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TOXICOLOGY 707

HEMATOLOGY IN TOXICOLOGY STUDIES Dr. Gregory Travlos Fall 2007

Introduction:

Blood can be a target organ of a toxic insult and hematotoxicity can result in the altered number and/or function of circulating blood cells. Thus, hematology evaluations, the study of blood and blood forming tissues, are useful in detecting, monitoring, and understanding toxic processes. Hematology evaluations are commonly recommended in animal toxicology and safety assessment studies and several national and international regulatory agencies provide guidelines for hematological testing in nonclinical toxicity and safety studies. Measurement of hematological variables affords the toxicologist some advantages compared to standard histopathology evaluations. Advantages include detection of hematopoietic system-specific effects, identification of a toxic mechanism or metabolic injury, serial sampling, and assistance in establishment of the no effect level. Blood has been extensively studied and these notes are a brief overview. The purpose of this review is to provide general information concerning hematology and the individual components of a complete blood count, the most commonly used method for evaluating the hematopoietic system. For detailed information regarding hematology and hematotoxicity refer to the references provided.

Hematopoiesis (Hemopoiesis):

Table 1. lists the wide variety of organs and tissues involved in the production of blood cells. In the fetus, hematopoiesis is first seen in the yolk sac. As the fetus develops blood production shifts to the liver and eventually the spleen. At birth, production in the liver and spleen is markedly reduced and the bone marrow becomes the primary blood cell producer. There are species differences. For example, the spleen is an active participant in blood cell production in the adult mouse. In a study on the response to hemorrhage, 50% of the increase in erythropoiesis in bled mice took place in the spleen. In man, most of the marrow cavities of long bones are inactive by the age of 20. At this stage, only the upper humerus and femur, ribs, sternum, vertebrae, and bones of the skull, remain in production. The hematopoietic microenvironment is very important. It must be able to recognize and retain hematopoietic stem cells and provide the factors required to support development and differentiation of stem cells along particular lines. In most animals, the microenvironment conducive to hematopoiesis exists in the bone marrow (and the spleen of some animals). The requirement of a hematopoietic microenvironment has been demonstrated by irradiation studies. When donor hematopoietic cells were infused into irradiated mice, hematopoietic colonies developed only in the bone marrow or spleen. Additionally, it has been shown that appearance of marrow stroma precedes development of extramedullary hematopoiesis. It also has been shown that transplanting marrow fibroblasts beneath the kidney capsule provides an area capable of retaining hematopoietic cells, illustrating the need for a particular tissue to provide the unique environment required to support hematopoiesis. Hematopoiesis is a continuous process, but can be separated into distinct stages (Figure 1). The first stage involves uncommitted (pluripotent) stem cells contained in the bone marrow. These pluripotent cells have two main functions: 1.) they can maintain there numbers by a process of self-renewal and 2.) they have the ability to give rise to all hematopoietic cells (erythrocytes, granulocytes, lymphocytes, monocytes, and platelets). Most of the understanding of hematopoietic proliferation and maturation has been derived using an irradiated syngeneic mouse model. Irradiated mice infused with donor cells give rise to hematopoietic foci in the spleen. Each focus has been shown to arise from a single pluripotent cell which has been termed the colony forming unit-spleen (CFU-S). Depending on need, the bone marrow microenvironment 2 and growth factors influence pluripotent stem cells to differentiate into committed stem cells of either the myeloid or lymphoid series (multipotential stem cells). These committed stem cells can be considered as being the second stage. They have a limited capacity for self-renewal, but have the potential to differentiate and develop mature progeny. The lymphoid stem cells eventually differentiate into pre-B or pre-T lymphocyte progenitors. Myeloid stem cells are the multipotential colony forming unit for granulocytes, erythrocytes, monocytes, and megakaryocytes (CFU- GEMM). The third stage is when committed stem cells, influenced by various growth factors, differentiate into lineage-specific progenitor cells. Progenitor cells exist in the bone marrow for megakaryocytes (CFU-Meg), lymphocytes, erythrocytes (BFU-E), eosinophils (CFU-Eos), and basophils (CFU-Baso). It appears neutrophils and monocytes arise from a common precursor (CFU-GM). In addition to microenvironment, the production, differentiation, and maturation of blood cells is regulated by humoral factors, some of which are listed in Table 2. Some factors (e.g., BPA/IL-3) act on the more primitive cells and have a general action, while others, (e.g., erythropoietin) act on later progenitors of a specific cell line. The sources of hematopoietic factors vary. Erythropoietin is produced primarily in the kidney with minor amounts from the liver. Burst promoting activity (BPA) is produced by T-lymphocytes and macrophages. IL-3 is produced by T-lymphocytes and myeloid cells and may be the same macromolecule as BPA. Colony simulating factors are produced by a variety of cells, including macrophages/monocytes, fibroblasts, endothelial cells, lymphocytes, and placenta. Most interleukins, B-cell growth factor, and B-cell differentiation factor are derived from T- lymphocytes. IL-1 is produced by macrophages.

