5 Sherilyn A. Gross This chapter describes toxicities affecting blood. The following subjects are covered: The hematopoietic system is the most dynamic organ system in the human body. All hematopoietic or blood cell lineage develop within the bone marrow microenvironment and each cell type is groomed toward maturation within the niches of the bone marrow by exposure to specific growth factors and cytokines, as well as cell-to-cell and cell-to-stroma interactions. Once the hematopoietic cell reaches a specific stage of maturation, the marrow releases it into the peripheral blood where it further matures into a terminally differentiated cell that performs a unique cell function. For example, one cell function for a mature red blood cell (RBC) is to carry oxygen from the lung through the peripheral blood and deliver it to the tissues. A cell function for a platelet is to release proteins necessary for clot formation and a mature neutrophil’s primary function is travel to inflamed tissues where they protect the body against bacterial infection. Despite the common origin of hematopoietic cells, there is a quantitative and qualitative equilibrium between all blood cell lineages under normal physiological conditions. If the cell does, indeed, reach terminal differentiation, each cell lineage has a normal quantitative range of mature cells in the peripheral blood and a finite lifespan. For example, RBCs normally range from 4.2 to 5.9 × 106 cells/μl with an average lifetime of 120 days. The normal range for platelets in peripheral blood is 140–440 × 103 cells/μl and a lifespan of 14 days, whereas neutrophils have a normal range of 35–85 cells/μl in peripheral blood with a lifespan of only 12 h. Therefore, the bone marrow is continually producing all blood cell lineages in varying quantities in order to maintain physiological homeostasis. The continuous proliferation and development of all blood cell lineages within the niches of the bone marrow microenvironment makes the bone marrow susceptible to toxins, especially toxins that target proliferating cells. Toxic influences such as chemical agents, irradiation, or severe infection usually lead to a greater or lesser degree of suppression of all blood cell lineages. The most extreme result of a toxic effect is the suppression of all hematopoietic cell series. An examination of hematopoiesis will aid in understanding the necessity of continuous proliferation, and, subsequently, the vulnerability of the hematopoietic system to toxic effects. Hematopoiesis is the process by which mature blood cells of distinct lineages (e.g., platelets, erythrocytes, granulocytes, monocytes, and lymphocytes) are produced from pluripotent hematopoietic stem cells (HSCs). It is a dynamic process with respect to both lineage decisions and origin during development. During fetal development, hematopoietic activity sequentially progresses from the yolk sac to the liver, from the liver to the spleen, and from the spleen to the bone marrow. By age 20, most of the long bones have lost their hematopoietic ability; thus continuous cell development in the adult occurs in the marrow of the vertebrae, sternum, ribs, and pelvis. The bone marrow fills a three-dimensional architecture created by the cancellous tissue normally present in the interior of the bone. The trabecular pattern is an irregular meshwork of stress-related struts within the cancellous bone. The bone marrow vasculature is derived from bone artery, which branches into a capillary network of thin-walled sinusoids. The sinusoids are lined with endothelial cells supported by a discontinuous layer of reticular cells that synthesize reticulin fibers, which form an addition layer of meshwork to support hematopoietic cell development. The discontinuous layer of reticular cells is thought to provide a route for mature hematopoietic cells to enter the bloodstream. The extracellular matrix contains collagen, laminin, and fibronectin, which help to facilitate adhesion of hematopoietic cells to bone marrow stroma. If you can envision the architecture within the bone marrow, then it is easy to imagine this three-dimensional space as a perfect home for the formation of individual niches devoted to the development of specific hematopoietic cell lineage. For additional reading on niche regulation of bone marrow cells, see Renstrom et al. (2010). The stimulus for understanding and characterizing HSC was the clinical need for cells capable of protecting humans exposed to lethal doses of irradiation. Death due to radiation poisoning was associated with bleeding and infections; however, the mechanism by which these symptoms occurred was initially unknown. The first insight came with the observation that lead shielding of hematopoietic tissue prevented death from lethal doses of radiation. Following this observation, scientists noted that intravenous infusions of syngeneic marrow after radiation also prevented death by repopulating the bone marrow with all hematopoietic cell lineages. In 1961, the first assay for HSC, known as the colony-forming unit–spleen (CFU-S) assay was developed so that scientists could examine the functional characteristics of HSC. Using this assay, investigators observed that primitive bone marrow progenitors could give rise to hematopoietic colonies in the spleen of lethally irradiated mice. Through a series of experiments, the results suggested that bone marrow may contain highly proliferative progenitor cells capable of forming colonies of myeloid, erythroid, and megakatyocytic cells. Thus scientists proposed that the HSC must exist within the bone marrow and that these cells are capable of multilineage differentiation as well as self-renewal. These findings lead to the current view that in order for HSC to sustain hematopoiesis throughout an individual’s lifetime, HSC must be capable of (i) maintenance in a noncycling state, (ii) self-renewal to generate additional HSCs, and (iii) production of progenitor cells with more limited developmental potential. Due to the fact that the process of self-renewal and differentiation are tightly coupled within the progenitor cell pool, several models have been proposed for hematopoiesis. The models for self-renewal consider various scenarios. First, there may be multiple lineages of HSC with intrinsic differences in self-renewal and differentiation in response to the microenvironment. Second, the stem cell pool may be organized into at least three distinct populations with discrete self-renewal potential. Finally, the stem cell pool may represent a continuum of HSC with the potential