveals characteristic megaloblastic changes in the appearance of erythrocytes and WBCs. The development of neutrophils is also affected, which results in large cells with hypersegmentation (more than three nuclear lobes).17 A mild pancytopenia (decreased numbers of all blood elements) also occurs. The usual laboratory test results associated with vitamin B12 deficiency are listed in Table 16-2.

In the past, vitamin B12 concentrations were measured using a microbiologic assay and a cobalamin-dependent organism. The assay has largely been replaced by a competitive displacement assay using radioactive cobalamin and intrinsic factor. Unfortunately, because of cross-reactivity with other cobamides, approximately 5% of patients have cobalamin concentrations that appear to be within the normal range yet can be shown to have hematologic/neurologic signs of deficiency. Metabolic intermediates homocysteine and methylmalonate may be more sensitive indicators of B12 deficiency. In the presence of inadequate B12, these two compounds accumulate because of the cobalamin dependence of their metabolizing enzymes, methionine synthase and methylmalonyl-CoA-mutase, respectively. An elevated methylmalonate and homocysteine concentration in the presence of normal RBC folate is strongly indicative of a pure deficiency of B12.

Historically, the Schilling test, which involves oral administration of radiolabeled B12, was used to determine if impaired absorption is the reason for the cobalamin deficiency. However, with the advent of assays to measure autoantibodies targeting intrinsic factor and/or gastric parietal cells, this test is now rarely used. The availability of intramuscular injections of vitamin B12 obviates the need to specify the defect in B12 absorption, and such injections are favored in patients with impaired B12 absorption, regardless of the cause.

Pernicious anemia is a specific disease associated with B12 deficiency characterized by atrophic gastritis associated with antibodies against intrinsic factor and gastric parietal cells. In addition to causing B12 deficiency, pernicious anemia is associated with gastric cancer. Gastrectomy (removal of all or part of the stomach) can also lead to vitamin B12 deficiency because the procedure removes the production site of intrinsic factor. Achlorhydria from gastrectomy or drugs such as proton pump inhibitors can decrease the release of B12 from food. Defective or deficient absorption of the intrinsic factor–vitamin B12 complex can be caused by inflammatory disease of the small bowel, ileal resection, and bacterial overgrowth in the small bowel.1416 Administration of colchicine, neomycin, and para-aminosalicylic acid can also lead to impaired absorption of vitamin B12 (Table 16-3).15,16,18

Folic Acid Deficiency

Folic acid is also called pteroylglutamic acid. Folates refer to folic acid or reduced forms of folic acid that may have variable numbers of glutamic acid residues attached to the folic acid molecule. The folates present in food are mainly in a polyglutamic acid form and must be hydrolyzed in the intestine to the monoglutamate form to be absorbed efficiently. The liver is the chief storage site. Adult daily requirements are approximately 50 mcg of folic acid, equivalent to about 400 mcg of food folates. Folate stores are limited, and anemia arising from a folate-deficient diet occurs in 4 to 5 months.1416

Inadequate dietary intake is the major cause of folate deficiency. Folates are found in green, leafy vegetables such as spinach, lettuce, and broccoli. Inadequate intake can have numerous causes: alcoholics often have poor nutritional intake of folic acid; certain physiologic states such as pregnancy require an increase in folic acid; malabsorption syndromes (mentioned in the section on vitamin B12) can lead to defective absorption of folic acid; and celiac sprue can lead to folate malabsorption. Patients with chronic hemolysis, such as in sickle cell disease, and patients undergoing hemodialysis also may develop folate deficiency15,16

Certain medications (eg, methotrexate, trimethoprim–sulfamethoxazole, and triamterene) can act as folic acid antagonists by interfering with the conversion of folic acid into its metabolically active form, tetrahydrofolic acid. Phenytoin and phenobarbital administration can interfere with the intestinal absorption or use of folic acid (Table 16-3).15,16,18

Folic acid is required as the intermediate for one-carbon transfers in several biochemical pathways, including the thymidine required for DNA synthesis. After absorption, folate is reduced to tetrahydrofolate, and a carbon in one of several oxidation states is attached for transfer. The formation of methyltetrahydrofolate requires vitamin B12 as a cofactor for the methyl group transfer. Methyltetrahydrofolate is required for the conversion of homocysteine to methionine, which is subsequently used as a methyl donor in many synthetic pathways that include the production of critical neurotransmitters and amino acids.

Clinical and laboratory diagnosis

Because folic acid is necessary for DNA synthesis, a deficiency causes a maturation retardation in the bone marrow similar to that caused by vitamin B12 deficiency. Folic acid deficiency is also characterized by a macrocytic, megaloblastic anemia.1416 However, with folic acid deficiency, pancytopenia does not develop as consistently as it does with vitamin B12 deficiency.

Folate supplementation in patients with a folate deficiency provides folate for the nonmethyl transfer steps that do not require vitamin B12. High doses of folic acid can often, at least partially, reverse megaloblastic anemia in patients with B12 deficiency but do not reverse the neurologic sequelae. Although folate deficiency is more common and easily treated, it is critical to correctly identify the cause of a megaloblastic anemia so that any vitamin B12 deficiency is appropriately treated.

Folate Concentration

Normal range: serum folate 5 to 25 mcg/L (11.33 to 56.65 nmol/L); RBC folate 166 to 640 mcg/L (376.16 to 1450.24 nmol/L)

The folate concentration in both serum and in RBCs is used to assess folate homeostasis. A low serum folate indicates negative folate balance and can be expected to lead to folate deficiency when hepatic folate stores are depleted. Although in most patients the serum folate alone is adequate for assessment, in patients who were recently administered folate supplementation, the RBC folate concentration may be more indicative of folate status.

Microcytic Anemia

Iron Deficiency

Microcytic anemia, or anemia with abnormally small erythrocytes, is most commonly caused by iron deficiency. Decreased MCV is a late indicator of the deficiency (Figure 16-2). Daily requirements are approximately 1 mg of elemental iron for each 1 mL of RBCs produced, so daily iron requirements are approximately 20 to 25 mg for erythropoeisis.1921 Most iron needed within the body is obtained by recycling metabolized Hgb. RBCs have an average lifespan of approximately 120 days. When old or damaged erythrocytes are taken up by macrophages in the liver, spleen, and bone marrow, the Hgb molecule is broken down and iron is extracted and stored with proteins. Only about 5% of the daily requirement (1 mg) is newly absorbed to compensate for losses caused by fecal and urinary excretion, sweat, and desquamated skin (Minicase 2).

Menstruating women require more iron because of increased blood losses. Iron requirements vary among women but average 2 mg/day. Orally ingested iron is absorbed in the GI tract, which should permit just enough iron absorption to prevent excess or deficiency. Typically, 5% to 10% of oral intake is absorbed (normal daily dietary intake: 10 to 20 mg).21

Dietary iron exists primarily in the ferric state. Because ferrous iron is more bioavailable, dietary ferric iron is reduced by gastric acid to ferrous iron. Patients with inadequate gastric acid secretion due to underlying diseases or medications such as proton pump inhibitor may develop iron deficiency due to decreased absorption.21

Recent research indicates that hepatic hepcidin22 is the primary controller of GI iron absorption, with an inverse relationship between hepcidin level and iron absorption. Hepcidin blocks the transmembrane iron transporter ferroportin. Hepcidin levels are low in the presence of iron deficiency and increase with iron therapy. Hepcidin levels are increased in the presence of inflammatory states. Hepcidin is being studied as a biomarker for the diagnosis of iron deficiency to optimize oral iron replacement therapy and predict failure of oral iron therapy.