Blood Cells:

A. Erythrocytes Production of erythrocytes (RBC's) occurs extravascularly in the bone marrow parenchyma. Tissue hypoxia is the major stimulus for erythropoiesis and blood hemoglobin concentration is the primary determinant of the degree of tissue oxygenation. Other factors affecting tissue oxygenation include cardiac output, pulmonary function, oxygen tension of inspired air, alterations in the oxyhemoglobin dissociation curve, and tissue blood distribution. Under the influence of erythropoietin, CFU-E's differentiate (no division) to rubriblasts. The rubriblast is the first cell in the erythroid series that is recognizable morphologically and has proliferation capability. As erythrocyte precursors mature and divide they become smaller, their nuclei shrink and condense, and division stops when an appropriate amount of hemoglobin is reached in the cytoplasm (usually the late rubricyte stage). The nucleus is extruded at the late metarubricyte stage and an anucleate reticulocyte is generated (the reticulocyte still contains mitochondria and aggregates of RNA in the cytoplasm). Reticulocytes and mature erythrocytes migrate into the bone marrow venous sinuses and enter the peripheral circulation. It takes about 5 days from the time the stem cells are stimulated before reticulocytes are released to the peripheral blood. Reticulocytes mature in the bone marrow or in the spleen, and maturation takes about 2-3 days. 16 RBC's are produced from each rubriblast. The life span of circulating RBC's varies for each species. In man, the life span is ~120 days; in rats, ~50 days; in mice, ~30 days; in cows, ~160 days. RBC numbers also vary between species. In man, there are about 4-6 x 106 RBC's per microliter of blood; in rats, ~7-9 x 106 RBC's/L; in goats, ~13 x 106 RBC's/L. In most mammalian species, mature RBC's are anucleate, biconcave discoid cells. This shape gives the RBC's a high surface area to volume ratio (maximal gas exchange) and allows deformability (required for circulation). There are species variations in RBC size (e.g., man, ~7-8 m dia. and ~110 fl; rat, ~5-6 m dia. and ~55 fl). The primary purpose of RBC's is to produce, carry, and protect hemoglobin (Hb) for oxygen transport. Hemoglobin accounts for ~95% of the RBC's total protein content. There are benefits to packaging Hb in RBC's; it decreases Hb turnover (the half-life of free Hb is a few hours), it keeps Hb close to the metabolic mechanisms necessary to maintain a functional status (e.g., maintenance of Hb iron in the Fe+2 state), and it eliminates the osmotic effect free Hb would 3 have in plasma. Besides oxygen transport, Hb also is the most important protein buffer in the blood (~6x the buffering capacity of plasma proteins).

B. Leukocytes Blood leukocytes (WBC's) consist of five cell lines (neutrophils, monocytes, eosinophils, basophils, and lymphocytes). In man, there are ~4-11 x 103 WBC's/l blood (in rats, ~6-10 x 103 WBC's/l). Granulocytes (neutrophils, eosinophils, and basophils) comprise ~50-75% of the total number of circulating WBC's. Lymphocytes comprise ~20-40% and monocytes ~8%. There are species differences in cell distribution. For example, rats have ~10-25% granulocytes, ~70-85% lymphocytes, and ~1-5% monocytes. All granulocytes (neutrophils, eosinophils, and basophils) share common stages of development and morphology. The first recognizable granulocytic precursors are myeloblasts. They pass through succeeding generations as progranulocytes, myelocytes, metamyelocytes, bands, and segmenters (mature granulocytes). The different stages of neutrophil development are separated into three pools within the bone marrow. Myeloblasts, progranulocytes, and myelocytes are capable of division and form the proliferative pool. The maturative pool, consists of metamyelocytes and bands. These stages do not proliferate but stay in the bone marrow to mature. Finally, there is a storage or reserve pool of mature neutrophils in the bone marrow and are ready for release when tissue demand increases. The emergence time in blood, from myeloblasts to mature neutrophils, is about 6-14 days (man, ~14 days). The life span of neutrophils in the circulation is very short (~6 hrs.). Once neutrophils leave the circulation they do not return. Granulocytes are 10-20 m in dia., have segmented nuclei, and cytoplasmic granules. Classification of granulocytes into neutrophils, eosinophils, and basophils is based on staining characteristics of their primary granules in Romanowsky-stained blood smears. The cytoplasmic granules in granulocytes contain biologically active substances that are important mediators of inflammation and allergic responses. Basophils contain a variety of mediators of inflammation including, heparin and histamine, and are primarily involved with allergic reactions (immediate-type hypersensitivity). Eosinophils attack parasites and also are involved with hypersensitivity reactions (they contain substances that can inactivate mediators released from mast cells and basophils). Neutrophils seek out, phagocytize, and destroy pathogenic bacteria. They also play an important role in the inflammatory process. Monocytes are 15-30 m in dia., with large irregular nuclei, and abundant bluish-gray cytoplasm. They are derived from the CFU-GM stem cells, as are neutrophils. Although blast cells with monocytic characteristics are observed (termed "monoblasts"), no maturation sequence for monocytic cells has been identified in the bone marrow. Monocytes circulate in the blood for about 3 days, then migrate into the tissues (macrophages) where they live for about 3 months. Macrophages have two primary functions, phagocytosis and antigen presentation. They have a weaker chemotactic response than neutrophils, but are important against bacteria, fungi, foreign bodies, viruses, parasites, and tissue debris. Antigens processed by macrophages are presented on the cell surface and made available to T-lymphocytes. This activates the T-cells which in turn signal B-lymphocytes to produce antibodies against the antigen. Macrophages also produce monokines (for example, INF, TNF, IL-1, GM-CSF, etc.) which are important for a wide variety of effects. Lymphocytes are derived by the formation of lymphoid stem cells from pluripotent cells in the bone marrow. Formation of lymphoid stem cells is followed by differentiation into pre-B or pre-T cell precursors. Maturation of B-cells occurs within the bone marrow. Eventually, mature B-cells leave the marrow and migrate to lymphoid organs such as lymph nodes and spleen. T- cells are different in that they complete their maturation outside the bone marrow (maturation occurs in the thymus). Only a small fraction (~1%) of the early T-cells reach maturity. B-cells and T-cells are morphologically indistinguishable in the peripheral blood. They are ~8-15 mm, with a round dense nucleus and a scant rim of bluish cytoplasm. About 70% of the circulating lymphocytes are T-cells. Lymphocyte life span varies, small lymphocytes in bone marrow and thymus are short-lived, having a turnover time of about 2-4 days. In the peripheral tissues and blood the life span is much greater (months-years). Lymphocytes have the ability to recirculate. Maturing T-cells develop into unique cell types with different functions and names. Cytotoxic/suppresser T-cells (CD8 cells) seek out intracellular pathogens (ex. viruses) and 4 regulate activity of other cells in the immune system. Helper T-cells (CD4 cells) mediate inflammatory reactions and delayed-type hypersensitivity. When activated by antigen, B-cells mature into immunoglobulin-producing plasma cells and are important in humoral immunity.