for self-renewal. In addition to self-renewal, inductive and stochastic models have been proposed to interpret hematopoietic commitment and differentiation. The inductive model proposes that the binding of hematopoietic growth factors to their receptors induce differentiation, and that lineage choice is determined by multipotent hematopoietic progenitor cells (HPC) by activating the transcription of lineage-specific gene programs. In contrast, the stochastic model proposes that multipotent HPC do not require exposure to external stimuli to undergo lineage commitment. Instead, this model suggests that hematopoietic growth factors simply permit the proliferation of intrinsically committed cells and the subsequent expression of mature phenotype. It is possible that both the inductive and the stochastic models represent HPC commitment and differentiation. Regardless of the model, it is well established that growth factors and growth factor receptor expression play a key role in hematopoietic lineage commitment, growth, and differentiation. The typical language used to describe terminally differentiated cells in the peripheral blood includes RBCs, platelets, and white blood cells (WBCs). However, the differentiation process and terminology associated with blood cell maturation is more complicated. Blood cell maturation is referred to as poiesis—“the formation or production of.” Myelopoiesis is the production of granulocytes, erythrocytes, macrophages, and megakaryocytes from the multipotent granulocyte erythrocyte–macrophage megakaryocyte-colony-forming unit (GEMM-CFU). Erythropoiesis refers to the formation of RBCs (also called erythrocytes). The hormone erythropoietin, produced in the kidneys, controls the process of erythropoiesis. Erythropoiesis occurs within the niches of the bone marrow architecture and, as discussed previously, originates from the multipotent GEMM stem cell. Further commitment to the erythroid lineage includes the blast-forming unit–erythrocyte (BFU-E), which then matures into the colony-forming unit–erythrocyte (CFU-E). The proerythroblast is the earliest distinguishable red cell precursor in the bone marrow and is characterized by a dense nucleus that turns deep blue with Romanowsky staining. The daughter cell is termed a basophilic erythroblast, which has a smaller nucleus than the parent cell. These erythroid precursors only occur in the peripheral blood under pathological conditions. Mitosis generates more mature red blood precursor cells called polychromatic erythroblasts characterized by grayish blue cytoplasm and the potential to divide. Orthochromatic erythroblasts are distinguished from the parent cell by cytoplasm with a pink hue (i.e., contains hemoglobin (Hgb)) and are no longer able to divide. The nuclei of the orthochromatic erythroblast gradually condense into small black spheres without structural definition that are eventually expelled from the cell. The now enucleated young erythrocytes contain an abundant amount of ribosomes that precipitate into reticular (“net-like”) structures, hence the name reticulocytes. Reticulocytes are still considered immature erythrocytes and are only in the peripheral blood for approximately 1 day before they transform into mature erythrocytes. An increased number of reticulocytes in the blood is known as recticulocytosis and is indicative of a condition that has stimulated erythropoiesis such as anemia or blood loss. As previously stated, erythrocytes have a life span of approximately 120 days before their membranes weaken and the reticuloendothelial phagocytic cells in the spleen remove them from the blood. Mature erythrocytes have a discoid shape and are loaded with the heme-containing protein, Hgb, which is responsible for their unique function. Erythrocytes’ function is to transport oxygen to tissues in the arterial vasculature and carry carbon dioxide back to the lungs for elimination in venous vasculature. Erythrocytes also provide a blood pH buffering capacity by converting carbon dioxide to carbonic acid via the enzyme carbonic anhydrase. Erythrocytes possess an outer membrane that is supported by a complex cytoskeletal system essential for squeezing through the smaller capillaries of the vascular system. Not surprisingly, if this cytoskeleton is damaged in response to reactive oxygen species, chemical attack, or hereditary disease, the changes may lead to membrane fragility and hemolysis. Thrombopoiesis is the technical term for the development of thrombocytes (platelets). The hormone thrombopoietin, produced by the liver, bone marrow stromal cells, and various other organs, is the primary regulator of platelet production. Platelets are derived from the multipotent GEMM stem cell and thrombopoiesis proceeds independently toward the development of megakaryoblast cells when the granulocyte–macrophage stage of maturation is complete. The megakaryoblast reduplicates its nuclear and cytoplasm components up to seven times without cell division, and each reduplication causes an increase in the number of complete chromosome sets, nuclear lobulation, and cell size. Thus the megakaryocyte is an extremely large, lineage-committed precursor cell that is easily recognized by its size and multilobed nucleus within the niches of the bone marrow. Platelets are formed from the cytoplasmic fragmentation of the megakaryocyte within the bone marrow and are identified in the peripheral blood as small disc-shaped anucleated cells. The normal number of platelets in the peripheral blood ranges from 140 to 440 × 103 cells/μl with a lifespan of 14 days. Mature platelets have a well-developed cytoskeleton with bands of microtubules. In addition, the mature cytoplasm has granules that contain coagulation factors, serotonin, acid hydrolases, and peroxidase. These components aid in the platelets’ ability to serve as the first line of defense against damage to blood vessels. Once the epithelial lining of a blood vessel is compromised, platelets adhere to collagen by interacting with coagulation factor. Platelet actin, myosin, and microtubules cause reversible platelet adhesion along a broad surface of the damaged endothelium. Platelets release the contents of their granules and begin the synthesis of thromboxane. Combining with thromboxane, ADP and Ca2+ ions mediate the adhesion of other platelets. Platelet phospholipids activate the blood clotting cascade, leading to the formation of fibrin. Neutrophils, eosinophils, and basophils are all considered as granulocytes because at maturation the cytoplasm of these cell types contains prominent granules. All