Anemia and Iron Stores

Denise T. is a 25-year-old woman seen in a community health clinic for a routine checkup. Her family history includes a sister with sickle cell disease. She has not been affected personally but has not been tested to determine her sickling genotype. She describes painful menstrual periods and takes aspirin for them. She also admits to a pica of ingesting cornstarch throughout the day. The following laboratory test results are obtained:

TEST NAME

RESULT

REFERENCE RANGE

RBC

3.3 × 106 cells/μL

4.1–5.1 × 106 cells/μL for women

WBC

5.1 × 103 cells/μL

4.4–11.3 × 103 cells/μL

Hgb

8.3 g/dL

12.3–15.3 g/dL for women

Hct

26%

36% to 45% for women

MCV

78 fL/cell

80–96 fL/cell

RDW

16.1%

11.5% to 14.5%

Platelets

195,000 cells/μL

150,000–450,000 cells/μL

Neutrophils

52%

45% to 73%

Bands

3%

3% to 5%

Monocytes

2%

2% to 8%

Eosinophils

1%

0% to 4%

Basophils

0%

0% to 1%

Lymphocytes

42%

20% to 40%

Serum iron

44 mcg/dL

50–150 mcg/dL

TIBC

451 mcg/dL

250–410 mcg/dL

Transferrin saturation

14%

20% to 50%

Serum ferritin

5.2 mcg/L

10–20 mcg/L

QUESTION: What hematologic abnormalities are apparent from these results?

DISCUSSION: This patient demonstrates an anemia as manifested by the decreased RBC, Hgb, and Hct. Her WBC and platelet counts are normal. The RDW is elevated, indicating increased variability of erythrocyte size (anisocytosis). Because the MCV is low, this microcytic, hypochromic form of anemia is most likely due to iron deficiency. This is corroborated by the iron studies, which indicate a low serum iron and transferrin saturation. Serum ferritin is also decreased, indicating that her iron stores are markedly reduced. The TIBC is increased both because of increased transferrin production and decreased iron available to bind to the protein.

There may be multiple causes of her iron deficiency. Most commonly, the combination of low dietary iron and blood loss from menstruation increases the frequency of iron deficiency anemia in women. An additional possibility is occult blood loss from GI ulcerations caused by aspirin. An exacerbating factor for this woman is her starch pica (craving for unusual food). In addition to the high caloric intake associated with this particular pica, the starch decreases the bioavailability of ingested iron, decreasing the ability of the patient to absorb dietary or supplemental iron. Given the pica, parenteral iron may be considered.

Iron deficiency is usually due to inadequate dietary intake in children and increased iron requirements in adults. Poor dietary intake, especially in situations that require increased iron (eg, pregnancy), is a common cause. Other causes of iron deficiency include the following factors:

  • Blood loss due to excessive menstrual discharge

  • Peptic ulcer disease

  • Hiatal hernia

  • Gastrectomy

  • Gastritis due to the ingestion of alcohol, aspirin, and nonsteroidal antiinflammatory drugs

  • Bacterial overgrowth of the small bowel

  • Inflammatory bowel disease

  • Occult bleeding from GI cancers

  • Starch or clay pica

Ionized, soluble iron is toxic because of its ability to mediate the formation of oxidative species. Iron is therefore bound to proteins both in and outside of cells. Ferritin is the iron-protein storage complex (Figure 16-3). In the normal adult, approximately 500 to 1,500 mg of total body iron is stored as ferritin and 2,500 mg is contained in Hgb.19 When the total quantity of extracted iron exceeds the amount that can be stored as ferritin, the excess iron is stored in an insoluble form called hemosiderin.

FIGURE 16-3.
FIGURE 16-3.
Intake, loss, and recycling of iron and iron storage forms.

The serum ferritin concentration reflects total body iron stores and is the most clinically useful method to evaluate patients for iron deficiency. Because ferritin is an acute phase reactant, serum ferritin concentrations can be increased by critical illness, chronic infections, fever, and inflammatory disorders such as rheumatoid arthritis, hepatitis, and malignancies. The transport of iron in plasma and extracellular fluid occurs with two ferric ions bound to the protein transferrin, which when not binding iron or other metals, is termed apotransferrin. Transferrin binds to specific membrane transferrin receptors where the complex enters the cell and releases the iron. Apotransferrin is released when the apotransferrin-receptor complex returns to the surface of the cell.

The tendency of ferritin to be falsely elevated with inflammatory processes has led to recent interest in using soluble transferrin receptor concentrations as an alternative marker of iron deficiency. The circulating receptor fragment is considered to reflect total body receptor expression and is elevated in times of increased erythropoiesis such as sickle cell anemia, thalassemias, and chronic hemolysis. If such causes of increased erythropoiesis can be excluded, elevated concentrations of circulating transferrin receptor are thought to reflect iron deficiency. The use of transferrin receptor concentrations may help determine if decreased ferritin concentrations are due to iron deficiency or anemia of chronic (inflammatory) disease.19

Clinical and laboratory diagnosis

The first change observed in the development of iron deficiency anemia is a loss of storage iron (hemosiderin). If the deficiency continues, a loss of plasma iron occurs. The decrease in plasma iron stimulates an increase in transferrin synthesis. When enough iron has been depleted such that supplies for erythropoiesis are inadequate, anemia develops. The RDW rises, often before the MCV decreases, to a notable degree. If the iron deficiency persists, the RBCs become smaller than usual (microcytic—low MCV) and not as heavily pigmented as normal RBCs because they contain less Hgb than normal erythrocytes (hypochromic-low mean hemoglobin content and MCHC). Patients present with progressively worsening weakness, fatigue, pallor, shortness of breath, tachycardia, and palpitations. Numbness, tingling, and glossitis also may exist.13,21 Laboratory results for iron deficiency anemia are listed in Table 16-2. With adequate iron therapy, the maximal daily rate of Hgb regeneration is 0.3 g/dL, or approximately 1%/day in Hct.

Serum Ferritin

Normal range: >10 to 200 ng/mL (>10 to 200 mcg/L)

Loss of storage iron (hemosiderin) was traditionally evaluated by iron-stained bone marrow aspirate. Serum ferritin has largely replaced these invasive tests as an indirect measure of iron stores. Serum ferritin concentrations are markedly reduced in iron deficiency anemia (3 to 6 mcg/L) and elevated in the setting of inflammation.

Serum Iron and Total Iron-Binding Capacity

Serum iron normal range: 50 to 150 mcg/dL (9 to 26.9 μmol/L); TIBC normal range: 250 to 410 mcg/dL (44.8 to 73.4 μmol/L); transferrin saturation 20% to 50%

The serum iron concentration measures iron bound to transferrin. This value represents about one-third of the total iron-binding capacity (TIBC) of transferrin.20 The TIBC measures the iron-binding capacity of transferrin protein and is an indirect indicator of iron stores. In iron deficiency anemia, TIBC is increased due to a compensatory increase in transferrin synthesis. This increase leads to a corresponding decrease in the percent transferrin saturation that can be calculated by dividing the serum iron by the TIBC and then multiplying by 100. For example, a person with a serum iron concentration of 100 mcg/dL and a TIBC of 300 mcg/dL has a transferrin saturation of 33%. Iron deficient erythropoiesis exists whenever the percent transferrin saturation is 15% or less.

Other disease states besides iron deficiency that can alter serum iron and TIBC are critical illness, infections, cancers, and inflammatory diseases7,23. Anemia from these diseases is sometimes called anemia of chronic disease or anemia of inflammation. Serum iron and TIBC both decrease in these disorders, unlike in iron deficiency anemia in which serum iron decreases but TIBC increases (Minicase 2).