C. Platelets Platelets are the derived from the myeloid stem cell. Their development starts when a CFU-GEMM stem cell differentiates into CFU-Meg. The later transforms into a megakaryoblast which eventually matures into a megakaryocyte. Megakaryocytes are found in low numbers in bone marrow (~0.05% of the marrow cells). Megakaryocytopoiesis differs from erythropoiesis and granulopoiesis in that it involves endomitosis (nuclear division without cytoplasmic division). The megakaryocytes become polyploid (16-32N), and are the largest cells in the bone marrow (~40-50 m). A mature megakaryocyte has a single large multilobed nucleus, abundant pale cytoplasm, and numerous small azurophilic cytoplasmic granules. As the megakaryocyte matures, demarcation membranes form organizing the cytoplasm into platelet fractions. A mature megakaryocyte extends a pseudopod (proplatelet) across the marrow endothelial barrier into the marrow venous system. Cytoplasmic fragments break off at the demarcation lines and platelets are formed. Platelets are anucleate and are the smallest formed element in blood (~1-4 m, ~3-15 fl) with a life span of about 4-10 days. In most species, counts range between 200,000-500,000/L (rats, ~800,000/L ; mice, >1,000,000/L). The primary function of platelets is to maintain hemostasis. More specifically, they form a plug at the sites of endothelial lining injury. However, platelets also play a role in coagulation (PF3, coagulation factors, clot retraction), as mediators of inflammation (chemotactic substances, vasoactive amines, and cationic proteins), and in phagocytosis of small particles and bacteria. 5

Evaluating the Hematopoietic System:

Fundamental physiology and pathophysiology of blood is similar in most mammalian species and the hematological variables examined are identical for laboratory animals and human clinical patients. However, hematological tests and instrumentation were developed to aid the physician diagnose human disease and generating data from laboratory animals presents several practical problems. Obtaining a representative blood sample, blood volume required for testing, processing large numbers of samples in a timely manner, appropriate instrumentation and methods, blood features unique to individual species, and establishment reference values are examples of considerations that must be addressed by the laboratory investigator before samples are brought to the hematology laboratory. In 1986, the National Toxicology Program (NTP) revised its procedures for clinical pathology (hematology and clinical chemistry) evaluations in 13-week toxicity studies. Objectives of this were to standardize the approach to clinical pathology testing, produce relevant information on chemicals selected for study, and generate an extensive data base for the Fischer 344 rat and B6C3F1 mouse. Frequently, little information exists concerning the toxicity of the compounds selected for study. So a "core" of clinical pathology tests was established, permitting evaluation of several organ systems. The use of a core removes guess work from study design and prevents inadvertent omission of important endpoints. However, the use of a core profile does not preclude the inclusion of other relevant clinical pathology tests in a study. A complete blood count (CBC) is the most commonly used method for evaluating the hematopoietic system. It contains a variety of variables, providing quantitative (cell numbers) and qualitative (morphology and production) information concerning the functional status of the hematopoietic system. Access to automated instruments that perform large numbers of assays rapidly, reliably, and at a reasonable cost are readily available. Most instruments can be modified for use with animal blood and usually do not require large sample volumes.

