Cellular Ultrastructure and Organization (PDF)

CELLULAR ULTRASTRUCTURE AND ORGANIZATION Cells, as the smallest organized units of living tissues, have the ability to individually perform all the functions essential for life processes. Although the range of morphological features varies widely, all cells conform to a basic model (Fig. 3.1A). Larg...

CELLULAR ULTRASTRUCTURE AND ORGANIZATION Cells, as the smallest organized units of living tissues, have the ability to individually perform all the functions essential for life processes. Although the range of morphological features varies widely, all cells conform to a basic model (Fig. 3.1A). Large cellular structures are observable on stained preparations with the light microscope. Smaller ultrastructures or organelles must be viewed with an electron microscope.  FIGURE 3.1 Cellular organization. Most of the organelles depicted in (A) are visible only with electron microscope examination. A. Cellular ultrastructures. B. Fluid mosaic model. The unique positioning of the phospholipids and free-floating proteins is characterized by the fluid mosaic model of the cellular membrane. ER, endoplasmic reticulum. Description CELLULAR MEMBRANES Structure Cellular membranes provide a semipermeable separation between the various cellular components, the organelles, and the surrounding environment. The cytoplasmic membrane, or outer membrane, defines the boundaries of the cell, while being resilient and elastic. Differences in membrane thickness reflect the various functional properties of specific cell types or organelles within the cell. Chemically, membranes consist of proteins, phospholipids, cholesterol, and traces of polysaccharide. The most popular hypothesis to explain the arrangement of these molecular components is the fluid mosaic model (Fig. 3.1B). According to this model, the cell membrane is a dynamic fluid structure with globular proteins floating in lipids. The lipids, as phospholipids, are arranged in two layers. The polar (charged) phosphate ends of the phospholipids are oriented toward the inner and outer surfaces, whereas the nonpolar (fatty acid) ends point toward each other in the interior of the membrane. Protein molecules may be either integral (incorporated into the lipid bilayer) or peripheral (associated with either the outer or the inner surface of the membrane). Polysaccharides in the form of either glycoproteins or glycolipids can be found attached to the lipid and protein molecules of the membrane. Membrane Functions The lipid bilayer is directly responsible for the impermeability of the membrane to most water-soluble molecules. Proteins within the membrane act as transport molecules for the rapid penetration of polar and non–lipid-soluble substances. Additionally, protein molecules determine and protect the shape and structure of the membrane, often through attachment to underlying microtubules and microfilaments. In human blood cells, the extrinsic protein, spectrin, and the protein, actin, form a contractile network just under the cell membrane and provide the cell with the resistance necessary to withstand distorting forces during movement through the blood circulation. Membrane-bound carbohydrates act as surface antigens, which function in the process of cellular recognition and interaction between cells. Each type of blood cell has a unique repertoire of surface markers at various stages of differentiation and maturation. An international system, the cluster of differentiation (CD) system, exists to identify blood cell surface antigens. As a unit, the cytoplasmic membrane maintains cellular integrity of the interior of the cell by controlling and influencing the passage of materials in and out of the cell. This function is accomplished through the major membrane processes of osmosis, diffusion, active transport, and endocytosis. The term osmosis is used to describe the net movement of water molecules through a semipermeable membrane (Fig. 3.2). Normally, water molecules move in and out of the cell membrane at an equal rate, producing no net movement. If a concentration gradient exists, the movement of water molecules will be greater from areas of low solute (e.g., sodium and chloride ions) concentration to areas of higher solute concentration. Osmosis is the basic principle underlying the previously popular erythrocyte fragility test (see Chapter 32 Laboratory Manual) that demonstrates changes in the erythrocytic membrane. Alterations in the erythrocytic membrane, such as the loss of flexibility, can be observed by placing erythrocytes in solutions with varying solute concentrations. FIGURE 3.2 Effects of osmosis on red blood cells in different concentrations: isotonic, hypotonic, and hypertonic solutions. (Reprinted with permission from