Patients with renal failure often have anemia because of inadequate renal production of erythropoietin.24 These patients, particularly those receiving hemodialysis, may have iron deficiency in addition to the anemia caused by their renal disease. In these patients, the transferrin saturation (TSat) is used to determine if the patient has iron deficiency.

Normochromic, Normocytic Anemia

This classification encompasses numerous etiologies. Three causes are discussed: acute blood loss anemia, hemolytic anemia, and anemia of chronic disease.

Acute Blood Loss Anemia

Patients who suffer from acute hemorrhage may experience a dramatic drop in their whole blood volume. In this situation, the Hct is not a reliable indicator of the extent of anemia. It is a measure of the amount of packed RBCs per unit volume of the blood, not the total body amount of RBCs. The total whole blood volume may be markedly reduced, but in the acute phase of the hemorrhage, the Hct may be normal or even slightly increased. Usually, the Hgb and Hct are decreased by the time a CBC is obtained. Also, patients with hemorrhage often receive IV crystalloid fluids such as normal saline or lactated Ringer’s solution to maintain intravascular volume. The drop in these patients’ Hgb and Hct is partly iatrogenic, caused by dilution of the patient’s RBCs with the IV fluid.

In patients with normal bone marrow, the production of RBCs increases in response to hemorrhage, resulting in reticulocytosis. If the patient is transfused, each unit of packed RBCs administered should increase the Hgb by 1 g/dL if the bleeding has stopped. Table 16-2 shows the usual laboratory findings in acute blood loss anemia (Minicase 3).

Anemia and Low Platelet Count

Michael T., a 50-year-old man with a long history of alcohol abuse, cirrhosis, and esophageal varices, is brought to the emergency department (ED) by concerned family members. The family says that he suddenly began coughing up bright red blood. As he is moved to a bed in the ED, he begins coughing and vomiting large amounts of bright red blood. A stat CBC reveals the following values:

TEST NAME

RESULT

REFERENCE RANGE

RBC

2.91 × 106 cells/μL

4.5–5.9 × 106 cells/μL for men

WBC

6.6 × 103 cells/μL

4.4–11.3 × 103 cells/μL

Hgb

8 g/dL

14–17.5 g/dL for men

Hct

28.2%

42% to 50% for men

MCV

92.4 fL/cell

80–96 fL/cell

RDW

14.1%

11.5% to 14.5%

Platelets

75,000 cells/μL

150,000–450,000 cells/μL

QUESTION: What does this CBC indicate?

DISCUSSION: The presence of bright blood (as opposed to dark, “coffee ground” material) in the emesis indicates an acute and active bleed—either from a gastric ulcer or from esophageal varices. The CBC is consistent with acute blood loss. At the onset of bleeding, the RBC, Hgb, and Hct may show minimal changes. Here, the RBC, Hgb, and Hct are all moderately decreased, and the red cell indices are within normal limits, supporting a recent history of significant blood loss. The platelet count is also decreased, which may have led to the increasing risk of bleeding. Esophagogastroduoedenoscopy revealed that the bleeding was caused by ruptured esophageal varices.

Hemolytic Anemia

Hemolysis, the lysis of RBCs, often leads to irregularly shaped or fragmented erythrocytes, termed poikilocytosis. If hemolysis is rapid and extensive, severe anemia can develop, yet RBC indices (MCV and MCHC) remain unchanged in the short term. Patients with normal bone marrow respond with an increase in erythrocyte production to replace the lysed cells, and reticulocytosis is present. Specialized tests, called antiglobulin tests, can be useful in determining immune causes of hemolytic anemia.

Plasma (free) Hgb measures the concentration of Hgb circulating in the plasma unattached to RBCs. It is almost always elevated in the presence of intravascular hemolysis. Haptoglobin, an acute-phase reactant, binds free Hgb and carries it to the reticuloendothelial system. In the presence of intravascular hemolysis, the serum haptoglobin concentration is decreased. Concomitant corticosteroid therapy may confound interpretation because many diseases associated with in vivo hemolysis are treated with corticosteroids. Serum haptoglobin may be normal or elevated in hemolysis if the patient is receiving steroids. If the increase in serum haptoglobin is from steroids, other acute-phase reactants, such as hepcidin or ferritin, will also be elevated. Serum haptoglobin is also elevated in patients with biliary obstruction and nephrotic syndrome. It is variably decreased in folate deficiency, sickle cell anemia, thalassemia, hypersplenism, liver disease, estrogen therapy, and pregnancy.8

Immune hemolytic anemias are caused by the binding of antibodies and complement components to the erythrocyte cell membrane with subsequent lysis.25,26 The method used to detect autoantibodies already bound to erythrocytes is a direct antiglobulin test (DAT), sometimes referred to as the direct Coombs test. The DAT is performed by combining a patient’s RBCs with rabbit or goat antihuman globulin serum, which contains antibodies against human immunoglobulins and complement.23 If the patient’s RBCs are coated with antibody or complement, the antibodies in the antiglobulin serum bind to the immunoglobulins coating the RBCs, leading to the agglutination of the RBCs. The DAT is the only test that provides definitive evidence of immune hemolysis.25 The DAT can also be used to investigate possible blood transfusion reactions.

The method used to detect antibodies present in serum is an indirect antiglobulin test (IAT, indirect Coombs). Patient serum is combined with several types of normal erythrocytes of known antigenic expression. Any antibodies able to bind to the antigens expressed on these sample RBCs adhere after the serum is washed away. Antihuman immune globulin is then added and binds to any of the patient’s immune globulin that is present on the erythrocytes, followed by agglutination.25

The antiglobulin tests are sensitive, but a negative result does not eliminate the possibility of antibodies bound to erythrocytes. A low concentration of antibodies may give a false-negative reaction. Numerous conditions and medications can be associated with immune hemolytic anemia (Table 16-4).26,27 Medications can induce antibody formation by three mechanisms2527 that result in a hemolytic anemia.

TABLE 16-4.
Causes of Immune Hemolytic Anemia

Neoplasm

 Chronic lymphocytic leukemia

 Lymphoma

 Multiple myeloma

Rheumatologic disorders

 Systemic lupus erythematosus

 Rheumatoid arthritis

Medication

Autoimmune type

  Levodopa, mefenamic acid, methyldopa, procainamide

Innocent bystander type

  Cefotaxime, ceftazidime, ceftriaxone, chlorpromazine, doxepin, fluorouracil, isoniazid, quinidine, quinine, rifampin, sulfonamides, thiazides, chlorpropamide

Hapten type 1

  Cephalosporins, penicillins

Infections

Mycoplasma

 Viruses

Source: Adapted with permission from References 2529.
Autoimmune type

Methyldopa and procainamide are infrequently used cardiovascular drugs that may induce the formation of antibodies directed specifically against normal RBC proteins. This autoimmune state can persist for up to 1 month after drug administration has been discontinued. This mechanism is known as a true autoimmune type of antibody formation and is detected using the DAT.

Innocent bystander type

Antibodies to the drugs quinine and quinidine are examples of the immune complex (innocent bystander) mechanism. Each drug forms a drug–protein complex with plasma proteins to which antibodies are formed. This drug–plasma protein–antibody complex attaches to RBCs and fixes complement, which leads to lysis of the cells.25,26 In this situation, the RBC is an innocent bystander. Examples of other drugs implicated in causing this type of hemolytic anemia are listed in Table 16-4.

Hapten type 1

The hapten (penicillin) type 1 mechanism is involved when a patient has produced antibodies to penicillin. If the patient receives penicillin at a future date, some penicillin can bind to the RBC membrane. The antipenicillin antibodies, in turn, bind to the penicillin bound to the RBC, and hemolysis can result.