Currently, the National Toxicology Program has a core CBC that includes:

Automated analyses Manual analyses RBC count Manual microhematocrit WBC count Reticulocyte count (can be automated) Platelet count WBC differential (can be automated) Hematocrit Morphological evaluation of blood cells Hb concentration Mean corpuscular volume Mean corpuscular Hb Mean corpuscular Hb concentration

A. Evaluation of the circulating red cell mass RBC count, hematocrit (Hct), and Hb concentration are all used as estimates of the circulating RBC mass. They provide information concerning the oxygen-carrying capacity of the blood and the bone marrow erythropoietic activity. The RBC count is a quantitative measure of circulating RBC numbers. The Hct measures the ratio of blood volume occupied by RBC's and is reported as a percentage. The Hb concentration is a quantitative measure of total blood hemoglobin and is reported in g/dL of blood. Low values occur with decreased RBC mass (anemia), while increased values indicate an increase in RBC mass (erythrocytosis). Alterations in the three variables may be disproportionate if cell size and/or Hb content are altered. The spleen is a reservoir for RBC's and splenic contraction can cause a transient increase (spurious erythrocytosis). Changes in plasma volume affects all three measurements and hydration status must be considered when interpreting the data.

B. Evaluation of the red cell indices Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) are morphological measures and are useful in the classification of anemias (Table 3.). They are calculated values derived from the RBC count, 6

Hct value, and Hb concentration. Therefore, the validity of the indices depends on the accuracy of the data required for calculation. MCV expresses, in femtoliters (fl), the average RBC volume of a population of erythrocytes and is an indicator of RBC size. MCV is increased (macrocytic cells) in anemias in which there is an increased bone marrow release of immature RBC's (reticulocytes) or as a result of deficient nucleic acid synthesis (e.g., vitamin B12 and folate deficiencies). Reticulocytes are larger than mature RBC's and a reticulocytosis causes a transitory increase in MCV. Altered nucleic acid synthesis, decreases DNA production causing fewer cell divisions during development; fewer divisions results in larger RBC's. Iron deficiency causes a decreased MCV (microcytic cells). Iron deficiency slows hemoglobin production, allowing extra cell divisions before the critical level of Hb is reached; smaller are RBC's the result. MCV is represented by the formula:

MCV = [Hct (%)/RBC count (millions)] X 10

MCH expresses, in picograms (pg), the weight of Hb in an average RBC in a population of cells. It is represented by the formula below and assumes all Hb measured is in RBC's. Iron deficiency causes a decreased MCH due to decreased Hb production. Reticulocytosis causes a normal to slightly increased MCH. Hemolysis (in vivo or in vitro) can cause an increased MCH due to decreased RBC numbers.

MCH = [Hb (g/dL)/RBC count (millions)] X 10

MCHC expresses, in percent, the ratio of Hb weight to the volume of an average erythrocyte in a population of cells. For most mammalian species the MCHC is approximately 33%. The MCHC is the most accurate of the indices because it does not require the RBC count. MCHC is decreased in iron deficiency and reticulocytosis. MCHC is increased with in vivo and in vitro hemolysis, spherocytosis, and significant RBC shrinkage (eg., excess EDTA). High MCHC values do not occur physiologically; Hb crystallizes when a concentration of >36% is achieved. Therefore, when an increased MCHC occurs hemolysis (in vivo or in vitro) or altered RBC count (eg., lab error) should be considered.

MCHC = [Hb (g/dL)/Hct (%)] X 100

C. Evaluation of erythropoietic activity The reticulocyte count is a semiquantitative indicator of bone marrow erythropoietic activity and is estimated by evaluating the presence of immature RBC's in the peripheral blood. It is one of the CBC variables not routinely performed using an automated instrument. Counting reticulocytes may be performed by flow cytometry or by microscopic examination of smears prepared with supravitally stained blood. A reticulocyte count is most useful for assessing the marrow response to anemia. A regenerative (responsive) anemia is characterized by a decreased RBC mass with increased reticulocyte numbers. This indicates the bone marrow has the ability to respond and suggests the anemia is not related to a bone marrow defect or suppression. A nonregenerative (nonresponsive) anemia is characterized by a decreased RBC mass with normal to decreased reticulocyte numbers and suggests the marrow ability to produce RBC's has been compromised. Reticulocyte counts are often reported as a percentage of RBC's (a relative number). An increased reticulocyte percentage in very anemic blood may overestimate the degree of marrow response. Therefore, absolute reticulocyte counts should be calculated and used instead of percentages.

Absolute reticulocyte count = RBC count (millions) X Reticulocyte %

An increase in absolute reticulocyte numbers is termed a reticulocytosis and indicates a responding bone marrow. Following the occurrence of anemia, reticulocytosis does not become evident until 2-3 days and should reach maximum in 7 days. There are species differences in normal reticulocyte numbers. For example, in rats and mice reticulocytes range between 100- 7

300 X 103/l; dogs and cats, reticulocytes are usually < 60 x 103/l. Horses do not have circulating reticulocytes even during a marked erythropoietic response.

D. Evaluation of the circulating platelet mass The platelet count is a component of the instrument performed analyses and is used for evaluating quantitative platelet disorders. Thrombocytopenia (decreased platelet numbers) is the most frequent quantitative abnormality encountered, and is probably the most common cause of hemorrhagic diathesis. In most species, spontaneous hemorrhage due to thrombocytopenia requires platelet counts below ~25,000/L. Counts below 50,000 result in increased bleeding subsequent to an injury (e.g., trauma). Thrombocytosis (increased platelet numbers) does occur and is often related to a physiologic or reactive response (e.g., epinephrine-induced physiologic thrombocytosis).