granulocytes are derived from a common myeloid-committed progenitor cell and the first recognizable granulocytic precursor cell is the myeloblast. Myeloblast maturation into a neutrophil takes approximately 7–8 days. The differentiation process includes the subsequent stages of promyelocyte, myelocyte, metamyelocyte, band cell, and finally, the segmented neutrophil. The band cell has a horseshoe-shaped nucleus whereas the segmented neutrophil can be identified by its multilobed nucleus and abundant cytoplasm. Although no further division takes place, cellular acquisition of chemotactic ability, receptor expression, and complement are features of neutrophil maturation. Structurally mature neutrophils remain in the bone marrow for an additional 5 days prior to their release into the bloodstream. Neutrophils only circulate in the peripheral blood for approximately 6 h before they migrate into tissues where they can survive for an additional 2–5 days. Interestingly, some neutrophils are stored in the bone marrow and are capable of quick mobilization in response to bacterial infection. Neutrophils have a major role in the phagocytosis of bacteria and dead cells; therefore, they contain acid hydrolase, myeloperoxidase, alkaline phosphatase, and antibacterial and digestive agents, among many other substances. Neutrophils are attracted to areas of infection or tissue damage by the release of degrading cellular chemicals (i.e., chemotaxins). They migrate through the endothelium and, once in the tissue, the neutrophils move by way of actin filaments. Neutrophil phagocytosis expends all the neutrophils’ cellular energy, and they die soon after. The collection of dead neutrophils, tissue fluid, and degradation material is termed pus. Basophils share a common bone marrow precursor cell with the other granulocytic cells up to the myeloblast stage. Basophilic differentiation is analogous to neutrophil maturation. Mature basophils are characterized by large, basophilic (blue-stained) granules and are considered precursors to tissue mast cells. Mast cells migrate to supportive tissue, like below the epithelia, around blood vessels, and in the lining of serous cavities. They are long-lived and can proliferate in tissue in response to T lymphoctyes. The granules of both basophils and mast cells contain sulfated protoglycans, heparin, chondroitin sulfate, histamine and leukotriene 3. Both basophils and mast cells have surface receptors that are highly specific for allergens. Exposure to allergens results in rapid exocytosis of their granules and release of histamine and vasoactive mediators resulting in an immediate hypersensitivity reaction. Eosinophils also share a common progenitor cell with other granulocytes and their differentiation pathways diverge after the myeloblast stage as well. Eosinophils are distinguishable from neutrophils at the myelocyte stage by the appearance of eosinophilic (i.e., red-stained) granules. Eosinophils remain in the bone marrow for several days and then circulate in the peripheral blood for 3–8 h before migrating into the skin, lungs, and gastrointestinal tract. Interestingly, eosinophils have a marked diurnal pattern with maximum numbers circulating in the peripheral blood in the morning and minimal numbers in the afternoon. Eosinophilic granules contain hydrolytic lysosomal enzymes and peroxidase, acid phosphatase, large concentrations of aryl sulfatase, and an alkaline protein referred to as major basic protein. Perhaps one of their main functions of eosinophils is that they increase in number in response to parasitic infection. They can also phagocytize small molecules or undergo degranulation in response to bacterial products and complement components. In addition, eosinophils are attracted to products of mast cells and respond to the localized destruction of mast cell granules by neutralizing histamines and releasing prostaglandins thought to inhibit mast cell degranulation. Monocytes are derived from the multipotent GEMM stem cell in the bone marrow, and differentiation proceeds independently toward the development of macrophage when the granulocyte–macrophage stage of maturation is complete. The monoblast and the promonocyte are two morphological distinguishable precursors. At least three cell divisions occur before the monocyte maturation is reached. Mature monocytes leave the bone marrow soon after maturation and circulate in the peripheral blood for approximately 3 days before migrating to the tissues. Circulating monocytes are large, motile, phagocytic cells, and contain numerous lysosomal granules and cytoplasmic vacuoles. The granules contain acid phosphatase, aryl sulfatase, and peroxidase analogous to the components found in neutrophilic granules. Monocytes have numerous small pseudopodia extending from their cellular membrane and respond chemotactically to necrotic material, invading microorganisms, and inflammation. When monocytes leave the bloodstream, they are referred to as macrophages and are unable to reenter the circulation. The monocyte–macrophage system of circulating monocytes and tissue macrophages are found in both the free and the fixed forms. This cell system also includes the Kupffer cells of the liver, pulmonary alveolar macrophages, and dendritic antigen presenting cells. Multiple growth factors contribute to cellular maturation of each lineage at various stages of cell differentiation. These growth factors were defined historically, by trial and observation of the type of lineage-committed HPC that were stimulated to produce colonies. A great deal of information has been accumulated regarding chromosome mapping, protein structure, cellular origin, and cellular functions of these growth factors, as well as other interleukin cytokines and their respective receptor expression—all of which contribute to lineage commitment, cellular differentiation, and cell function. A detailed discussion of these growth factors, cytokines, and receptor expression are beyond the scope of this book chapter. For additional information, please refer to Jandl’s (1996) Textbook of Hematology. The bone marrow is also the site for production of both B and T cell lymphocytes. A common myeloid and lymphoid stem cell was demonstrated in irradiated mice transplanted with HSCs and evidenced by the repopulation of the irradiated bone marrow with all hematopoietic cell lineages. Although both the myeloid and the lymphoid cell lineages are thought to originate from a pluripotent HSC, commitment toward the lymphoid lineage is thought to occur in a pluripotent lymphopoietic bone marrow cell. T