Glucose-6-Phosphate Dehydrogenase Deficiency Anemia

Glucose-6-phosphate dehydrogenase (G6PD) is an intracellular enzyme that forms the nicotinamide adenine dinucleotide phosphate needed by the erythrocyte to synthesize the antioxidant glutathione. Variants of this enzyme are more commonly found in African Black (GdA-), Mediterranean, and Asian populations (GdMed) than in white populations. These variants have an impaired ability to resist the oxidizing effect of drugs and collateral oxidative exposure to the granulocytic response to infections. Thus, exposure of patients with G6PD deficiency to oxidizing drugs or an infection can lead to a dramatic, nonimmunologic hemolysis.28 Examples of drugs that can lead to hemolysis in G6PD-deficient patients include dapsone, primaquine, rasburicase, phenazopyridine, methylene blue, and nitrofurantoin. A more complete list of offending medications and their relative risk can be found at https://www.g6pd.org/G6PDDeficiency/SafeUnsafe.aspx.

Assessment of at-risk patients for signs of hemolysis (anemia, hemoglobinemia, dark urine, and back pain) is appropriate. Routine genotyping of patients aids in drug selection and monitoring; for example, it aids before prescribing dapsone for Pneumocystis juroveci prophylaxis in a patient with human immunodeficiency virus (HIV) infection.

Anemia of Inflammation (Anemia of Chronic Disease)

Mild-to-moderate anemia often accompanies various infections, inflammatory illnesses, or neoplastic diseases that last more than 1 to 2 months.23,30 Chronic infections include pulmonary abscesses, tuberculosis, endocarditis, pelvic inflammatory disease, and osteomyelitis. Chronic inflammatory illnesses (eg, rheumatoid arthritis and systemic lupus erythematosus), solid tumors, and hematologic malignancies (eg, Hodgkin disease, leukemia, and multiple myeloma) are also associated with anemia. Because these disorders as a group are common, anemia due to chronic disease (also called anemia of chronic inflammation) is also common. Although anemia of chronic disease is more commonly associated with normocytic, normochromic anemia, it can also cause microcytic anemia. Table 16-2 shows the usual laboratory results found in anemia of inflammation.

The pathogenesis of this anemia is not totally understood. Various investigations have found that the erythrocyte lifespan is shortened and that the bone marrow does not increase erythrocyte production to compensate for the decreased longevity. While iron stores are normal or even elevated, iron use is impaired. Although erythrocytes are frequently normal size, microcytosis can develop. One distinguishing feature between early iron deficiency anemia and microcytic anemia of chronic disease is the normal serum ferritin that is present in the latter.23,30

In patients with chronic kidney disease (CKD), anemia is associated with decreased renal production of erythropoietin, decreased RBC lifespan, impaired RBC production, and, in patients receiving hemodialysis, blood loss with subsequent iron deficiency and folate deficiency.24,30 In the presence of sufficient iron stores, erythrocyte-stimulating agents (ESA), such as epoetin alfa and darbepoetin, may be used to decrease a patient’s need for blood cell transfusions. In patients with CKD and anemia, a trial of IV or oral iron is recommended regardless of whether they are receiving an ESA, particularly if the TSat is ≤30% and the ferritin is ≤500 ng/mL. In pediatric patients, thresholds of TSat ≤20% and ferritin ≤ 100 ng/mL are used to determine the need for a trial of iron replacement.24 More conservative use and dosing of ESAs is recommended by recent guidelines based on evidence that ESAs increase the risk for serious adverse cardiovascular events. ESA use should be individualized to use the lowest dose of ESA sufficient to reduce the need for transfusion. Patients with CKD who are not on dialysis should consider starting ESA treatment only when the Hgb level is <10 g/dL and reduce or stop the ESA dose if the Hgb level exceeds 10 g/dL. For patients on dialysis, ESA treatment should be initiated when the Hgb level is <10 g/dL, and the ESA dose should be reduced or interrupted if the Hgb level approaches or exceeds 11 g/dL. Monitoring of Hgb levels should be done at least weekly until stable and then monitored monthly.24

Use of ESA therapy in patients with cancer has become controversial because of the increased risk of thromboembolism and mortality. Recent guidelines recommend use of ESA therapy only with great caution in patients with malignancy, such as in patients with incurable malignancies and Hgb <10 g/dL.24,31 As with patients with CKD, dosing should be individualized to use the lowest dose of ESA sufficient to reduce the need for transfusion.

Anemia of critical illness32 is common in critically ill patients and is similar to anemia of inflammation, but in a compressed time frame. Critically ill patients have decreased RBC life span and production, with the additional problem of blood loss due to frequent phelobotomies and potential hemorrhagic losses in surgical and trauma patients. Anemia of critical illness is associated with adverse patient outcomes, and it is not clear if transfusion and/or ESA therapy improves outcomes.

Hemoglobinopathies

Several diseases arise from abnormal synthesis of the α or β subunits of Hgb. The most common types of anemias related to these hemoglobinopathies include sickle cell trait/disease and thalassemias. Sickle cell disease is caused by the substitution of a valine amino acid for glutamate on the β chain of Hgb. The heterozygous (trait) carrier state involving valine substitution on one β chain is thought to provide a resistance to clinical manifestations and sequelae of malaria. Homozygous persons with both β chains carrying the valine substitution are at increased risk of developing a sickling of RBCs. This occurs most commonly under circumstances of hypoxia, infection, dehydration, or acidosis. Deoxygenated Hgb molecules polymerize into rod-like structures within the RBC, deforming the cell into an arched, rigid sickle-shaped cell. These erythrocytes are not able to deform and pass through the capillaries or reticuloendothelial system. Hypoxia, ischemia, and even infarction occur in tissues downstream of these sites of impaired blood flow. Severe pain is usually present during these “sickle crises,” and pain management is often needed in addition to hydration, transfusion, and other treatments. Diagnosis is made by inspection of the peripheral blood smear and by electrophoresis of the patient’s Hgb.33

Thalassemias are a more diverse group of hemoglobinopathies most commonly associated with persons of ancestry arising in the Mediterranean, Middle East, South Asia, and Asia regions. Unlike the chemical change caused by the valine substitution in sickle cell patients, thalassemias are characterized by a deficiency or absence of one of the subunits of the Hgb. Because there are two α and two β Hgb subunits in the normal Hgb tetramer, an inability to produce adequate amounts of one of the subunits would clearly lead to difficulty in synthesizing intact, complete Hgb molecules.34

Thalassemias are often diagnosed by a peripheral blood smear, which shows small, pale erythrocytes, sometimes in very high numbers. Some of the RBCs are nucleated, reflecting the intense pressure on erythropoiesis in the bone marrow to provide oxygen-carrying capacity to the body even if it requires releasing immature, nucleated erythrocyte precursors. The type of thalassemia present is determined using electrophoresis.

WHITE BLOOD CELL COUNT AND DIFFERENTIAL

White blood cells are divided into two general classifications:

  1. Granulocytes or phagocytes (leukocytes that engulf and digest other cells)

  2. Lymphocytes (leukocytes involved in the recognition of nonself cells or substances)

The functions of these general leukocyte classes are interrelated. For example, immunoglobulins produced by B lymphocytes are needed to coat or opsonize encapsulated bacteria so that T cells and neutrophils can more effectively identify, adhere, and destroy them.