Braun CA. Pathophysiology, Baltimore, MD: Lippincott Williams & Wilkins, 2007.) Description Diffusion is an important process in overall cellular physiology, such as the physiological activities of the erythrocyte. This passive process through a semipermeable membrane may also be referred to as dialysis. Substances passively diffuse, or move down a concentration gradient from areas of high solute concentration to areas of low solute concentration, by dissolving in the lipid portion of the cellular membrane. Diffusion through the membrane is influenced by the solubility of molecules in lipids, temperature, and the concentration gradient. Lipid- soluble substances diffuse through the lipid layer at rates greater than through the protein portions of the membrane. Small molecules, such as those of water or inorganic ions, are able to pass down the concentration gradient via hydrophilic regions. These hydrophilic regions are associated with the points where some of the membrane’s protein molecules create a polar area, resulting in pore-like openings. However, movement of molecules through these regions is affected by electrical charges along the surface of the region, the size of the region, and the specific nature of the protein. Calcium ions affect the permeability of membranes. An increase in the concentration of calcium ions in the fluid surrounding the cell, or accumulation of calcium ions in the cytoplasm, can decrease the permeability of the membrane and has been demonstrated as a factor in the aging process of erythrocytes. Active transport is another essential membrane function. Because the cellular membrane also functions as a metabolic regulator, enzyme molecules are incorporated into the membrane. One such enzyme, particularly important as a metabolic regulator, is sodium-potassium-adenosine triphosphatase (Na-K-ATPase). This enzyme provides the necessary energy to drive the sodium-potassium pump, a fundamental ion transport system. Sodium ions are pumped out of the cells into extracellular fluids, where the concentration of sodium is higher than it is inside the cell. This movement of molecules is referred to as moving against the concentration gradient. The energy-producing activities of the mitochondria are heavily dependent on this process. Endocytosis (Fig. 3.3) is the process of engulfing particles or molecules, with the subsequent formation of membrane-bound vacuoles in the cytoplasm. Two processes, pinocytosis (the engulfment of fluids) and phagocytosis (the engulfment and destruction of particles), are forms of endocytosis. The vesicles formed by endocytosis either discharge their contents into the cellular cytoplasm or fuse with the organelles and the lysosomes. Phagocytosis is an important body defense mechanism and is discussed in more detail in Chapter 8. FIGURE 3.3 Vesicular transport. A. Endocytosis. B. Exocytosis. (Reprinted with permission from Premkumar K. The Massage Connection Anatomy and Physiology, Baltimore, MD: Lippincott Williams & Wilkins, 2004.) Description Cell Volume Homeostasis Maintenance of a constant volume despite extracellular and intracellular osmotic challenges is critical to the integrity of a cell. In most cases, cells respond by swelling or shrinking by activating specific metabolic or membrane transport processes that return cell volume to its normal resting state. These processes are essential for the normal function and survival of cells. Cells respond to volume changes by activating mechanisms that regulate their volume. The processes by which swollen and shrunken cells return to a normal volume are called regulatory volume decrease and regulatory volume increase, respectively. Cell volume can only be regulated by the gain or loss of osmotically active solutes, primarily inorganic ions such as sodium, potassium, and chloride or small organic molecules called organic osmolytes.  Regulatory loss and gain of electrolytes are mediated by membrane transport processes. In most animal cells, regulatory decreases in volume are accomplished by the loss of potassium chloride as a result of the activation of separate potassium and chloride channels or of the K+/Cl– cotransporter. Regulatory increases in volume occur through the uptake of both potassium chloride and sodium chloride. Certain ion transport systems have multiple roles, participating in volume regulation, intracellular pH control, and transepithelial movement of salt and water. Organic osmolytes are found in high concentrations in the cytosol of all organisms, from bacteria to humans. These solutes have key roles in cell volume homeostasis and may also function as general cytoprotectants. The accumulation of organic osmolytes is mediated either by energy-dependent transport from the external medium or by changes in