E. Evaluation of the circulating blood leukocyte mass The leukogram consists of the total and differential WBC counts. It is a means of evaluating leukocyte responses to a microbial infection or chemicals. The total WBC count is part of the instrument analysis and should always be corrected for nucleated RBC's (NRBC's). In some species, a marked erythropoietic response, some toxins (e.g., lead), and occasionally neoplasia involving bone marrow can cause increased release of NRBC's to the circulation. During instrument counting NRBC nuclei are erroneously counted as WBC's resulting in falsely elevated total WBC counts. The number of NRBC's are counted during performance of the WBC differential count and reported as #NRBC's per 100 WBC's.

Corrected WBC count = [100/100+#NRBC's per 100 WBC's] X Automated WBC count

The WBC differential count is performed by some automated cell counters or by microscopic evaluation of a Romanowsky-stained blood film. For microscopic evaluations, 100 WBC's are typically, examined and identified. Individual cell lines are reported as percentages (relative numbers). Relative numbers should not be interpreted and WBC counts should be converted to absolute numbers.

Absolute differential WBC count = Corrected WBC count X Differential WBC %

Any interpretation of a leukogram must take into consideration the normal values for the species, age, and any species-specific responses. A leukocytosis is used to describe an increase in total WBC numbers and may be related to increases in all WBC cell lines or specific cell lines (e.g., leukocytosis as a result of a neutrophilia). A leukocytosis may be physiologic (e.g., endogenous release of epinephrine or corticosteroids), pathologic (e.g., inflammatory leukocytosis), or neoplastic (leukemia). Leukopenia describes a decrease in total WBC numbers is considered a pathologic event.

F. Evaluation of the blood cell morphology Blood cell morphology provides important structural information about the hematopoietic system. It is performed by microscopic evaluation of a Romanowsky-stained blood film and blood cells are examined for abnormalities in size, shape, color, and presence of inclusions. Morphological examination can determine the intensity (or lack thereof) of a bone marrow response to anemia or inflammation, provide evidence as to specific causes or pathophysiological processes occurring, and detect hematopoietic neoplasia.

G. Additional tests The use of a CBC has become part of the routine evaluations used for compound toxicity screening and safety assessment studies. There are, however, a wide variety of other tests available for the evaluation of blood and bone marrow, including:

Bone Marrow Total count Differential count 8

Histopathology Stem cell culture

Red Blood Cells Methemoglobin Carboxyhemoglobin Enzymes and metabolites of carbohydrate metabolism Enzymes and metabolites of metabolic protection against oxidants Enzymes and metabolites of heme production Osmotic fragility Coomb's (antiglobulin) test RBC cholinesterase RBC production and turnover studies (eg., 59Fe)

White Blood Cells Cell isolation (eg., ficoll-hypaque) Adherence (eg., glass, nylon fibers, plastic) Chemotaxis, in vitro and in vivo Phagocytosis (eg., Staph sp.) Enzymes and metabolites of respiratory burst T- and B-cell subsets 9

Hematotoxicology

Hematotoxicity is manifested by altered number and/or function of circulating blood cells. A reduction in cell numbers is a common finding associated with hematotoxicity and lesions are usually classified by the cell type affected. For example, anemia is a relatively common occurrence in toxicity studies. It indicates a decrease in the circulating RBC mass and is characterized by an absolute decrease in the Hct, Hb concentration, and RBC count. Anemia is associated with excessive RBC loss, destruction, or suppressed production and can be caused by numerous mechanisms. In this context, anemia occurring in toxicity studies is a "sign" not a "diagnosis". Additionally, hematotoxicity may occur without altered cell numbers. This section will focus on examples of anemia, illustrating different mechanisms involved. Keep in mind that anemia is only one of the many effects certain chemicals and drugs have on the bone marrow and blood.

Anemia As mentioned previously, anemia is a decrease in circulating RBC mass that can be caused by excessive loss or destruction of erythrocytes, or bone marrow suppression. It is one of the most common hematology findings in toxicity studies. Bone marrow suppression (hypoproliferative) or failure (aplasia): This refers to a reduction in circulating blood cells resulting from a suppression/failure of the bone marrow precursor cells to produce mature cells. Decreased production can involve the pluripotent stem cells resulting in a decrease in all cell lines (pancytopenia) or the committed cell lines, resulting in a single-cell cytopenia. Aplastic anemia is the severest form of bone marrow failure and is characterized by persistent pancytopenia with loss of functional bone marrow. A similar blood picture is seen with patients receiving certain antineoplastic agents (eg., alkylating agents) or radiation therapy. However, the aplasias related to these treatments are usually transient and the patients recover. A wide variety of compounds have been implicated in the development of aplastic anemia. Unfortunately, the pathogenesis is unknown in many instances where there is a strong association between compound exposure and aplastic anemia. Anemias related to decreased erythropoiesis (ineffective or hypoproliferative erythropoiesis) are typically less severe than aplastic processes and usually involve just the red cell series. Such anemias may result from nutritional deficiencies (eg., iron, vit. B12, folic acid), functional impairment (eg., anemia of chronic disease, lead toxicosis), decreased hematopoietic stimulation (eg., kidney disease, hypothyroidism), or a combination (eg., chronic alcohol ingestion). There are important differences between anemias of nutritional deficiencies and anemias secondary to toxicosis or organic disease. In the former, the erythropoiesis is suppressed due to lack of essential nutrients and rebounds as soon as adequate nutrients are supplied. In contrast, anemias related to toxic or organic diseases have the bone marrow suppressed or destroyed by the insulting agent and an anemia develops in the face of adequate nutrition. The damage incurred may be mild or severe, reversible or permanent depending on the primary cause. Suppression/failure anemias are nonregenerative and are characterized by the lack of or inadequate numbers of reticulocytes in the circulation. The MCV and MCHC are usually normal, but can be variable depending on the etiology. Frequently, other RBC changes are present (eg., anisocytosis, microcytosis, acanthocytes, basophilic stippling, NRBC's).