When a WBC count and differential is ordered for a patient, the resulting laboratory report provides the total WBCs in a given volume of blood plus the relative percentages each cell type that contribute to the total. Therefore, the percentages of the WBC subtypes must add up to 100%. If one cell type increases or decreases, percentages of all other types change in the opposite direction. Table 16-5 is a general breakdown of the different types of WBCs and their usual percentages in peripheral blood.

TABLE 16-5.

Normal WBC Count and Differential

CELL TYPE

NORMAL RANGE

Total WBC count

4.4–11.3 × 103 cells/μL

Polymorphonuclear neutrophils (“polys,” “segs,” PMN)

45% to 73%

Band neutrophils (“bands,” “stabs”)

3% to 5%

Lymphocytes

20% to 40%

Monocytes

2% to 8%

Eosinophils

0% to 4%

Basophils

0% to 1%

PMN = polymorphonuclear cells.

The WBC count and differential is one of the most widely performed clinical laboratory tests. Large, clinical laboratories commonly use automated methods for determining the WBC differential, but manual differential counts may still be used. Automated instruments count thousands of cells and can report not only the relative percentages of the various WBC types but also the absolute numbers, Hgb, RBC, platelets, and RBC indices. When reviewing a WBC differential, one must be aware of not only the relative percentages of cell types but also the absolute numbers. The percentages viewed in isolation can lead to incorrect conclusions.

Numerous cluster of differentiation (CD) surface markers have been characterized on leukocytes and their precursors. CD molecules are surface proteins or glycoproteins that are typically immunologically characterized by their unique epitopes. The function of only a minority of the hundreds of CD molecules identified on human cells have been determined; however, their expression on specific cell types can permit identification of abnormal cell types and allow targeted treatment at cells expressing the CD molecule.

Granulocytes

Granulocytes are phagocytic cells and derive their name from the presence of granules within the cytoplasm. The granules store lysozymes and other chemicals needed to oxidize and enzymatically destroy foreign cells. Granulocytic leukocytes include neutrophils, eosinophils, and basophils. Monocytes are phagocytic cells that mature into macrophages, which are predominantly found in tissue rather than in the circulation. When a peripheral smear of blood is prepared, three types of granulocytes are named by the staining characteristics of their cytoplasmic granules8,35:

  1. Neutrophils, which retain neutral stains and appear light tan

  2. Eosinophils, which retain acidic dyes and appear red-orange

  3. Basophils, which retain basic dyes and appear dark blue to purple

Granulocytes are formed in large numbers from the pluripotential stem cells in the bone marrow. They undergo numerous differentiation and proliferation steps in the marrow and are usually released into the peripheral blood in their mature form. A common exception is the appearance of banded neutrophil during an infection, as discussed later. Neutrophils, eosinophils, and basophils die in the course of destroying ingested organisms or particles, forming purulent material or pus. On the other hand, monocytes and macrophages do not usually need to sacrifice themselves when destroying target cells.

Neutrophils

Normal range: PMN leukocytes 45% to 73% or 0.45 to 0.73; bands 3% to 5% or 0.03 to 0.05

Neutrophils are also termed segmented neutrophils (“segs”) or polymorphonuclear cells (PMNs, “polys”). The less mature form of the neutrophil with a crescent-shaped nucleus is a band cell. Bands derive their name from the morphology of their nucleus, which has not yet segmented into multiple lobes. Less mature forms of the neutrophil, such as the metamyelocyte and myelocyte, are normally found in the bone marrow but not in the peripheral blood. The neutrophil is a phagocytic cell that exists to ingest and digest foreign cells and proteins (eg, bacteria and fungi).

The absolute segmented neutrophil count is the percentage of neutrophils and bands multiplied by the WBC count. The reference range for absolute counts can be estimated by multiplying the normal range of percentages for the particular type of WBC by the upper and lower limits of the total WBC count. Absolute neutrophil counts of <1,000/μL represent neutropenia, with counts of <500/μL and 100/μL considered severe and absolute neutropenia, respectively. Because of the risk of rapidly progressing, life-threatening infection, antimicrobials may be started after cytotoxic chemotherapy if the absolute neutrophil count is <500/μL and the patient develops a fever.36

Under normal conditions, about 90% of the neutrophils are stored in the bone marrow. When released, neutrophils normally circulate for several hours before eventually marginating by the adhering to vascular endothelium in the spleen and other organs. This dynamic process of margination, with the potential for demargination, causes large shifts in the measured neutrophil count because only the granulocytes that are circulating at the time are measured by a venipuncture. Neutrophils spend only about 6 to 8 hours in the circulation, after which they move through the endothelium into the tissue. Unless used to engage a foreign body or sustained by the cytokine milieu, neutrophils then undergo programmed cell death, a noninflammatory process termed apoptosis.

During an acute infection, there is an increase in the percentage of neutrophils because they initially demarginate from the endothelium and are released from the bone marrow.37,38 Demarginated neutrophils are mature, so initially the percent of band neutrophils will remain normal. However, as less mature neutrophils are released from the marrow, usually in response to bacterial infection, the percent of band neutrophils increases. The increase percent of band cells in infections is termed a left shift. This term may be due to the traditional order in which the manual differential count was reported. It may also arise from the use of a left-to-right sequence in figures describing the process of neutrophil differentiation from the stem cell (Figure 16-1).

When the neutrophils and bands are elevated, the percentage of lymphocytes decreases. Ratios of only 10% to 15% lymphocytes may appear in these patients, but this relative lymphopenia arises from the concomitant increase in total WBCs and likely does not reflect an absolute lymphopenia. An exception is a neutrophilia caused by glucocorticoid treatment, which will cause a drop in the absolute lymphocyte count because of its lymphotoxic effect while increasing the absolute neutrophil count due to demargination.

Eosinophils and Basophils

Normal range: eosinophils 0% to 4% or 0 to 0.04; basophils 0% to 1% or 0 to 0.01

The functions of eosinophils and basophils are not completely known. Eosinophils are present in large numbers in the intestinal mucosa and lungs, two locations in which foreign proteins enter the body. Eosinophils can phagocytize, kill, and digest bacteria and yeast. Elevations of eosinophils counts are highly suggestive of parasitic infections and allergic diseases, including some forms of asthma.

Basophils are present in small numbers in the peripheral blood and are the most long-lasting granulocyte in blood, with a circulating lifespan of approximately 2 weeks.2 They contain heparin, histamine, and leukotriene B4.39 Many signs and symptoms of allergic responses can be attributed to specific mast cell and basophil products. Basophils are probably involved in immediate hypersensitivity reactions (eg, extrinsic, or allergic, and asthma) in addition to delayed hypersensitivity reactions. Basophils may be increased in chronic inflammation and in some types of leukemia.

Monocytes/Macrophages

Normal range: monocytes 2% to 8% or 0.02 to 0.08

Monocytes leave the circulation in 16 to 36 hours and enter the tissues, where they mature into macrophages. Macrophages are present in lymph nodes, alveoli of the lungs, spleen, liver, and bone marrow, comprising the reticuloendothelial system.40 Macrophages, both those circulating and those that have migrated out of the blood, participate in the removal of foreign substances from the body. In addition to attacking foreign cells, they are involved in the destruction of old erythrocytes, denatured plasma proteins, and plasma lipids. Tissue macrophages also salvage iron from the Hgb of old erythrocytes and return the iron to transferrin for delivery to the bone marrow. Under appropriate stimuli, monocytes/macrophages are transformed into antigen-presenting cells (also termed dendritic cells). These transformed macrophages are an important component of both cell-mediated (T lymphocytes) and soluble (B lymphocyte) immune activity against antigens.40 Macrophages express a variety of chemokine receptors and secrete a variety of substances, including enzymes, a variety of interleukins, tumor necrosis factor-α, interferons, and a variety of tissue and vascular growth factors.