the rates of osmolyte synthesis and degradation. Volume accumulation induces a very rapid increase in the passive efflux of organic osmolytes. Generally, this process is slow. Cell swelling inhibits transportation of the genes coding for organic osmolyte transporters and the enzymes involved in osmolyte synthesis. As transcription decreases, levels of messenger RNA (mRNA) drop and the number of functional proteins declines over a period of many hours to days. The sensing mechanism for cell size is not yet understood. A number of volume signals have been postulated, including swelling- and shrinkage-induced changes in membrane tension, cytoskeletal architecture, cellular ion concentrations, and the concentration of cytoplasmic macromolecules. No one signaling mechanism can account for the volume sensitivity of the various genes and membrane transport pathways that reactivate or are inactivated in response to perturbations in cell volume. Recent evidence suggests that cells can detect more than simple swelling or shrinkage. Disruption of cellular osmoregulatory mechanisms can give rise to a diverse group of disease states and their complications. Reactive and Neoplastic Growth Processes The size and shape of particular cell types (Fig. 3.4) are constant. Individual cell features can vary because of infectious disease or malignancy, and groups of cells (tissues) can manifest a variety of changes as well. Terms that may be encountered in the study of hematological diseases include the following:  FIGURE 3.4 Adaptive cell changes. (Asset provided by Anatomical Chart Co.) Description Anaplasia–highly pleomorphic and bizarre cytologic features associated with malignant tumors that are poorly differentiated. Atrophy–decrease in the number or size of cells that can lead to a decrease in organ size or tissue mass. Dysplasia–abnormal cytologic features and tissue organization; often is a premalignant change. Hyperplasia–increase in the number of cells in a tissue. Hypertrophy–increase in the size of cells that can lead to an increase in organ size. Metaplasia–change from one adult cell type to another (e.g., glandular to squamous metaplasia). Necrosis–the death of groups of tissue cells caused by environmental factors such as lack of oxygen. NOTE: This is a good time to review Key Terms in the Glossary and the Navigate 2 Advantage course. It is also a good time to complete Review Questions related to the preceding content. CYTOPLASMIC ORGANELLES AND METABOLITES Organelles (see Fig. 3.1A) are functional units of a cell. Most of the smaller organelles must be viewed with an electron microscope. Staining techniques are valuable in the identification of larger organelles and soluble substances in the cytoplasm. Stains such as Wright’s stain (discussed in Chapter 2) aid in differentiating the features of cells found in the blood and bone marrow. The staining and morphological characteristics of blood cells are presented in the last section of this chapter. Specialized stains (discussed in Chapter 32) can be used to identify constituents such as lipids, glycogen, iron, enzymes, and nucleic acids in cells. In abnormal cells, the soluble substances in the cytoplasm can provide important clues to the cell’s identity. A detailed discussion of representative cytochemical staining is included in Chapter 32. The organelles and their respective functions are listed here. Centrioles are two central spots inside of the centrosomes. These paired structures are cylindrical, and the long axes are always oriented at right angles to each other. Internally, each structure consists of nine (triplet) groups of microtubules. The centrioles divide and move to the opposite ends of the cell during cell division. They serve as points of insertion of the spindle fibers during cell division. The endoplasmic reticulum (ER), an extensive lace-like network, is composed of membranes enclosing interconnecting cavities or cisterns. It is classified as either rough (granular) or smooth (agranular). The rough sections contain ribosomes. Rough ER is associated with protein production; smooth ER is thought to be the site of the synthesis of lipids such as cholesterol and also the site of the breakdown of fats into smaller molecules that can be used for energy. The Golgi apparatus appears as a horseshoe-shaped or hook-shaped organelle with an associated stack of vesicles or sacs. In stained blood smears, the Golgi apparatus appears as the unstained area next to the nucleus. Functionally, the Golgi apparatus is the site for concentrating secretions of granules, packing, and segregating the carbohydrate components of certain secretions. Part of the Golgi apparatus and adjacent portions of the ER appear to form lysosomes. The Golgi- associated endoplasmic reticulum lysosome (GERL) concept focuses on the coordination of these cellular components. Products of the Golgi apparatus are usually exported from the cell when a vesicle of the Golgi apparatus fuses with the plasma membrane. Lysosomes contain hydrolytic enzymes. Three types of lysosomes have been identified: primary, secondary, and tertiary. Lysosomes are responsible for the intracellular digestion of the products of phagocytosis or the disposal of worn-out or damaged cell components. In some instances, lysosomes fuse with vacuoles containing foreign substances engulfed by the cell. In this process, the lysosomes may rupture and these internal enzymes actually autolyze the entire cell.  