White Blood Cell Differential Count

CBC analyzers determine the total number of WBCs per microliter or liter of blood and perform an automated differential count in which the percentage and absolute number of each type of WBC is determined. If the instrument count is abnormal, or a sample is flagged for suspected abnormal or immature cells, a manual slide review and differential count is performed by a laboratory scientist. In addition, the morphology of all the cells is assessed, and the presence of immature and abnormal cells is noted.

The relative count is the percentage of a particular cell among all the WBCs counted. The absolute count for that cell type is determined by multiplying its percentage or relative count by the total WBC count. CBC analyzers automatically calculate the absolute counts for each type of WBC. Table 7-2 provides sample reference ranges for the relative and absolute counts of each type of WBC.

Because the relative count is influenced by increases or decreases of the other WBC types, the absolute count provides a more useful quantitative assessment. In a simplified example, if an adult patient had a total WBC count of 20 ×10⁹/L with a relative count of 85% neutrophils and 15% lymphocytes, the absolute counts would be 17 × 10⁹/L for neutrophils and 3 × 10⁹/L for lymphocytes. Comparing these results to the reference ranges in Table 7-2, there is both a relative and an absolute increase in neutrophils. On the other hand, although the relative count for the lymphocytes is decreased, the absolute count is well within the reference range.

White Blood Cell Functions

Neutrophils, eosinophils, and basophils are included in a general category of cells called granulocytes because of their prominent granules. However, they have different functions.

Segmented neutrophils (previously called polymorphonuclear leukocytes) are the most numerous cell type in the peripheral blood, constituting 50% to 70% of circulating WBCs. A band is a slightly less mature neutrophil in which the nucleus has not yet segmented. Bands normally represent only 0% to 5% of the circulating WBCs.

The primary function of neutrophils is to destroy invading microorganisms, foreign material, and dead cells. They accomplish this by phagocytosis, a process in which the neutrophil engulfs and destroys the particles through release of various enzymes and reactive molecules.

Neutrophils are produced and stored in the bone marrow, a process that takes 8 to 12 days. However, if the demand for neutrophils increases—as may occur in an acute bacterial infection— their time in the bone marrow may be shortened to as few as 2 days. In these cases, some immature neutrophils may be released. Once released into the peripheral blood, neutrophils have a very short half-life of 6 to 8 hours.

In the peripheral blood, neutrophils are continuously exchanged between two intravascular pools, with about half the cells in the circulating pool and half in the marginated pool. The circulating neutrophils are those counted in the CBC. Marginated neutrophils adhere to the walls of the blood vessels and are not counted. Neutrophils are able to rapidly shift from one pool to the other based on physiologic conditions (discussed subsequently).

Eosinophils are a type of granulocyte with large granules that stain bright orange or pink, whereas basophils have large granules that stain dark blue or purple. Eosinophils normally constitute 1% to 3% of WBCs, whereas basophils are even more rare at 0% to 1%. Both cell types are involved in immune system regulation, control of parasitic infections, and allergic reactions. Eosinophils also accumulate at the site of allergic reactions.

Lymphocytes constitute 20% to 45% of circulating WBCs in adults; in healthy children, the relative and absolute lymphocyte count is higher. Lymphocytes are particularly important in the body’s defense against foreign microorganisms and cells. There are three major types of lymphocytes: T cells, B cells, and NK (natural killer) cells. T and B cells participate in the body’s adaptive or specific immune response by recognizing foreign antigens and tagging them for destruction. T cells are involved in cell-mediated immunity, which is particularly important in eliminating viruses and other intracellular organisms. B cells are involved in humoral immunity and develop into antibody-producing cells. NK cells are able to destroy certain virally infected cells and tumor cells.

The different lymphocyte types appear similar on a peripheral blood smear. Enumeration of the different types of lymphocytes is not done routinely but can be accomplished using monoclonal antibodies. Approximately 85% of circulating lymphocytes are T cells, with B cells making up 10% to 15% and NK cells less than 2%. T lymphocytes also can be separated into subcategories. For example, knowledge of the CD4⁺ T-lymphocyte cell count provides important information about the severity and prognosis of patients with acquired immunodeficiency syndrome (AIDS; see later discussion of lymphocytopenia).