Lymphocytes and Plasma Cells

Normal range: lymphocytes 20% to 40% or 0.2 to 0.4

Lymphocytes make up the second major group of leukocytes. They are characterized by a far less granular cytoplasm and relatively large, smooth nuclei. These cells give specificity and memory to the body’s defense against foreign invaders.41 There are three subgroups of lymphocytes:

  1. T lymphocytes (T cells)

  2. B lymphocytes (B cells)

  3. Natural killer cells (NK cells)

Lymphocytes are not phagocytic, but the NK and T-cell subtypes are cytotoxic by virtue of complement activation and antibody-dependent cell-mediated cytotoxicity. Morphologic differentiation of lymphocytes is difficult; visual inspection of a blood smear cannot uniformly distinguish between T, B, and NK cells. Fortunately, lymphocytes can be distinguished by the presence of CD lineage-specific membrane markers. Thus, mature T cells have CD3 and CD5, B cells have CD20, and NK cells have CD56 membrane markers.41 Individual CD moieties may be surface proteins, enzymes, or adhesion molecules, to name a few. Labeled antibodies to specific CD molecules identify the lineage of the lymphocyte, either in blood or in tissue.

Identification of the subtype of lymphocytes is not a routine clinical hematology test at present; they are reported simply as lymphocytes by automated counting instruments. However, in research applications and for the diagnosis and guidance of targeted treatment of leukemias and lymphomas, subtypes can be both counted and sorted by an automated process termed fluorescence-activated cell sorting. The WBC layer is separated by centrifugation and exposed to one or more CD antibodies tagged with fluorescent dyes. The labeled cells are given an electrostatic charge and then flow individually past one or more lasers that induce the labeled cells to glow at wavelengths specific to the dye staining each cell. This method is general and can be used to count and sort virtually any cell that can be labeled with a fluorescent tag.42

With the help of T cells, B cells recognize foreign substances and are transformed into plasma cells, capable of producing antibodies (discussed later). Table 16-6 lists the types of disorders in which lymphocytes are increased or decreased.

TABLE 16-6.

Quantitative Disorders of White Blood Cells35,41,43,47,50,51,34

WBC ABNORMALITY

TYPICAL THRESHOLD (CELLS/μL)

POSSIBLE CAUSES

Neutrophilia

>12,000

Acute bacterial infection

Trauma

Myocardial infarction

Chronic bacterial infection

Epinephrine, lithium, G-CSF, GM-CSF, glucocorticosteroids

Neutropenia

<1,500

Radiation exposure

Medications:

 Antineoplastic cytotoxic agents

 Captopril

Carbamazepine

 Cephalosporins

Chloramphenicol

 Clozapine

Diclofenac

 Ganciclovir

Levamisole

 Methimazole

Penicillins

 Phenothiazines

Procainamide

 Propylthiouracil

Ticlopidine

 Vancomycin

Zidovudine

 Tricyclic antidepressants

 Sulfamethoxazole–trimethoprim

Overwhelming acute bacterial infection

Vitamin B12 or folate deficiency

Salmonellosis

Pertussis

Eosinophilia

>350

Allergic disorders/asthma

Parasitic infections

Leukemia

Medications

Angiotensin-converting enzyme inhibitors

Antibiotics (or any allergic reaction to a drug)

Eosinopenia

<50

Acute infection

Basophilia

>300

Chronic inflammation

Leukemia

Monocytosis

>800

Recovery state of acute bacterial infection

Tuberculosis (disseminated)

Endocarditis

Protozoal or rickettsial infection

Leukemia

Lymphocytosis

>4,000

Infectious mononucleosis

Viral infections (eg, rubella, varicella, mumps, cytomegalovirus)

Pertussis

Tuberculosis

Syphilis

Lymphoma

Lymphopenia

<1,000

HIV type 1

Radiation exposure

Corticosteroids

Lymphoma (Hodgkin disease)

Aplastic anemia

G-CSF = granulocyte-colony stimulating factor; GM-CSF = granulocyte-macrophage colony-stimulating factor.

Source: Adapted with permission from references 34,35,41,43,47,50,51.

T Lymphocytes

T lymphocytes are responsible for cell-mediated immunity and are the predominant lymphocytes in circulation and in tissue. They require partial maturation in the embryonic thymus, hence, the name T cell. In addition to identifying infections, they oversee delayed hypersensitivity (seen with the skin test for tuberculosis) and rejection of transplanted organs.41,43 For a foreign antigen to be recognized by T cells, it must be “presented” by macrophages or dendritic cells on one of two complex, individualized molecules termed major histocompatibility complexes (MHC1 and MHC2).

T cells can be further divided into helper and cytotoxic (or suppressor) cells, which, respectively, express the CD4 and CD8 markers. CD4 helper cells are not cytotoxic but on recognizing an antigen will activate and produce cytokines such as IL-2, which stimulate nearby immune cells, including macrophages and CD8 T cells, B cells, and NK cells.

CD4 T-helper cells can again be divided into T1H and TH2 subtypes. The T1H subtype mediates the activation of macrophages and the delayed hypersensitivity response, while the TH2 subtype appears primarily responsible for B-cell activation. The cellular specificity of these subtypes appears to arise primarily from their distinct pattern of cytokine production.

Human immunodeficiency virus binds specifically to the CD4 receptor but does not elicit the desired antiviral response in most patients. This infection leads to destruction of this subset of T cells and a reversal of the CD4/CD8 ratio (normally >1). The CD4 lymphocyte count and viral load measured by viral RNA are inversely related and correlate with overall prognosis. Although the CD4 count remains a useful surrogate marker in monitoring the course and treatment of patients infected with HIV, viral loads are routinely measured. The lack of adequate numbers of active T-helper cells that activates other immune cells leads to an increased susceptibility to numerous opportunistic infections, cancer, and progression to acquired immune deficiency syndrome.44,45 T cells are the primary mediator for host rejection of transplanted solid organs,47 such as heart, lung, kidney, liver, and pancreas grafts. The perioperative and postoperative treatment of solid organ graft recipients is directed toward minimizing the antigraft T-cell response, while not ablating the T-cell population to the point of causing life-threatening infections. In practice, this is a narrow path plagued by viral and fungal infections that cause substantial morbidity and mortality in graft recipients.

Typically, T-cell populations in graft recipients46 are not measured, and drug titration is based on biopsies of the transplanted organ, drug concentrations of the immunosuppressants, and blood counts. Anti–T-cell treatments employed in transplant recipients include corticosteroids; Muromonab-CD3 (OKT3), an anti-CD3 antibody directed against the CD3 marker found on T cells; antihuman lymphocyte immunoglobulin; and inhibitors of T-cell activation such as cyclosporine, tacrolimus, or mycophenolate. Because these immunoglobulin products are typically obtained from nonhuman species, they can cause severe allergic reactions and are usually effective for only a short period.

B Lymphocytes

B cells are named after similar avian lymphocytes that required maturation in an organ termed the Bursa of Fabricius. There is no equivalent organ in humans, and maturation of B lymphocytes occurs in the bone marrow. Quiescent, circulating B cells express one form of antibody, immunoglobulin M (IgM). When stimulated by activated T cells or antigen-presenting cells, B cells are transformed into plasma cells that will produce one of five immunoglobulin types: IgA, IgD, IgE, IgG, or IgM.47

The two antibodies most commonly associated with the development of immunity to foreign proteins, viruses, and bacteria are IgM and IgG. IgE is associated with the development of immediate hypersensitivity reactions, such as anaphylaxis and allergic diseases, including asthma. IgA is secreted into the lumen of the GI tract and helps avoid sensitization to foods, and IgD is bound to the lymphocyte cell membrane.47 Abnormal immunoglobulins can typically be detected using serum and/or urine protein electrophoretic gels and urine immunofixation. Monoclonal hyperimmunoglobulinemias are identified by single electrophoretic peaks and are typically associated with plasma (B) cell premalignant or malignant disorders. Polyclonal hyperimmunoglobulinemias can be associated with infections and inflammatory reactions.