lymphocytes migrate from the fetal bone marrow to the thymus where they undergo three to four additional divisions before their release into the peripheral blood and lymph nodes. Mature T lymphocytes are responsible for cell-mediated immune response. In contrast to T lymphocytes, B lymphocyte progenitor and precursor cells reside and differentiate in the bone marrow. Most B cells leave the bone marrow before full maturation and travel to the spleen and lymph nodes where they acquire specific receptors and surface immunoglobulins such as IgM and IgD. Adult bone marrow retains very few B lymphocytes. Mature B lymphocytes are responsible for humoral immunity mediated by the secretion of antibodies. Lymphocytes are the most numerous type of white cell in the peripheral blood. Most lymphocytes are small with only 3% considered large activated lymphocytes on route to target tissues. The lymphocyte has a kidney-shaped nucleus with dense chromatin. It is important to note that small lymphocytes in the peripheral blood cannot be distinguished as B or T lymphocytes by morphology or staining and are not the functional end form, but undergo transformation in response to specific immunological stimuli. However, plasma cells, which are a differentiated form of B lymphocytes, can be visualized in the peripheral blood by the morphological trademark “pin–wheel” chromatin pattern, which is reflective of active immunoglobulin synthesis. Toxicity associated with B and T lymphocytes is technically termed immunotoxicity and is usually discussed independent of hematoxicity. Lymphopoiesis is discussed here only for the purpose of inclusion of lymphocytes as a component of WBCs in the peripheral blood. Immunotoxicity will not be discussed in this chapter. There is a quantitative and qualitative equilibrium between all blood cells under normal conditions. This equilibrium is regulated by a variety of growth factors, cytokines, and cellular communication in order to ensure physiological balance between blood cell production and degradation. There are a number of assays that are routinely used to determine the status of blood cell equilibrium. For example, a complete blood count (CBC) and a blood chemistry profile are common automated assays performed with peripheral blood as part of a wellness examination in order to establish baseline parameters for a multitude of body processes (e.g., red and white cell counts, clotting factors, liver enzymes, serum lipids, electrolyte levels, and metabolic by-products). In turn, disturbances in bone marrow function are accompanied by changes in the composition of peripheral blood cells and blood proteins. CBC and blood chemistry studies are also used for clinical diagnosis when disorders of the blood and bone marrow are suspected. In addition to routine hematology testing, the World Health Organization introduced the Classification of Tumours of the Haematopoietic and Lymphoid Tissues in 2001 (Jaffe, 2001). The objective was to provide pathologists, oncologists, and geneticists with unified criteria for diagnosis of human neoplasms defined according to a combination of composition of the blood and bone marrow, cellular morphology, immunophenotype, genetic features, and clinical features. This established criterion was based on the state of the science at that time and the use of state-of-the-art diagnostic tools. Since 2001, the scientific knowledge of cell biology and disease has evolved and the resolution of diagnostic tools has improved. As a result, in 2008, WHO published a second edition of the classification system for tumors of the hematopoietic and lymphoid tissue (Swerdlow, 2008).The complete references for Jaffe (2001) and Swerdlow (2008) are found in the reference section of this chapter. A CBC is an automated measurement of the various cell lineages circulating in the peripheral blood at a given moment. The automated cell count has largely replaced the “manual” cell count as an inexpensive, easy, and rapid way to obtain information about the hematological system as well as other organ systems. Normal CBC values are affected by several factors, including age, gender, metabolic activity, circadian rhythms, and nutritional status, as well as blood sampling technique, storage, and counting method. For this reason, laboratories provide a normal range in which 95% of the values are found in a clinically normal group of individuals. Overlap between normal and pathological data is expected to some degree. Therefore, values that fall within the borderline areas must be interpreted with respect to the reference range of normal individuals that closely resemble the patient. For example, the reference range for leukocytes varies between newborns, toddlers, young children, and adults. Once the appropriate reference range is established, the CBC provides the current status of erythrocytes, WBCs (leukocytes), and platelets. The CBC is usually accompanied by a differential blood cell count performed with a peripheral blood smear. The differential measures the percentage of each type of leukocyte present in the same specimen. The peripheral blood smear is prepared manually by spreading one drop of blood sample across a glass slide to achieve a thin smear. The blood is smeared from one end of the glass slide to the other, ending in a “feathered” edge. Dried peripheral blood smears are stained with a mixture of acidic and basic stains so individual WBCs can be distinguished from each other by their cellular components. The cells are counted manually under a microscope. Table 5.1 contains an example of a CBC and accompanying differential. The significance associated with each test is discussed separately. Table 5.1 Normal Range and Mean Values for CBC in Adults Hct, hematocrit; Hgb, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; RDW, red blood cell distribution width. Reference range: 95% of the population. a SI units give the measurements per liter. The leukocyte count refers to the total number of WBCs in 1 mm3 of peripheral blood. As with the CBC in general, the normal range for leukocytes varies with age. The normal range for leukocytes in a newborn is 9,000–30,000 × 106/l and in toddlers the range is from 6,200 to 17,000 × 106/l. Although there is a wide range for total number of leukocytes, there are many causes for abnormal values. In an adult, the critical values may be below 2,500 106/l (leukopenia) and above 10,000 106/l (leukocytosis). An increase in total WBC usually indicates infection, inflammation, necrotic tissue, or leukemia neoplasm, although emotional stress or physical trauma