Monocytes are the largest WBCs normally seen in the peripheral blood and constitute 2% to 11% of circulating WBCs. In tissues, the monocyte is known as a macrophage. The primary functions of the monocyte are phagocytosis of organisms and other foreign material invading the body and initiation and regulation of the specific immune response with T lymphocytes. In the lung, alveolar macrophages play a key role in clearing inhaled particulate matter.

Nonmalignant White Blood Cell Abnormalities

Leukocytosis is an increase in the WBC count above the reference range, whereas leukopenia is a decrease in the WBC count below the reference range. Abnormalities in the WBC count can be defined more specifically by referring to the specific cell type that is increased or decreased using similar terminology. For example, an increase in neutrophils is called neutrophilia, and a decrease is called neutropenia. Increases in the cell counts may be primary (a result of uncontrolled proliferation of cells in the bone marrow) or secondary (a result of stimulation of the bone marrow secondary to other diseases or disorders). Similarly, decreases in cell counts may be caused by either primary bone marrow failure or increased destruction of the cells peripherally. Bone marrow failure can occur as a side effect of various drugs and disorders (secondary) or as a result of unknown causes (primary or idiopathic).

Neutrophilia is a common response to acute bacterial infections, such as bacterial pneumonia (Box 7-1). Neutrophilia also may occur in response to fungal, parasitic, or early viral infections. In addition, neutrophilia also occurs in inflammation due to autoimmune disorders or tissue damage that occurs after surgery, burns, myocardial infarction, or traumatic injury. Acute hemorrhage or hemolysis, metabolic disorders (such as acidosis or uremia), and certain drugs, toxins, and chemicals also cause neutrophilia. When the bone marrow is stimulated to release neutrophils at a rate faster than it can produce them, it releases them at increasingly more immature stages. This is called a left shift. The degree of the left shift and the severity of the neutrophilia usually correlate with the severity of the infection.

Pseudoneutrophilia, also called physiologic neutrophilia, occurs when marginated neutrophils are shifted to the circulating pool and are counted in the CBC. This type of neutrophilia is immediate and typically transient, lasting less than an hour. Because these are mature neutrophils, there is no spike in band-type cells as seen in pathologic neutrophilia.

Neutropenia may occur as a result of decreased bone marrow production or increased destruction of circulating neutrophils (Box 7-2). Many drugs, some chemicals, and radiation therapy can cause neutropenia by destroying the hematopoietic cells in the bone marrow, thus decreasing bone marrow production. One of the most common causes of neutropenia is cancer chemotherapy. Because of their short half-life, the neutrophils are the first blood cell type to decrease with chemotherapy. The absolute neutrophil count is closely monitored during chemotherapy, and adjustments in therapy are made if it drops too low. Other causes of decreased production include acquired and hereditary defects in hematopoietic stem cells, infiltration of cancer cells into the bone marrow, and deficiencies of vitamin B₁₂ or folate. Neutrophils can also be quickly depleted by overwhelming infections or be destroyed as a result of the production of antibodies against them. Pseudoneutropenia is a transient shift of the neutrophils in the circulating pool to the marginated pool. It may be caused by bacterial endotoxins or hypersensitivity reactions.

Neutrophils are the first-line defense against microorganisms, so patients with neutropenia have a high risk for developing life-threatening bacterial or fungal infections. The lower the absolute neutrophil count and the longer the duration of the neutropenia, the greater the risk for serious infection.

Eosinophilia (increase in eosinophils) is often seen in parasitic infestations and allergic states (such as hay fever, dermatitis, and drug reactions). Patients with extrinsic or atopic asthma often have eosinophilia. Basophilia (increase in basophils) is usually associated with myeloproliferative neoplasms (discussed later in the chapter).