Lymphopenia and hypogammaglobulinemia (a decrease in the total quantity of immunoglobulin) are seen as a consequence of corticosteroid treatment, transplant rejection prophylaxis, and anticancer treatment, but they can also paradoxically arise from leukemias. In general, lymphopenia is more common in chemotherapy regimens that include high doses of corticosteroids, which bind to a receptor on lymphocytes and are lymphotoxic, even to the point of initiating cellular apoptosis.47 Interestingly, although HIV-1 infections lead to lymphopenia, other viral infections (eg, infectious mononucleosis, hepatitis, mumps, varicella, rubella, herpes simplex, herpes zoster, and influenza) often increase the number of circulating lymphocytes (lymphocytosis)48,49 (Minicase 4). Severe malnutrition also may result in lymphopenia.

Natural Killer Cells

Natural killer cells (NK) are derived from T-cell lineage but are not as restricted in requiring MHC identification of the target cell. NK cells are thought to be particularly important for cytotoxic effects on virally infected cells and cancer cells.

Leukocyte Disorders

Patients can suffer from three major classes of leukocyte disorders: functional, quantitative, and myeloproliferative. Functional disorders involve defects in recognition, metabolism, cytotoxic effects, signaling, and other related activities. Routine laboratory values are not intended to evaluate these abnormalities and are not discussed further here.

Quantitative disorders involve too few or too many leukocytes. Possible causes are listed in Table 16-6. Neutropenia is usually considered to exist when the neutrophil count is <1,500 or 1,000 cells/μL.36,50 When the neutrophil count is <500 cells/μL, normal defense mechanisms are significantly impaired, and the patient is at increased risk of bacterial and fungal infections. A neutrophil count <100/μL is termed absolute neutropenia or agranulocytosis. This is often encountered after cytotoxic chemotherapy is administered and after regimens intended to ablate the bone marrow in preparation for a stem cell transplant. An infection is probable if agranulocytosis is prolonged or severe, so patients at risk are monitored closely for infection and administered broad spectrum antimicrobials when fever or other signs of infection are seen. When infections do occur in such patients, they can be difficult to successfully treat—even with normally effective antibiotics—because the number and phagocytic activity of the neutrophils are impaired.

Agranulocytosis51 may be seen as a specific toxic drug effect (such as seen with propylthiouracil) or as part of a broader myelopoietic disorder (such as aplastic anemia). Aplastic anemias (inadequate production of blood cells by the bone marrow) have multiple causes including drug, toxin, or radiation exposure; congenital defect; or age-related fatty or fibrotic bone marrow replacement. The word anemia in this term is misleading because production of other blood cell types can also be decreased resulting in pancytopenia. Because some cases of aplastic anemia are autoimmune in nature, some patients are treated with immunosuppressive therapy.

Myelodysplastic anemias are characterized by abnormal maturation of RBCs and WBCs. These are typically classified by the World Health Organization system52 based on the marrow morphology identified from a bone marrow aspirate. The usual treatment course is supportive care (ie, transfusions or stem cell transplant in patients for whom this is feasible).

Neutrophilia (increased circulating neutrophils) is caused by both an increased release from the bone marrow and a shift of marginated cells into the circulation. This rapid rise in the number of circulating cells can be caused by acute infections, trauma, or administration of epinephrine or corticosteroids. Neutrophilia exceeding 50,000 cells/μL is termed “leukemoid reaction” and can be seen with a variety of underlying inflammatory conditions. While sometimes mistaken for leukemia, the neutrophils in leukemoid reactions typically are mature cells rather than the highly immature cells seen in leukemias.

Myeloproliferative Disorders

Leukemias

Neoplasms of the bone marrow cells most commonly involve a leukocyte line and are termed leukemias. Leukemias are broadly classified as being acute or chronic, and leukemias are either of myeloblastic (granulocytic lineage) or lymphoblastic (lymphocytic) lineage.52;53 The clinical course and biology of various leukemias varies. Almost all leukemias fall within one of four categories:

  1. Acute myelogenous

  2. Acute lymphoblastic

  3. Chronic myelogenous

  4. Chronic lymphocytic

Anemia and Lymphopenia

Donna L. is a 55-year-old woman with a history of rheumatoid arthritis and type 2 diabetes mellitus who presents to her physician for a routine physical examination. She is feeling well and has no complaints, other than the soreness routinely associated with the arthritis in her hands. She has normal vital signs, and other than the stigmata of her moderate rheumatoid arthritis, she has a normal physical examination. She takes the following oral medications routinely:

  • Prednisone 5 mg once daily with dinner

  • Metformin 750 mg once daily with dinner

  • Methotrexate 10 mg weekly

  • Acetaminophen 650 mg q 6 hr PRN for hand pain

The physician draws a comprehensive metabolic panel and a CBC with differential and platelet count. The results of the CBC with differential and platelet count are as follows:

TEST NAME

RESULT

REFERENCE RANGE

RBC

4 × 106 cells/μL

4.1–5.1 × 106 cells/μL for women

WBC

9.6 × 103 cells/μL

4.4–11.3 × 103 cells/μL

Hgb

13.3 g/dL

12.3–15.3 g/dL for women

Hct

37.9%

36% to 45% for women

MCV

105.5 fL/cell

80–96 fL/cell

MCH

39.2 pg/cell

27–33 pg/cell

RDW

15%

11.5% to 14.5%

Platelets

304,000 cells/μL

150,000–450,000 cells/μL

Neutrophils

76%

45% to 73%

Bands

5%

3% to 5%

Monocytes

7%

2% to 8%

Eosinophils

2%

0% to 4%

Basophils

1%

0% to 1%

Lymphocytes

9%

20% to 40%

QUESTION: What abnormalities are present, and what is their cause and resolution?

DISCUSSION: This patient has somewhat low RBC count and Hgb as well as elevated MCV and MCH, indicating a macrocytic anemia. She also has a high WBC count with increased neutrophil and decreased lymphocyte counts. She is not showing signs of infection. The macrocytic anemia could be caused by vitamin B12 or folate deficiency. Treatment with methotrexate, an antifolate drug, is the likely cause. The differential diagnosis can be made by obtaining blood assays for vitamin B12 and folate. Many clinicians prescribe 5 mg oral folate daily except for methotrexate dosing days, and this would be an appropriate recommendation for this patient as well.

The lymphopenia and neutrophilia are likely caused by the prednisone therapy. Glucocorticoids are known to cause demargination of neutrophils from the vascular endothelium, leading to a relative neutrophilia. Glucocorticoids are also lymphotoxic, typified in their use for treatment of lymphocytic malignancies. No treatment is indicated in this patient, but monitoring for opportunistic infections, such as candidiasis, needs to be ongoing. The corticosteroid-induced changes in lymphocyte and neutrophil counts are expected to return to normal after the cessation of the steroid dosing.