can also cause an increase in WBC. A decrease in total number of leukocytes is most often associated with bone marrow failure induced by antineoplastic agents, radiation, diseases that infiltrate the bone marrow, overwhelming infections, nutritional deficiencies, and autoimmune diseases. The differential WBC count refers to the percentage of each subtype of leukocyte within the total number of leukocytes in a given sample. Leukocytes range from 8 to 20 μm in size. There are five types of leukocytes that compose the WBC differentiation, and these cell subtypes are characterized by the presence or absence of granules. The agranulocytes are lymphocytes and monocytes. The granulocytes are the neutrophils, eosinophils, and basophils. An increase in the percentage of one cell type leads to a decrease in the percentage of another cell type. Neutrophils and lymphocytes make up 75–90% of the total number of leukocytes. When neutrophil production is stimulated, immature forms of neutrophils (i.e., bands) enter the circulation resulting in less mature forms of neutrophils. In the clinical setting, this is referred to as a shift to the left. Increased production of eosinophils is associated with allergic reaction, as these cells are capable of phagocytosis of antigen–antibody complexes. When the allergic reaction subsides, the eosinophil count decreases. Increase in eosinophils is also seen with drug-induced hypersensitivity. These drugs include various antibiotics, gold preparations, hydantoin derivatives, phenothiazines, and dextrans. Hypereosinophilia syndrome, with eosinophil values greater than 40% of the total leukocyte count, is seen with splenomegaly, heart defects, and pulmonary infiltrates. When hypereosinophilia presents clinically in combination with these diseases, it is classified as either an autoimmune disease or myeloproliferative disorders, or somewhere in between. Increase in eosinophil production is also associated with parasitic infection. Acute eosinophilic leukemia is rare. Increase in basophil production (<2–3%) is rare, although it can be associated with allergic reactions to food, drugs, or parasites and is usually seen in clinical cases when eosinophils are also involved. Basophilia is seen with infectious diseases such as tuberculosis and chickenpox and with metabolic diseases such as hyperlipidemia. Proliferation of basophils is associated with myeloproliferative neoplasms, although acute basophilic leukemia is rare. Monocytes function as phagocytic cells inside and outside of the circulatory system. An increase in the monocytic cell population above 7% in the differential blood cell count is indicative of an immune defense reaction. Monocytosis occurs in the case of infection although it usually presents near the end of the infection. Chronic monocytosis is seen especially in brucellosis and tuberculosis. Monocytosis can also be part of a noninfectious response such as Crohn’s disease and ulcerative colitis. Interestingly, monocytosis often occurs in response to a disseminating neoplasm such as carcinoma of the bronchi and breast. Perhaps not surprisingly, monocytosis is prominent in myeloproliferative disorders such as chronic myelomonocytic leukemia (CMML) and acute monocytic leukemia. In fact, monocytic leukemia is associated with a sharp rise in peripheral blood monocyte counts accompanied by a drop in absolute counts in other cell types noted in the differential. Alterations in lymphocyte counts are most often associated with viral infections or in diseases of the lymphatic system. A spontaneous decrease in lymphocyte count is seen only in some rare congenital diseases such as agammaglobulinemia and chromosome 22q11 deletion syndrome. Further, low lymphocyte counts are seen in some systemic diseases such as Hodgkin’s disease and active AIDS. As previously stated, an increase in the percentage of one cell type leads to a decrease in the percentage of another cell type. An example of this is a toxic response in the neutrophil cell series that manifests as a benign increase in the absolute number of lymphocytes. The technical term for this abnormality is agranulocytosis, which refers to an increase in WBCs without granules. There are several different classifications for neutropenia and agranulocytosis, such as drug hypersensitivity reaction, infection, autoimmune neutropenia, congenital or familial neutropenia, and neutropenia secondary to a bone marrow disease. The Hgb concentration is the measure of the total amount of Hgb in the peripheral blood and is a reflection of the number of erythrocytes. The hematocrit (Hct) is a measure of the percentage of total blood volume that is made up of RBCs and is a close reflection of both the Hgb and the RBC value. The Hct value is usually three times the Hgb concentration. A decrease in both Hgb and Hct values are associated with anemia; an increase is associated with erythrocytosis. The erythrocyte count represents the number of RBCs in 1 mm3 of peripheral blood. The normal range for erythrocytes tends to decrease with increasing age. The normal range for erythrocytes in a newborn spans from 4.8 to 7.1 × 1012/l and in young children from 4.0 to 5.5 × 1012/l. An adult female has normal RBC values ranging from 4.2 to 5.4 whereas an adult male ranges from 4.7 to 6.1 × 1012/l. Other causes of variations in RBC counts include a decreased count during pregnancy, an increased count at high altitudes, and an increased or decreased count depending upon hydration status. Intravascular abnormalities can shorten the lifespan of RBCs by causing trauma to the blood cell membrane. These abnormalities include artificial heart valves and peripheral vascular atherosclerosis. An enlarged spleen can also cause an early, inappropriate destruction of erythrocytes. When the RBC values decrease by 10% of the expected normal value, the individual is labeled anemic. Additional causes of low RBC values are hemorrhage, hemolysis, hemaglobinopathy, advanced cancer, bone marrow fibrosis, chemotherapy, renal failure, multiple myeloma, leukemia, and dietary deficiencies. Abnormal increases in RBC values are related to congenital heart disease, polycythemia vera (PV), and pulmonary fibrosis. RBC indices provide information about RBC size (MCV and RDW), weight (mean corpuscular hemoglobin (MCH)), Hgb concentration (mean corpuscular hemoglobin concentration (MCHC)), and maturation (reticulocyte count). RBCs are approximately 8 mm in size, which allows them to pass through the tiny capillaries. Cell size is indicated by terminology such as normocytic, microcytic, and