Lymphocytosis (increase in lymphocytes) is typically seen in viral infections and certain bacterial (pertussis) and parasitic (toxoplasmosis) infections. In some viral infections, especially infectious mononucleosis, many of the lymphocytes are enlarged and have a characteristic appearance, being labeled as reactive or variant lymphocytes. Interpretation of lymphocytosis should take into account the patient’s age because children normally have higher counts than adults. Lymphocytopenia (decrease in lymphocytes) is seen in acquired and congenital immune deficiency states and in various conditions such as acute inflammation, malnutrition, and after treatment with chemotherapy, radiation, or corticosteroids. Lymphocytopenia is an important feature of human immunodeficiency virus (HIV) infection, the virus that causes AIDS. The virus infects and destroys the CD4⁺ helper T lymphocytes, resulting in their depletion. As the CD4⁺ cells decline, the immune system becomes more compromised, leading to increased risk for certain cancers and infections. Peripheral blood CD4⁺ counts, along with the HIV viral load, are used to monitor the progression of the disease.

Monocytosis (increase in monocytes) is characteristic of certain infections, including tuberculosis, syphilis, typhoid fever, and subacute bacterial endocarditis. A monocytosis often signals the recovery stage of an acute infection. Monocytosis also may occur in inflammatory conditions and autoimmune states.

Malignant White Blood Cell Abnormalities

Leukemias, myeloproliferative neoplasms, and myelodysplastic syndromes are the primary hematologic malignancies involving the bone marrow and peripheral blood. Leukemias result from the uncontrolled proliferation (growth) of a specific type of WBC and may be either acute or chronic. Myeloproliferative neoplasms encompass a spectrum of diseases resulting from an abnormality in stem cells (the precursor of all blood cells). Myeloproliferative neoplasms may have a variable blood picture as the disease progresses, and they sometimes terminate as an acute leukemia. Myelodysplastic syndromes are characterized by decreases in WBCs, RBCs, and platelets in the peripheral blood due to a stem cell defect.

In acute leukemia, there is a mutation that causes a maturation arrest in a blood cell precursor at an early stage of development. Typically, blasts, which are the most immature stage of a cell type, accumulate in the bone marrow and peripheral blood. These blasts quickly replace all other cells in the bone marrow and, if left untreated, will cause a rapid death of the patient. WBC counts are variable, but the key feature in the WBC differential is the presence of blasts. The normal WBCs, along with RBCs and platelets, are decreased. Acute lymphoblastic leukemia usually occurs in young children, whereas acute myeloid leukemia most often occurs in infants and older adults. Acute leukemias are classified by the World Health Organization according to the presence of recurrent genetic abnormalities in the leukemia cells.

Chronic leukemias result from a slower proliferation and accumulation of more mature cells in the peripheral blood and bone marrow. Chronic lymphocytic leukemia is the most common type of leukemia and occurs predominantly in elderly people. The WBC count is increased, and the differential shows a preponderance of mature lymphocytes. Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm that occurs predominantly in middle-aged adults. It is caused by a mutation in a stem cell that results in uncontrolled proliferation of granulocytes. The WBC count is increased, and the bone marrow and peripheral blood contain vast numbers of neutrophils, with immature neutrophils in all stages of maturation, as well as eosinophils and basophils. Platelets are also increased, and there is progressive anemia. Without treatment, CML terminates in acute leukemia, known as blast transformation.

Other myeloproliferative neoplasms include polycythemia vera, primary myelofibrosis, and essential thrombocythemia. Polycythemia vera is characterized by a proliferation of granulocytic, erythrocytic, and platelet precursors in the bone marrow with an increase in WBCs, RBCs, and platelets in the peripheral blood. Myelofibrosis is characterized by defective hematopoiesis caused by the excessive growth of fibrous tissue (fibrosis) in the bone marrow. Essential thrombocythemia primarily involves the excessive proliferation of megakaryocytes, with increased platelets in the peripheral blood. Myelodysplastic syndromes result from stem cell mutations that cause ineffective blood cell production in the bone marrow and a progressive decrease in WBCs, RBCs, and platelets in the peripheral blood. They occur predominantly in the elderly.