Although the clinical course varies among these neoplasms, a common denominator is the proliferation of the neoplastic cell line and displacement of normal hematopoiesis. The neoplastic cells may arise from cells of varying levels of differentiation of either a granulocytic or lymphocytic lineage. Morphology and CD membrane markers vary among individuals but are fairly uniform throughout the disease course in a given patient. The morphology and CD membrane markers of cells obtained from the diagnostic bone marrow aspirate and flow cytometry, respectively, are used to assign a French-American-British (FAB) classification of M0 through M7 to subtype acute myelogenous leukemia or to diagnose acute lymphoblastic leukemia. Other morphologic features and surface marker combinations are used to characterize the other leukemias.

Multiple (plasma cell) myeloma is notable in that it is a plasma cell neoplasm of the bone marrow. The monoclonal neoplastic plasma cells produce a single immunoglobulin isotype (IgG, IgA, light chain only, IgD, IgE, or rarely IgM). This single, monoclonal protein is referred to as the M-protein. The M-protein is usually identified using serum protein electrophoresis. The specific immunoglobulin type can be defined with a subsequent step of serum immunofixation with protein-specific antibody (eg, anti-IgG). Other laboratory findings associated with multiple myeloma include Bence Jones protein (light chain) in urine, hypercalcemia, increased ESR, normochromic, normocytic anemia, and coagulopathy.54

Chronic myeloproliferative disorders55 involve an abnormal proliferation of more mature bone marrow cells. Excessive or uncontrolled proliferation of all cell lines leads to polycythemia vera, a malignancy when erythrocyte overproduction is the most prominent abnormality. Chronic myelogenous leukemia is characterized by a chromosomal translocation [t(9:22), “Philadelphia chromosome”] that creates a fusion product (BCR/ABL) resulting in autonomous tyrosine kinase activity, a growth-signaling enzyme. Patients with chronic lymphocytic leukemia present with increased numbers of circulating mature B lymphocytes, which are monoclonal.

Patients with chronic leukemias may live for several years with minimal treatment because of the indolent nature of the disease. At some point, a patient typically develops a transformation of the disease into a life-threatening accelerated phase or blast crisis. Fortunately, with the development of tyrosine kinase inhibitors and other targeted medications, this fatal complication can now often be substantially delayed. Although the chronic leukemias are less aggressive than the acute leukemias, they are less curable with chemotherapy, and stem cell transplantation is appropriate in selected patients.

Lymphomas

A lymphoma is a neoplasm of lymphocytic lineage, which typically predominates in lymph nodes forming tissue masses rather than being primarily located in the bone marrow. The lymphomas are classified into two main groups: (1) non-Hodgkin lymphoma (NHL), and (2) Hodgkin lymphoma. The pattern of tissue involvement—termed either diffuse or follicular (nodular)—and the cytology of the neoplastic lymphoid cells (primarily the size and appearance of the cell nucleus) are used to morphologically subclassify non-Hodgkin lymphoma.56 The World Health Organization classification57 of NHL also uses CD surface markers, cytogenetics, and molecular and genetic studies to further define subcategories of NHL. Non-Hodgkin lymphomas can also be practically divided into aggressive and indolent forms. The aggressive lymphomas grow and spread quickly but are generally more likely to be eradicated with current, intensive chemotherapy. In contrast, the slower-growing, indolent lymphomas are not as responsive and are more difficult to cure, but these often have a long disease course. Hodgkin lymphoma is generally a more treatable lymphoma. The neoplastic cellular element is termed the Reed-Sternberg cell. This is a large cell with a lobulated nucleus and prominent nucleoli. It is typically surrounded by a nonneoplastic population of lymphocytes, eosinophils, neutrophils, plasma cells, and macrophages.

Lymphomas predictably involve T-lymphocyte or B-lymphocyte precursors, and many express CD marker characteristics of mature lymphocytes. Identification of the CD20 marker on B-cell lymphomas provides an opportunity to treat these patients with recombinant antibodies specific to this surface marker. Differentiation between a T-cell leukemia and a peripheral T-cell lymphoma likely requires the identification of CD phenotypes.

SUMMARY

This chapter presents a brief characterization of the lineage and function of RBCs and WBCs. Normal laboratory values have been presented, but it is important to realize that normal ranges vary slightly depending on the laboratory conducting the analysis and the population being studied. Hematologic conditions are common, resulting in widespread use of hematologic tests such as the CBC in all patient care settings. Proper interpretation of these commonly used tests is important for the clinician to provide a correct assessment of the patient’s condition, choose the most appropriate therapy, and monitor the outcomes of that therapy.

ACKNOWLEDGMENTS

The authors and editors would like to acknowledge the contributions of Dr. Paul R. Hutson, who authored this chapter in previous editions of this textbook.

LEARNING POINTS

1. How do iron deficiency and nutrient deficiency (folate and vitamin B12) differ in their presentation in a CBC?

ANSWER: As expressed by the term anemia, in each of these circumstances, the Hgb and Hct are low. Iron deficiency is characterized by small (microcytic, low MCV) and pale (hypochromic) RBCs. In contrast, both folate and vitamin B12 deficiency classically present with larger (macrocytic, elevated MCV) erythrocytes. Patients with vitamin B12 deficiency may also have abnormalities in WBC and platelets.

2. What are the roles of transferrin, ferritin, TIBC, and TSat, and how are these laboratory values interpreted?

ANSWER: Transferrin’s primary role is to transport iron to the bone marrow for erythrocyte synthesis, while in the process protecting intervening tissue from the reactivity of the metal ion. Ferritin serves as the storage form of iron. Ferritin protein not bound to iron is termed apoferritin. Most of the iron-binding protein in the plasma is transferrin, and the serum TIBC is an indirect measure of the transferrin concentration. With iron deficiency, the liver synthesizes more transferrin. Thus, the residual, unbound capacity of the transferrin (and thus TIBC) is increased, while the percentage saturation of receptors on the transferrin molecules (transferrin saturation, TSat) is decreased. With less tissue stores of iron, the ferritin level is decreased in iron deficiency. In anemia of chronic disease, the plasma iron and transferrin concentrations are both low, and the TSat may be decreased or normal. Liver disease or malnutrition can also slow the production of transferrin, which may complicate the interpretation of the TIBC.

3. What are typical reasons why WBC counts are elevated, and how can the differential cell count help clarify the cause?

ANSWER: A sustained elevation of the WBC count is typically due to metabolic stress, infections, certain medications, or leukemias. Infections, corticosteroids, epinephrine, and exercise cause a demargination of neutrophils from the endothelium, causing a transient, increased percentage of neutrophils, but in most cases a normal absolute lymphocyte count. Corticosteroids cause neutrophil demargination but are also lymphotoxic, so the absolute lymphocyte count decreases. Bacterial infections are associated with an increase in the percentage and absolute number of neutrophils and to the release of less mature neutrophils (band cells) from the bone marrow.

4. What are common, unintended drug-induced alterations in RBC and WBC counts and function?

ANSWER: RBC counts can be reduced by nonsteroidal antiinflammatory drug–induced GI bleeding or by hemolytic anemia in patients with G6PD deficiency treated with various oxidizing drugs. RBC and WBC (and platelet) counts are commonly decreased following cytotoxic chemotherapy, but the impact on WBCs is greater, especially for neutrophils, because of their faster turnover and shorter lifespan. Macrocytic, hypochromic anemia can be caused by treatment with antifolates such as methotrexate, or chronic treatment with antibiotics inhibiting DNA synthesis such as trimethoprim. Corticosteroids are lymphotoxic and decrease the lymphocyte count but also lead to a higher apparent neutrophil count due to their drug-induced demargination from the endothelium. Some medications, such as propylthiouracil and clozapine, can cause a sudden, dramatic reduction in neutrophils, resulting in agranulocytosis.

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