macrocytic. In turn, Hgb content is indicated by the terms normochromic, hypochromic, and hyperchromic. Extremely elevated WBCs and abnormal number of immature RBCs circulating in the peripheral blood can actually affect the RBC indices. Mean corpuscular volume (MCV) is a measure of the average size of a single RBC and is used in classifying anemias. An increase in MCV is associated with abnormally large RBCs typically seen with liver disease, antimetabolite therapy, alcoholism, pernicious anemia, and folic acid deficiency. A decrease in MCV is associated with abnormally small RBC and is seen with iron deficiency anemia and thalassemia. Red blood cell distribution width (RDW) value is indicative of the variation in RBC size. The RDW is important in determining the degree of variability and abnormality in RBC size (e.g., anisocytosis). An increase in RDW is associated with iron deficiency anemia, B12 or folate deficiency anemia, hemolytic anemia, and posthemorrhagic anemias. MCH is a measure of the average amount of Hgb within an RBC. Macrocytic cells have more Hgb content whereas microcytic cells have less Hgb. As such, an increase in MCH is associated with macrocytic and hyperchromic anemia whereas a decrease in MCH is associated with microcytic and hypochromic anemia. MCHC is a measure of the average concentration percentage. This value is used to determine whether the RBCs have increased, decreased, or whether there are normal concentrations of Hgb (e.g., hypochromic, hyperchromic, normochromic). Increases in MCHC are associated with spherocytosis, intravascular hemolysis, and cold agglutins. A decrease in MCHC is associated with iron deficiency anemia and thalassemia. A reticulocyte is an immature RBC and the reticulocyte count can be used to determine bone marrow function and erythropoietic activity. Normally, there are a few reticulocytes circulating in the peripheral blood. An increase in reticulocytes is indicative of the bone marrow producing an increased number of RBCs, which usually occurs in response to anemia. However, if Hgb is normal, then an increase in the number of reticulocytes in the peripheral blood can be associated with increased production of RBCs in response to ongoing loss of red cells from hemolysis or hemorrhage. A normal or low reticulocyte count in an anemic patient indicates that the bone marrow’s response to the anemic condition is an inadequate production of RBCs or the inadequate production of RBCs is causing the anemia (e.g., aplastic anemia, iron deficiency, Vitamin B12 deficiency). Platelet count is the number of platelets per 1 mm3 of peripheral blood. Platelet counts of 150,000–400,000 mm3 are considered within normal range. Counts less than 100,000 mm3 indicate thrombocytopenia, whereas counts that are greater than 400,000 mm3 are referred to as thrombocytosis. Hemorrhage may occur with severe thrombocytopenia especially when platelet counts drop below 20,000 mm3. Thrombocytopenias develop secondary to bone marrow failure or from tumor infiltration of the bone marrow. They can also arise from the sequestration of platelets by the spleen or consumption of platelets due to coagulation within the vasculature. In addition, thrombocytopenias are caused by the accelerated destruction of platelets secondary to antibodies, drugs, or infections. Thrombocytopenia due to reduced platelet production is also associated with chronic alcoholism, cytostatic drugs, virus infection, and vitamin deficiency. Spontaneous thrombocytosis is often associated with malignancies such as leukemias, lymphoma solid tumors, PV, rheumatoid arthritis, and iron deficiency anemia. A platelet count greater than 1,000,000 mm3 is referred to as thrombocythemia. Platelet counts can vary with high altitude and strenuous exercise. Oral contraceptives can increase platelet count, whereas menstruation and some over-the-counter drugs such as acetaminophen, aspirin, and cimetidine can decrease platelet count. Individuals with a low platelet count may have bruising, petechiae (i.e., tiny purple or red spots on the skin resulting from small hemorrhages), nose bleeds, and bleeding of the gums. The mean volume varies with total platelet production. For example, normal bone marrow will react to thrombocytopenia associated with sequestering of platelets by the spleen by releasing larger, immature platelet cells in an effort to maintain a normal platelet count. Therefore, the mean platelet volume will increase. In contrast, if the production of platelets in the bone marrow is inadequate, the released platelets are small and pyknotic (old). For further information on peripheral blood parameters see Wintrobe’s Clinical Hematology, Mossby’s Diagnostic and Laboratory Test References, and Mosby’s Medical Dictionary. The bone marrow cellularity, myeloid:erythroid (M:E) cell ratio, and a bone marrow differential cell count are simple diagnostic tools used to determine the extent of bone marrow involvement in hematotoxicity. Bone marrow aspirate and biopsy are usually obtained from the upper part of the posterior iliac crest (i.e., back of the hip bone). For the core biopsy, a deep local anesthesia is used to numb the area and then a small incision is made on the skin. The core biopsy is collected using a Jamshidi, which is a combination of an obturator, a sharp hollow needle, and a hollow histology cylinder that is at least 1.5 cm in length. The instrument is twisted back and forth through the soft tissue and approximately 30 mm into the bone marrow space. The needle is removed with a twisting motion and then the compact core biopsy is extruded with a plunger. The procedure preserves the bone marrow architecture. The bone marrow aspirate can be collected independently using a cytology needle that is guided by a stylus, which is slowly pushed through the through the soft tissue and the compact of the bone into the marrow space. Removal of the stylus is followed by withdrawal of the plunger, filling the syringe with bone marrow cells in suspension. The distribution of cell types within the bone marrow is fairly constant. A semi-quantitative assessment of the bone marrow cellularity is determined using both the bone marrow aspirate and sections of the core biopsy. There are three crude categories used to describe the relative proportions of bone marrow cells, but the differences between the categories are somewhat arbitrary. For example, bone marrow cellularity is referred to as hypocellular (<40% cellular), normocellular (40–60% cellular), and hypercellular (>60% cellular). Significant changes in cellularity as a whole or in