Microbodies are small, intracytoplasmic organelles, limited by a single membrane that is thinner than the lysosome. Microbodies contain enzymes. These organelles are especially likely to contain oxidase enzymes that produce hydrogen peroxide. Their function, related to oxidative activity, is an important aspect of phagocytosis. Microfilaments are solid structures, consisting of the protein actin and the larger myosin filaments. Microfilaments are the smallest components of the cytoskeleton. These structures are responsible for the amoeboid movement of cells, such as the phagocytic cells. In cytokinesis, the plasma membrane pinches in because of the contraction of a ring of microfilaments. Microtubules are small, hollow fibers composed of polymerized, macromolecular protein subunits, tubulin. They are narrow and have an indefinite length. The formation of tubules occurs through rapid, reversible self-assembly of filaments. Microtubules are associated with cell shape (the cytoskeleton) and the intracellular movement of organelles and may have a passive role in intracellular diffusion. The mitotic spindle is composed of microtubules. Mitochondria are composed of an outer smooth membrane and an inner folded membrane. Cells contain from hundreds to thousands of these rod-shaped organelles; however, mature erythrocytes lack mitochondria. The inner membrane functions as a permeable barrier. Each of the membranes has distinct functional differences. The cristae contain the enzymes and other molecules that carry out the energy-producing reactions of the cell. The granules of the matrix function as binding sites for calcium and contain some (DNA) and some ribosomes that are similar to those found in microorganisms. The reaction located on the inner membrane of the mitochondria is enzyme-controlled, energy-producing, and electron transfer- oxidative. Ribosomes, small dense granules, show a lack of membranes and are found both on the surface of the rough ER and free in the cytoplasm. They contain a significant proportion of ribonucleic acid (RNA) and are composed of unequally sized subunits. Ribosomes may exist singly, in groups, or in clusters. The presence of many ribosomes produces cytoplasmic basophilia (blue color) when a cell is stained with Wright’s stain. The complex of mRNA and ribosome serves as the site of protein synthesis. Numerous cytoplasmic ribosomes with few associated membranes suggest significant protein synthesis activity for internal use, such as in growing and dividing cells or in erythrocytic precursors in which hemoglobin is retained as it is synthesized. Cells, such as the plasma cell, that synthesize proteins for use outside of the cell tend to have greater amounts of rough ER except in the Golgi area. Cellular Inclusions and Metabolites Cells contain a variety of inclusions. Some of these structures are vacuoles with ingested fluids or particles, stored fats, and granules of glycogen and other substances. Numerous soluble cellular metabolites are present in the cytoplasm, but few have a clearly defined ultrastructural identity. Two metabolites of importance to hematologists are glycogen and ferritin. Glycogen is a long-chain polysaccharide, a storage form of carbohydrate that is detectable with a special stain, the periodic acid-Schiff (PAS) stain (refer to Chapter 26). The size of these particles is about twice that of a ribosome. The beta form of glycogen is found in single particles in the neutrophilic leukocytes. Undoubtedly, increased glycogen concentrations in cells such as the neutrophilic leukocyte are related to the needs of the cells for a high energy reserve to carry out their body defense functions. Ferritin is a common storage form of iron. Ferritin measures approximately 9 nm in diameter, which makes it substantially smaller than a ribosome. It is often found in iron-rich dense bodies referred to as telolysosomes. The term siderosome is used to refer to iron-saturated telolysosomes. Histologists refer to granular, iron-rich brown pigment as hemosiderin. Ferritin can be found in the macrophages of the spleen