Chloramphenicol: This antibiotic is a nitrobenzene compound that was released for general use in the late 1940's. As many as 26% of all acquired aplastic anemia cases have been associated with chloramphenicol therapy. The actual risk is about 1 in 20,000-30,000. The mechanism is unknown. However, the toxic effect appears to involve multiple processes. There is a dose-dependent, reversible marrow suppression that is seen in all patients (suggested to be related to mitochondrial damage). In some patients, a persistent aplastic anemia develops weeks to months after exposure. This appears to be an idiosyncratic reaction and there are no dose or duration relationships for this syndrome. A minority of the affected individuals develop severe aplastic anemia at doses tolerated by the majority of patients without bone marrow suppression. There is some evidence to link chloramphenicol-induced aplastic anemia with certain 10 histocompatibility antigens and genetic predisposition may play a role. Additionally, mechanisms involving immune regulation and altered microenvironment have been suggested.

Cytotoxic agents and radiation: Bone marrow suppression is a common transient response of cancer chemotherapy. The effects are linked cell cycling and related to the high susceptibility of proliferative cells versus resting cells to these agents. The cell cycle can be divided into several stages: 1) Resting/non-cycling phase (G0 phase); 2) A period of RNA and protein synthesis, but no DNA synthesis (G1 phase); 3) Active DNA synthesis (S phase); 4) A period post-DNA synthesis where the cell contains twice the diploid amount of DNA (G2 phase); 5) Cell division/mitosis (M phase). Usually these agents target rapidly dividing cells and are termed cycle-specific agents (eg., nitrogen mustard, busulphan, nitrosoureas, actinomycin-D, ionizing radiation). These agents affect cycling cells in any phase but there can be variation in response. For example, busulphan and the nitrosureas target resting and proliferating pluripotent stem cells, reducing their number. The response can be latent and severe bone marrow damage may not be seen for 1-2 months post-administration. The mechanism is thought to involve direct alkylation of DNA and RNA. Cyclophosphimide, 5-fluorouracil, and methotrexate affect DNA synthesis (S phase) and target cells with low self renewal capabilities but high proliferative rates. Ionizing radiation causes single and double strand DNA breaks and DNA cross-linking that can affect cells in any cycle phase. In general, cells in G2 are more sensitive for radiation injury. Additionally, erythroid cells are more sensitive than the granulocytic or megakaryocytic series. Some compounds are phase specific. For example, the vinca alkaloids (vincristine and vinblastine) interfere with microtubule assembly and mitotic spindle formation which causes cells to arrest in G2/M.

Benzene: Benzene is an aromatic hydrocarbon that has been used extensively as a solvent and in the synthesis of other chemicals. Benzene causes aplastic anemia through biotransformation of the parent compound to toxic metabolites. Microsomal enzymes convert benzene to phenol. Further hydroxylation of the phenol produces hydroquinone, and its terminal oxidation product p-benzoquinone. It is these metabolites that are thought to responsible by interfering with microtubule assembly and arresting proliferating cells in G2/M. Benzene metabolites can also covalently bind DNA (formation of a chemical-DNA adduct) in nuclei and mitochondria and is a potential mechanism for cell replication inhibition.

Alcohol: In man, chronic alcohol ingestion, especially in malnourished alcoholics, causes of sideroblastic and/or megaloblastic anemias. There are multiple mechanisms involved. Alcohol has a direct affect on hematopoiesis. Anemia is common and alcohol appears to inhibit erythroid progenitor growth. Thrombocytopenia also occurs frequently, but the defect appears to be related to altered megakaryocyte maturation. To a lesser extent, alcohol can decrease granulocyte production. Suppression occurs at the level of the CFU-GM by inhibiting GM-CSF. Sideroblastic change occurs due to the inhibitory effect of alcohol on several enzymes of heme production (eg., ALA-dehydratase, uroporphyrinogen decarboxylase, coproporphyrinogen oxidase, and ferrochetolase). Megaloblastic change is related to folate deficiency and is nutritional in origin. The exception is beer drinkers; beer has a significant folate content.