specific cell types help to support a diagnosis. A normal M:E ratio is approximately 3:1 and is valid only as a crude index of relative cellularity. A change in this ratio could mean depression of erythropoiesis or enhancement of myelopoiesis. The M:E ratio is determined in combination with additional assays to help develop a total picture of the bone marrow status. A normal bone marrow differential is a morphological representation of all cell series in varying stages of maturation. Manual differential counts are prepared with microscope slides of bone marrow aspirate and high-powered oil emersion microscopy. An example of a normal bone marrow differential is illustrated in Table 5.2. Table 5.2 Normal Bone Marrow Differential Advances in cell biology have greatly improved knowledge of cell growth and differentiation. Additionally, there have been advances in the identification of molecules that appear both on the cell surface and intracellularly during the normal process of differentiation and maturation. As a result, changes in the normal morphology, histochemistry, cytology, and cytogenetics are important diagnostic markers used to determine the extent of bone marrow involvement in hematotoxicity and/or hematopoietic disease. Using routine staining techniques, cell histology allows for examination of cellular structures in paraffin-fixed bone marrow tissue samples, the most common of which is the H&E stain. In addition, certain dyes have affinity for chemical groups on cellular molecules. For example, lipid moieties are detected by lipid-soluble stains such as Sudan Black. Polysaccharides are identified by periodic acid–Schiff (PAS), which oxidizes glucose residues into aldehydes. A Schiff reagent (Fuchsin) is added, which reacts with the aldehyde to produce purple fuchsin. Enzyme activity can also be identified using an enzyme substrate, a cofactor, and visualizing agents. Immunocytochemistry methods are base on antibodies raised with affinity for specific cellular molecules. Detection of the primary antibody is performed using a second antibody linked to an enzyme called horseradish peroxidase. A well-established antibody detection method, the horseradish peroxidase is developed as a colored reaction and visualized under light microscopy. Immunofluorescence techniques are based on the same antibody affinity, except fluorescence label antibodies are used to identify cellular molecules with fluorescence microscopy. Immunophenotyping is based on fluorescently labeled antibodies with affinity for cluster designation (CD) molecules on the cell surface. Over the last two decades, numerous CD molecules have been identified as cellular protein configurations that are expressed on the cell surface and further characterized by functional activity. These surface molecules include, but are not limited to, growth factor and cytokine receptor molecules, adhesion molecules, ion channels, and lineage-specific molecules. Expression of many of these surface molecules occurs in all cell types and is responsible for normal cellular function (e.g., movement across ion channels). Some surface proteins alter expression as the cell matures (e.g., adhesion molecules, growth factor receptors), and some of these surface molecules play a specific role in mature cell activity (e.g., lineage-specific growth factor receptors). Depending on the specific cell type and the cell’s stage in maturation, these surface molecules provide the phenotypic characteristic of the cell. It is important to note that the cellular phenotype remains fluid throughout lineage maturation until terminal differentiation. Immunophenotyping is the process of fluorescent labeling of these molecules on the cell surface to determine a cell’s specific cell lineage in a specific stage of cellular maturation. Furthermore, noted changes in the normal cellular expression pattern are also used as a diagnostic tool to follow abnormalities in cellular proliferation and differentiation. Many fluorochrome-conjugated monoclonal antibodies with affinity for specific CD molecules are commercially available for analysis of bone marrow cells in suspension. With the use of compatible fluorochromes, multiple conjugated antibodies can be combined to further characterize subpopulations of cells and to minimize the number of cells required for analysis. Fluorescently stained cells are analyzed by flow cytometric techniques in which an argon laser is used to excite the fluorochromes so that each emits spectra at an individual wavelength. The signals from forward and right-angle side scatter are collected in a logarithmic amplifier and then analyzed with software designed specifically for flow cytometric methods. In addition to immunophenotyping, flow cytometric techniques identify intracellular protein expression, cell cycle progression, and apoptosis assays. Hematotoxins and diseases associated with the hematopoietic system can manifest as alterations in differentiation, disruption of the cell cycle, as well as induction and/or inhibition of apoptosis. Flow cytometric techniques are therefore useful in characterizing deviations from normal patterns of cell proliferation, cell differentiation, and cell death. For additional reading on flow cytometric techniques see Howard Shapiro’s Practical Flow Cytometry. The study of whole chromosomes is called karyotyping. In this assay, chromosomes are stained with a specific dye followed by chemically induced cell cycle arrest. Pairs of chromosomes are arranged by size and position of centromeres. Karyotypes are arranged with the short arm of the chromosome on top and the long arm on the bottom. The short and long arms are referred to as p and q, respectively. In addition, the differently stained regions and subregions are given numerical designations from proximal to distal on the chromosome arms. The normal human karyotypes contain 22 pairs of autosomal chromosomes and 1 pair of sex chromosomes. Normal karyotype for a female contains two X chromosomes and is denoted as 46,XX whereas a malecarries both an X and a Y chromosome, 46,XY. Chromosome abnormalities can be numerical, as in the presence of extra or missing chromosomes, or structural, as in translocations, inversions, large-scale deletions, or duplications. Regarding numerical abnormalities, the term ploidy refers to the number of chromosome pairs in a karyotype, and aneuploidy refers to abnormal number of chromosomes. Trisomy is the terminology used when three copies of a particular chromosome are present instead of two, and monosomy refers to a single chromosome copy. Chromosome