and bone marrow. The presence of ferritin in macrophages is indicative of the role these cells play in the recycling and storing of iron for hemoglobin synthesis (discussed in Chapter 5). NOTE: This is a good time to complete Review Questions related to the preceding content. Nuclear Characteristics Structure and Function The overall average size of the nucleus is 10 to 15 µm. This structure, which is the largest organelle, functions as the control center of the cell and is essential for its long-term survival. The nucleus is surrounded by a nuclear envelope, which consists of an inner and an outer membrane with a gap between them of approximately 50 nm. The outer membrane is probably continuous at scattered points with the ER. Many large pores extend through this membrane envelope. The nuclear pores are usually bridged by a diaphragm that is more diffuse than a membrane and prevents materials from passing in and out freely. Inside the nucleus, within the inner nucleoplasm, are the nucleoli (singular, nucleolus) and chromatin. Normally, the nucleus contains one or more small nucleoli that are not separated from the nucleoplasm by a specialized membrane. Morphologically, the nucleoli are irregularly shaped. Chemically, the nucleoli are composed mainly of RNA. Functionally, the nucleoli are the site of synthesis and processing of various species of ribosomal RNA. As the cell goes through various stages of growth and cellular division, the appearance of the nucleoli changes. These changes in chromatin appearance are related to the rate of synthesis of ribosomal RNA. Chromatin Characteristics The genetic material is composed of nucleic acids and protein (nucleoprotein), which is referred to as chromatin (Fig. 3.5). Despite the presence of protein in the chromatin, the DNA component stores genetic information. DNA has two functions: FIGURE 3.5 Understanding human DNA. The location of DNA (nucleolus); histone H1 backbone and nucleosome; DNA double helix. (Asset provided by Anatomical Chart Co.) Description 1. To dictate the nature of proteins that can be synthesized, thereby controlling the  function of the cell 2. To transmit information for cellular control from one generation to the next Proteins associated with the nucleic acids are divided into basic, positively charged histones and less positively charged nonhistones. The histones are believed to be essential to the structural integrity of chromatin. Histones may be important in facilitating the conversion of the thin chromatin fibers seen during interphase into the highly condensed chromosomes seen in mitosis. The nonhistone proteins are thought to play other roles, including genetic regulation. A general model of the organization of DNA and histones (Fig. 3.6) depicts a regular spacing arrangement. The fundamental, complete unit, the nucleosome, consists of a string of DNA wrapped around a histone core to form a bead-like segment consisting of about 180 base pairs of DNA.  FIGURE 3.6 Understanding human DNA: DNA wrapped around histone protein. (Asset provided by Anatomical Chart Co.) Description The chromatin arrangement within the nucleus demonstrates characteristic patterns when stained and viewed with a light microscope. These patterns are the most distinctive feature of a cell in terms of recognition of cell types and cell maturity. Chromatin is divided into two types: euchromatin (previously called parachromatin), the uncoiled, pale-staining areas, and heterochromatin (previously called chromatin), the condensed, dark-staining areas. Euchromatin is considered to represent unwound or loosely twisted gestions of chromatin that are transcriptionally active. Dark-staining heterochromatin is thought to represent tightly twisted regions of chromatin that are transcriptionally inactive. Younger or more active cells have more areas of euchromatin. Labeled RNA shows that active transcription occurs within the euchromatin areas. Heterochromatin may be in patches or clumped toward the nuclear envelope in a thin rim. Small patches of heterochromatin may be associated with the nucleolus. Chromatin in most primitive pluripotent stem cells, for example, embryonic stem cells, is in an open active state (euchromatin) and several genes are transcribed. During the cell differentiation process, the open type of euchromatin changes to the more condensed and genetically silent heterochromatin. In general, the more restricted the function of a cell, the more predominant the heterochromatin. For example, in the maturation of an erythrocyte, the chromatin distribution is very diffuse in the young cells with abundant euchromatin. As the erythrocyte matures, dense aggregates of heterochromatin predominate before the nucleus is lost in the mature cell. Several functional characteristics