Lead: Lead is toxic to most living things and has a high affinity for binding to sulfhydral-, imidazole-, and carboxyl-containing proteins, including enzymes and cell cytosolic and membrane proteins. Several enzymes of heme production (eg., ALA-dehydratase, ferrochetolase) are inhibited by lead and analysis of enzyme activity or endproducts are very sensitive indicators of lead exposure. Sideroblastic change can be seen with lead toxicosis and is related to the inhibition of heme synthesis. Even though heme production is decreased, development of anemia is variable and is related more to suppressed globin synthesis and/or increased turnover of altered erythrocytes. RBC basophilic stippling is a related to aggregates of undigested rRNA and is the result of pyrimidine-5-nucleotidase inhibition.

Hemolytic Anemia: This refers to an anemia that is a result of increased destruction of circulating erythrocytes. This can occur within the vasculature or extravascularly within the 11 mononuclear phagocyte system (eg., the spleen). In most instances, the bone marrow tries to compensate for the erythrocyte loss and increased marrow erythropoiesis is evidenced by a reticulocytosis. Morphologically, the reticulocytosis is evidenced by an increase in RBC polychromasia and possibly NRBC's. The MCV is increased due to the large size of the reticulocytes. The MCHC is increased. Hemolytic anemia may be induced by chemicals or drugs in two main ways. First, the compound may have a direct effect on the mature red cells causing an oxidative hemolysis. Second, the compound may induce an immune response to the parent compound, its metabolites, or stimulate production of autoantibody directed against red cell antigens.

Oxidative hemolysis occurs when a compound overwhelms or impairs the antioxidant systems of RBC's. Red blood cells are protected from oxidant stress by a variety of mechanisms, including superoxide dismutase, catalase, reduced glutathione, methemoblobin reductase, and vitamins C and E. Oxidative damage can involve hemoglobin or the red cell membrane. Reticulocytes are more resistant to oxidative injury than mature RBC's because they have increased activity of the enzyme systems involved in oxidant reduction.

An example of a chemical methemoglobin (MetHb) former is phenylhydroxylaminea. Phenylhydroxylamine (PHA) is a n-hydroxylamine that causes RBC oxidative stress oxidizing Hb to MetHb. Normally, DeoxyHb is in the Fe+2 state, while OxyHb is in the Fe+3 state; an e- is - - transferred to bound O2 giving bound superoxide ion (O2 ). During deoxygenation, the e returns to - - the iron moiety and O2 is released. Oxidation to MetHb occurs when O2 is replaced with Cl during deoxygenation (<3%/day). Some MetHb and Heinz body formers produce oxidative damage by donating e- to OxyHb.

3+ - 3+ 2- Hb : O2 + PHA  Hb :O2 + .PHA 3+ 2- + 3+ Hb : O2 + 2H  Hb + H2O2

- The drug free radical generated (.PHA) can now donate the unpaired e to molecular O2 to form superoxide or to another OxyHb to form more MetHb and H2O2. Free radicals achieve stability by extracting e- from -SH groups of Hb, enzymes, membrane proteins, GSH, by oxidizing membrane unsaturated fatty acids, or by oxidizing NADH or NADPH. When the tritratible SH groups of Hb are oxidized beyond the protective process (GSH) a conformational change occurs exposing more SH groups for oxidation. The end result is denaturation and precipitation of Hb. Not all MetHb formers cause Heinz bodies (e.g., nitrites). Some produce both (e.g., chloronitrobenzenes, copper). a From Harvey, J. W. (1989). In “Clinical Biochemistry of Domestic Animals” (J. J. Kaneko, ed.), 4th Ed., pp. 185-234. Academic Press, NY.

Immune-mediated hemolytic anemia is most often related to the antigenic ability of the chemical or drug. Usually the compound acts as a hapten. They bind to a cellular component forming a new antigen. This antigen is recognized by the immune system as foreign resulting in antibody production against the cell-compound combination. The antibody will not interact unless both are present. Cell lysis is usually mediated through activation of complement. However, cell destruction can also be mediated through opsonization of cells with IgG and cell removal by the mononuclear phagocyte system. Penicillin is an antibiotic that can act as a hapten and produce an immune-mediated hemolytic anemia. -Methyldopa, can cause an immune response against normal red cells without the addition of the drug or its metabolites. In this instance, the drug does not act as any part of the antigen. It has been suggested that the drug alters normal RBC surface antigens so that they are recognized as foreign. The antibody produced cross-reacts with normal antigens resulting in the hemolytic anemia. Immune-mediated thrombocytopenias and neutropenias can be caused by either mechanism. For example, procainamide can cause a drug antigen-related neutropenia and thrombocytopenia. Gold is considered to cause an autoimmune thrombocytopenia by the non- antigen mechanism. 12

Blood Loss Anemia: This type of anemia results from an increased loss of circulating erythrocytes (hemorrhage). Similar to hemolytic anemia, there is a compensatory regenerative response. Morphologically, there is an increase in RBC polychromasia (reticulocytosis). However, with prolonged hemorrhage iron stores are depleted and decreased iron availability dampens the erythropoietic response. The MCV is increased early, but as iron is depleted, the MCV will become normal or even decreased. The MCHC can be normal to decreased. Blood loss anemia can be differentiated from hemolytic anemia by decreased plasma protein and iron concentration, normal to decreased spleen size, and normal serum haptoglobin and hemopexin concentration (others).