Hematocrit (Packed Cell Volume)

The Hct is the ratio of the packed RBC volume to the volume of whole blood. A manual Hct is performed by centrifuging whole blood to pack the blood cells to the bottom of a small capillary tube and then measuring the percentage or fraction of the total blood volume occupied by the RBCs (Fig. 7-1). CBC analyzers determine the Hct using various automated technologies. Because it measures the volume of the hemoglobin-containing RBCs, one can estimate the Hct by multiplying the hemoglobin content times a factor of three.

Hemoglobin

Hemoglobin is the protein that carries oxygen to the tissues; it is the major component of RBCs. Hemoglobin also is important in maintaining acid-base balance by acting as a buffer and by carrying carbon dioxide (CO₂) from the tissues to the lungs. The hemoglobin molecule consists of four heme groups, each with an iron molecule capable of binding oxygen, and four globin chains.

Hemoglobin is reported in grams per deciliter or liter of blood. The reference ranges for the RBC count, Hct, and hemoglobin vary by gender, with men having higher levels than women because of the positive influence of testosterone on RBC production (see Table 7-1). Age also affects hemoglobin levels, with higher values in newborns and slightly lower values in elderly people.

Red Blood Cell Indices

Three erythrocyte indices are the mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC). They are calculated from the RBC count, hemoglobin, and Hct. The MCV is the average volume of the patient’s RBCs. A decrease in the MCV indicates the RBCs are smaller, or microcytic, whereas an increase in the MCV indicates the RBCs are larger, or macrocytic. A normal MCV indicates the RBCs are normal in volume, or normocytic, although the presence of an equal number of smaller and larger RBCs could produce a normal MCV. The MCH is the average weight of hemoglobin in the patient’s RBCs. The interpretation of the MCH correlates with that of the MCV. The MCHC is the concentration of hemoglobin in the patient’s RBCs. A decrease in the MCHC indicates the RBCs have less hemoglobin, called hypochromic, whereas a normal MCHC indicates the RBCs have a normal complement of hemoglobin, called normochromic.

Red Blood Cell Abnormalities

Anemia is a decrease in the oxygen carrying capacity of the blood due to a quantitative deficiency or a functional defect in hemoglobin. Although the RBC count and Hct also decrease, anemia is clinically defined by a decrease in the hemoglobin concentration below the reference range for an individual’s gender and age.

One of the body’s responses to hypoxia is an increase in secretion of erythropoietin by the kidneys, which accelerates the maturation and release of new RBCs into the circulation. The circulatory system also responds to anemia by increasing the heart rate, respiration rate, and cardiac output to deliver oxygen more quickly to the tissues.

General symptoms of anemia include fatigue and dyspnea, but various other symptoms may also occur depending on the cause of the anemia. In severe anemia, tachycardia and hypotension also occur, which if untreated can lead to cardiac failure. The severity of the symptoms depends on the degree of hemoglobin reduction, the state of the patient’s cardiovascular system, and the rate of development and duration of the anemia.

Anemias may be classified by pathophysiology into those caused by a decrease in RBC production and those caused by an increase in RBC destruction or loss. However, a more practical approach is to use the MCV to classify anemias into microcytic, macrocytic, or normocytic types.

The most common type of anemia worldwide is the result of iron deficiency. Iron deficiency anemia occurs when there is a dietary deficiency of iron, increased need for iron that is not met (pregnancy, infancy, adolescent growth spurts), malabsorption of iron, or chronic blood loss. In chronic blood loss, the iron contained in the RBCs slowly leaves the body instead of being recycled. Iron is required for hemoglobin synthesis, and with sustained, low iron levels, the bone marrow begins to produce smaller RBCs with a decreased amount of hemoglobin, and the RBC indices become microcytic and hypochromic.