distinguish heterochromatin from euchromatin. Heterochromatin has a low expression level of chromatin-modifying factors, that is, epigenetic or chromatin plasticity. Heterochromatin replicates later during the S phase of the mitotic cell cycle than does euchromatin. Epigenetics refers to stable changes in gene function that are transmitted from one cell to its progeny. Epigenetic changes play an important role in normal cellular development and differentiation and are associated with silencing genes and chromatin condensation into heterochromatin. Chromosomes The total genetic material stored in an organism’s chromosomes constitute its genome. This exists as diffuse elongated chromatin fibers during cellular interphase. However, during cellular division (mitosis), the individual strands condense into short visible structures, the chromosomes. The number of chromosomes in each cell is constant within each species. Humans have a complement of 46 chromosomes arranged into 23 pairs; one member of each pair is inherited from the father and the other from the mother. Each of the members of one chromosome pair is referred to as a chromosome homologue. Of the pairs, 22 are called autosomes; the remaining pair represents the sex chromosomes. Males have an X and a Y sex chromosomes, and females have two X chromosomes. The technique of staining cells to bring out the different parts more clearly was discovered around 1873. Basic dyes were used to stain the cells. The name chromosome was chosen because these structures showed the bright colors of the basic stain. Chromosomes were first seen in human cells by Flemming in 1882; however, there were so many, and they were so small that he could not accurately estimate the actual number. As a result of the squash technique developed in 1956, the entire chromosome complement of a cell can be spread out and flattened so that each chromosome can be seen clearly. Cells for chromosome studies can be taken from any area of the body including the bone marrow, circulating blood, and amniotic fluid. Most studies use leukocytes (white blood cells). Tissue culture technique allows these cells to be placed in a nutrient medium and stimulated to grow and divide very rapidly. Normally, mature blood cells do not divide, but the addition of a mitogen, such as colchicine, stimulates cell division in white blood cells of the lymphocyte type. Other cells such as red blood cells cannot divide because they lack a nucleus. The cell selected for chromosome analysis is usually in the metaphase stage of cellular division. Clinical Use of Cytogenetics Clinical cytogenetics contributes to understanding inborn or acquired genetic problems by providing a low-power screening method for detecting isolated or missing chunks of chromosomes. Chronic myelogenous leukemia (CML) was the first human malignancy to be consistently associated with a chromosome abnormality, the Philadelphia (Ph) chromosome. Today, molecular methods are used to identify changes ranging from a single chromosome disorder to alterations involving the interchange of DNA between chromosomes. Abnormalities of erythrocytes (sickle cell disease and α- and β- thalassemias), leukocytes (acute myelogenous leukemia [AML], acute lymphoblastic leukemia [ALL], CML, and lymphoma), and coagulation factors (hemophilia A, hemophilia B, and factor V Leiden defect) can be detected by molecular methods (Table 3.1). TABLE 3.1 Examples of Representative Chromosomal Translocations in Acute Leukemias Type of Leukemia or Lymphoma Translocation Leukemias Acute myelogenous leukemia (M2) t(8;21) Acute myelogenous leukemia (M3) t(15;17) T-cell ALL t(1;14) and variants B-cell ALL t(9;22) In 1961, the Denver system of identifying human chromosomes was established. Chromosome pairs were numbered according to relative size and the position of their centromeres (the constricted area of a chromosome) and placed in groups according to letters. This arrangement of chromosome constitutes a karyotype. Differential staining of chromosomes (Fig. 3.7) using cytological techniques was introduced in the early 1970s. Although most chromosome banding techniques are obsolete, one technique that continues to be used in the era of molecular techniques involves the digestion of chromosomes with the enzyme trypsin, followed by Giemsa staining, that produces G-bands (Fig. 3.8). Chromosome analysis is being performed today by other technologies (discussed in Instrumentation in Hematology and Molecular Techniques [Chapter 30 and Chapter 31]). FIGURE 3.7 G-band karyotype of a normal male. (Reprinted with permission from McClatchey KD. Clinical Laboratory Medicine, 2nd ed, Philadelphia, PA: Lippincott Williams & Wilkins, 2002.) Description  FIGURE 3.8 Chromosome banding. After the chromosomes