Coumarins/Indandiones: These compounds are vitamin K antagonists that have been used extensively as rodenticides. They inhibit the vitamin K cycle in the liver causing decreased hepatic production of functional clotting factors II, VII, IX, and X. There is a delayed onset coagulopathy that results in a hemorrhagic diathesis. 13

HEMATOLOGY REFERENCES

A. Texts/Books: 1. Veterinary Hematology, 5th edition; Feldman, B. F., Zinkl, J. G. and Jain, N. C. (eds.), Lippincott, Williams and Wilkins, Philadelphia, Pa., 2000.

2. Wintrobe’s Clinical Hematology, 10th edition, Lee, G. R., Foerster, J., Lukens, J. N., Paraskev, F., Greer, J. P., Rodgers, G. M. (eds.), Williams and Wilkins, Philadelphia, Pa., 1999.

3. Laboratory Medicine Hematology, 6th edition, Miale, J. B., C. V. Mosby Co., St. Louis, Mo., 1982.

4. Target Organ Toxicology Series: Toxicology of the Blood and Bone Marrow, Irons, R. D. (ed.), Raven Press, Ltd., N.Y., 1985.

5. The Natural Immune System: The Neutrophil, Abramson, J. S. and Wheeler, J. G. (eds.), Oxford University Press, N.Y., 1993.

6. Veterinary Laboratory Medicine, In Practice, The Compendium Collection, Veterinary Learning Systems Co., Inc., Trenton, NJ., 1993.

B. Journal Articles/Book Chapters: 1. Harvey, J. W., Erythrocyte Metabolism, in Clinical Biochemistry of Domestic Animals 4th edition; Kaneko, J. J. (ed.), Acedemic Press, Inc., N. Y., p. 185-234, 1989.

2. Kaneko, J. J., Porphyrins and Porphyrias, in Clinical Biochemistry of Domestic Animals 4th edition; Kaneko, J. J. (ed.), Acedemic Press, Inc., N. Y., p. 235-255, 1989.

3. Smith, J. E., Iron Metabolism and Its Diseases, in Clinical Biochemistry of Domestic Animals 4th edition; Kaneko, J. J. (ed.), Acedemic Press, Inc., N. Y., p. 256-273, 1989.

4. Conrad, M. E. and Umbreit, J. N., A Concise Review: Iron Absorption-The Mucin-Mobilferin- Integrin Pathway. A Competitive Pathway for Metal Absorbtion, American Journal of Hematology, 42, 67-73, 1993.

5. Smith, S. P. and Yee, G. C., Hematopoiesis, Pharmacotherapy, Supplement to vol. 12(2), 11s- 19s, 1992.

6. Phillips, R. A., Hematopoietic Stem Cells: Concepts, Assays, and Controversies, Seminars in Immunology, 3, 337-347, 1991.

7. Lemischka, I. R., Clonal, in vivo Behavior of the Totipotent Hematopoietic Stem Cell, Seminars in Immunology, 3, 349-355, 1991.

8. Smith, B. R., Regulation of Hematopoiesis, The Yale Journal of Biology and Medicine, 63, 371- 380, 1990.

9. Williams, D. E., Fletcher, F. A., Lyman, S. D., and de Vries, P., Cytokine Regulation of Hematopoietic Stem Cells, Seminars in Immunology, 3, 391-396, 1991.

10. Metcalf, D., Control of Granulocytes and Macrophages: Molecular, Cellular, and Clinical Aspects, Science, 254, 529-533, 1991.

11. Mansouri, A. and Lurie, A. A., Concise Review: Methemoglobinemia, American Journal of Hematology, 42, 7-12, 1993. 14

12. Jacob, H. S., Mechanisms of Heinz Body Formation and Attachment to Red Cell Membrane, Seminars in Hematology, 7(3), 341-354, 1970.

13. Smith, R. P., Toxic Responses of the Blood, in Casarett and Doull's Toxicology, 4th edition, Amdur, M. O., Doull, J., and Klaassen, C. D. (eds.), p. 257-281, Pergamon Press, 1991.

14. Suber, R. L., Clinical Pathology for Toxicologists, in Principles and Methods of Toxicology, 2nd edition, Hayes, A. W. (ed.), Raven Press, Ltd., N.Y., p. 485-519, 1989.

15. Irons, R.D., Blood and Bone Marrow, in Handbook of Toxicologic Pathology, Haschek, W. M. and Rousseaux, C. G. (eds.), Academic Press, Inc., N. Y., p. 389-419, 1991.

16. Olofsson, T. B., Growth Regulation of Hematopoietic Cells, Acta Oncologica, 30(8), p. 889- 902, 1991.

17. Luster, M. I., Wierda, D., and Rosenthal G. J., Environmentally Related Disorders of the Hematologic and Immune Systems, in The Medical Clinics of North America: Environmental Medicine, Upton, A. C. (ed.), 74(2), p 425-440, W. B. Saunders, Co., Philadelphia, Pa., 1990.