Anemia also can result from chronic inflammation that causes an impairment of iron regulation and RBC production in the bone marrow. The resulting anemia is often normocytic, but it can be microcytic in diseases of long duration. Macrocytic anemia is most often caused by either folate or vitamin B₁₂ deficiency. Low folate or vitamin B₁₂ levels impair DNA synthesis in the nucleus and result in ineffective blood cell production and macrocytic RBCs.

The hemolytic anemias constitute a large group of disorders in which excessive destruction of RBCs in the circulation exceeds the capacity of the bone marrow to replace the cells that are lost. Hemolytic anemia may be due to intrinsic defects in the RBCs (such as sickle cell anemia) or abnormalities extrinsic to the RBCs (such as hemolysis caused by antibodies, malaria, or drugs).

Hemoglobin may be converted to an inactive form through oxidation, denaturation, or other chemical reaction. The most common forms of inactive hemoglobins are carboxyhemoglobin (COHb) and methemoglobin (metHb). The measurement of these abnormal Hb combinations and their impact on oxygen transport are discussed in detail in Chapter 8.

Polycythemia is an increase in the RBC count, hemoglobin, and Hct and may be primary, secondary, or relative (spurious). Primary polycythemia is uncommon and is caused by an uncontrolled proliferation of hematopoietic cells within the bone marrow (polycythemia vera, previously discussed)

Secondary polycythemia is more common and is seen in patients who have chronic stimulation of the bone marrow to produce more RBCs secondary to some other disorder or condition. Examples include patients with chronic hypoxemia due to disease (e.g., chronic obstructive pulmonary disease [COPD]), obstructive sleep apnea, pulmonary fibrosis, chronic heart failure) or as compensation for the low oxygen pressures breathed by those living at high altitude. In these cases, chronic hypoxemia stimulates renal production of erythropoietin, causing the bone marrow to produce and release additional RBCs to compensate for the oxygen deficit.

Heavy smokers also may exhibit secondary polycythemia. The carbon monoxide associated with cigarette smoke binds tightly with the hemoglobin and reduces oxygen transport. This results in a functional anemia to which the bone marrow responds by increasing RBC production.

In the presence of significant hypoxia due to severe anemia, the maturation time of RBCs in the bone marrow is decreased, resulting in the release of immature nucleated RBCs (NRBCs), which are not normally seen in the blood of adults. As a result of the hypoxic conditions in utero, NRBCs are present in fetal blood and newborns but disappear 3 to 4 days after birth. NRBCs also occur in patients with some myeloproliferative neoplasms.

Both primary and secondary polycythemias involve an absolute increase in the total RBC mass. Sometimes, relative polycythemia occurs because of a decrease in the plasma volume. This is seen in patients who are dehydrated. Relative polycythemia does not represent a true increase in the number of circulating RBCs.

Although polycythemia is helpful in increasing the oxygen-carrying capacity of the blood, it can be detrimental to the heart and circulatory system. Polycythemia increases the viscosity of the blood, causing both an increased cardiac workload and a greater risk for clot formation and thrombosis. Treatment focuses on management of the underlying cause and in some patients may include restoring a normal Hct by controlled removal of whole blood through phlebotomy.

Reticulocyte Count

The reticulocyte is the final erythrocyte development stage before the RBC is fully mature. These slightly immature RBCs lack a nucleus and differ from mature RBCs in that they are slightly larger and have not yet assumed a biconcave shape. A reticulocyte count is performed by obtaining the percentage of reticulocytes among the RBCs. Normally about 1% of the circulating RBCs are reticulocytes, with slightly higher values in newborns. The reticulocyte count is also reported as an absolute count by multiplying the reticulocyte percentage by the RBC count. Reference ranges are provided in Table 7-1.

The reticulocyte count helps to assess the bone marrow response to an anemia. If the absolute reticulocyte count is high in a patient with anemia, it indicates that the bone marrow is responding to the anemia by increasing production of RBCs. In this case the cause of the anemia is likely due to excessive peripheral blood loss or destruction. However, if the reticulocyte count is low, then the anemia is likely the result of decreased bone marrow production.