banding employs different cytogenetic techniques to examine specific areas of the chromosome. Since each chromosome within the pair should have identical bands, comparing differences in banding patterns between chromosome pairs can pinpoint the area of the chromosome abnormalities. In G-banding, chromosomes are digested with trypsin and then identified with Giemsa stain, which binds to the phosphate regions of DNA and reveals both lightly and darkly stained chromosome sections. The lighter stained regions tend to be rich in guanine and cytosine (GC) base pairs whereas the dark regions are rich in adenine and thymidine (AT) base pairs. R-banding is the reverse of G-banding in that the dark regions are GC rich and the light regions are AT rich. Fluorescence in situ hybridization (FISH) techniques are used to map DNA locations on specific chromosomes using fluorescent-labeled probes that are specified to a DNA sequence in order to detect numerical and structural chromosome abnormalities. FISH analysis is usually performed in vitro using cultured cells of interest undergoing metaphase. A large number of FISH probes are commercially available with a variety of fluorochromes, allowing for simultaneous detection of multiple DNA loci. FISH probes are useful in the detection of known translocations, inversions, insertion, as well as microdeletions and chromosome breakpoints. For additional molecular genetics review read Wan and Ma (2012). For human cytogenetic nomenclature, read ISCN (2009). The colony-forming potential of HPC was established nearly 50 years ago. Colony-forming unit assays are an in vitro culture system that allows for an analysis of the colony-forming potential of normal, chemically treated, and diseased hematopoietic cells. This assay has evolved over time. Today, we know that different culture conditions are required for growth of primitive stem cells as compared to culture conditions required for relatively mature precursor cells. The late stages of hematopoiesis can be reproduced in vitro in a 14-day culture medium, whereas primitive HSCs require a two-step in vitro culture system: 5-week preincubation on feeder layer followed by 2 weeks in semisolid media. Colony-forming unit assays represent the intermediate state of hematopoiesis between repopulating HSC and the morphologically identifiable features of lineage differentiation. Lymphoid cells do not proliferate in culture systems that mimic the hematopoietic environment. Thus CFU assays allow for the detection of the specific lineage commitment of amyeloid precursor cell in an in vitro setting. In the clinical setting, CFU assays are used to evaluate deviation from normal colony-forming potential, such as an increase or decrease in overall colony growth, change in the M:E ratio, an increase in lineage-specific CFU, deficient hemoglobinization, and abnormally small quantities of hemoglobinized cells. While changes like these in an in vitro assay can be useful in differential diagnosis, the assay does not stand on its own as a definitive diagnostic tool for hematopoietic disease. For further reading, see Nissen-Druey (2005), Hematopoietic Colonies in Health and Disease, and Sornberger (2011), Dreams and Due Diligence. This section is not meant to be a comprehensive discussion or description of all diseases associated with the hematopoietic system. Instead, this section is limited to blood disorders commonly associated with hematotoxins. For the purposes of this book chapter, tables are used to help categorize classifications of hematopoietic disorders. For a comprehensive review of diseases of the hematopoietic system, please see Jandl (1996), Greer et al. (2008), Jaffe (2001), and Swerdlow (2008). Anemia, in the general sense, is a reduction in the concentration of RBCs and Hgb in the blood. Additional considerations associated with anemia are the total volume of RBCs as compared to the total volume of whole blood, that is, the ratio of the two parameters described earlier as the hematocrit. Other considerations include the rate of onset of anemia, the extent of reduction in Hgb concentration, and the body’s compensatory response. The cardiovascular system’s response to acute anemia is to increase the velocity of blood flow and to redistribute blood to organs that are most vulnerable to hypoxia. In chronic anemia, the same adaptive physiological changes occur so that the body can establish a new steady state. Anemias of the peripheral blood are classified into two primary categories: increased destruction (Table 5.3) or impaired production of RBCs (Table 5.4). Table 5.3 Increase Destruction of RBC: Hemolytic Anemia
TOXICITY OF THE HEMATOPOIETIC SYSTEM
5.1 HEMATOPOIESIS: ORIGINS OF HEMATOPOIETIC CELLS
Myelopoiesis
Erythropoiesis
Thrombopoiesis
Granulopoiesis
Neutrophil Maturation
Basophil Maturation
Eosinophil Maturation
Monopoiesis
Myelopoietic Growth Factors
Lymphopoiesis
5.2 HEMATOTOXICITY: ASSAYS USED FOR ANALYSIS
Complete Blood Count
Peripheral Blood Smear
Cell Type
Mean Value
Normal Range
Leukocytes (106/l)a
7000
4,300–10,000
Neutrophils bands (%)
2
0–5
Neutrophil segs (%)
60
35–80
Eosinophils (%)
2
0–4
Basophils (%)
0.5
0–1
Monocytes (%)
4
2–6
Lymphocytes (%)
30
20–50
Erythrocytes (1012/l)a
Male: 5.4
4.6–5.9
Female: 4.8
4.2–5.4
MCH (HbE pg)
29
26–32
MCV (fl)a
87
77–99
MCHC (g/dl)
33
33–36
RDW (µm)
7.5
Reticulocytes (%)
Male: 16
8–25
Female: 24
8–40
Hgb (10 g/dl)
Male: 15
14–18
Female: 13
12–16
Hct (%)
Male: 45
42–48
Female: 42
38–43
Platelets (103/µl)
180
140–440
Leukocytes (White Blood Cell Count)
Eosinophils
Basophils
Monocytes
Lymphocytes
Hemoglobin and Hematocrit
Erythrocytes (Red Blood Cell Count)
Red Blood Cell Indices
Mean Corpuscular Volume
Red Blood Cell Distribution Width
Mean Corpuscular Hemoglobin
Mean Corpuscular Hemoglobin Concentration
Reticulocyte Count
Thrombocytes (Platelets Count)
Mean Platelet Volume
Bone Marrow Aspirate and Biopsy
Bone Marrow Collection
Bone Marrow Cellularity
Myeloid:Erythroid (M:E) Ratio
Marrow Differential Count
Cell Types
Range %
Myeloblasts
<5
Promyelocytes
1–8
Myelocytes
Neutrophilic
5–15
Eosinophilic
0.5–3
Basophilic
<1
Metamyelocytes
Neutrophilic
15–25
Eosinophilic
<1
Basophilic
<1
Mature myelocytes
Neutrophilic
10–30
Eosinophilic
<5
Basophilic
<5
Mononuclear
Monocytes
<5
Lymphocytes
3–20
Plasma cells
<1
Megakaryocytes
<5
Normoblasts
25–50
Histochemistry
Flow Cytometry/Immunophenotyping
Chromosome Analysis
Karyotyping
Cytogenetics
Fluorescence In Situ Hybridization
Colony-Forming Units Assay
5.3 DISEASES OF THE HEMATOPOIETIC SYSTEM
Anemia
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