are stained with Giemsa stain, the areas of the chromosomes referred to as G-bands can be seen. The p (upper portion) and q (lower portion) are easily visible. Description The study of individual karyotypes and chromosome banding patterns is important to hematologists and geneticists. Supplementary information on hematological disorders, such as leukemias, can aid in establishing a diagnosis and can provide information about the probable outcome (prognosis) in some cases. Chromosomal Alterations Chromosomes sometimes break, and a portion may be lost or attached to another chromosome. Deletion and translocation are the terms used to describe these conditions. Deletion is defined as the loss of a segment of chromosome. Translocation is the process in which a segment of one chromosome breaks away (is deleted) from its normal location. Translocation can happen frequently between homologous chromosomes while they are paired in meiosis (discussed in the next section). An abnormality or aberration can result when the detached portion is lost or reattached. The Philadelphia chromosome, the first chromosomal abnormality discovered in a malignant disorder, is an example of a translocation from chromosome 22 to chromosome 9. Trisomy is another abnormality of chromosomes that is of interest to hematologists. In trisomy, one of the homologous chromosomes fails to separate from its sister chromatid. This failure to separate leads to a set of three chromosomes in place of the normal pair. Trisomy is encountered in a variety of hematological malignancies. Activities of the Nucleus Mitosis Mitosis (Fig. 3.9) is the process of replication in nucleated body cells (except ova and sperm cells). Cellular replication, or mitotic division, results in the formation of two identical daughter cells because the genes are duplicated and exactly segregated before each cell division. Originally, only two phases were recognized in mitosis: a resting phase or interphase (the period of time between mitoses) and M phase (the phase of actual cell division). FIGURE 3.9 Cell mitosis. (LifeART Super Anatomy Collection 2, CD-ROM, Baltimore, MD: Lippincott Williams & Wilkins.) SA202029. Description  Mitosis, particularly the interphase period in bone marrow cells, is important to hematologists because special staining and flow cytometry techniques (discussed in Chapter 26) now make it possible to perform DNA cell cycle analysis in these cells. This type of analysis is useful in the treatment of various hematological disorders because the optimum time for the administration of chemotherapeutic drugs can be determined. Interphase Since the introduction of isotope techniques, it has been documented that in cells capable of reproduction, DNA is replicated or doubled during the interphase. Interphase is now divided into three subphases (see Fig. 3.9): G1, first gap; S phase; and G2, second gap. Under normal conditions, the amount of time that a cell spends in interphase is relatively constant for specific cell types. 1. The G1 subphase lasts for approximately 6 to 8 hours. During this period, the nucleolus (nucleoli) becomes visible, and the chromosomes are extended and active metabolically. The cell synthesizes RNA and protein in preparation for cell division. As the G1 period ends, cellular metabolic activity slows. 2. The S subphase lasts for approximately 6 hours. This is the time of DNA replication, during which both growth and metabolic activities are minimal. However, all metabolic activities do not stop because not all the DNA is replicated at the same time. The shorter chromosomes are replicated first, and the others follow according to their length. Some DNA strands complete replication and resume the output of their messages, whereas others are still replicating. The protein portion of the chromosome is also duplicated, so that at the end of the S stage, each chromosome homologue has doubled but is held together by a single centromere (Box 3.1). BOX 3.1 Summary of DNA Structure and Activities A single strand of DNA is composed of a chain of phosphorylated deoxyribose sugars, each attached to a purine (adenine or guanine) or pyrimidine (cytosine or thymine) base to form nucleotides. The sugars are bonded together between the OH (hydroxyl) group of the 3′ carbon at one end and the PO4 group on the 5′ carbon of the next group. Every strand has a free 5′ phosphate on one end and a free 3′ hydroxyl on the other. This configuration results in a structural direction or polarity on each strand. Each strand of DNA is arranged in a linear sequence consisting of any of the four nucleotide bases (adenine, thymine, cytosine, and guanine). These nucleotide bases come in close contact with the complementary nucleotide bases on the opposite strand of the two DNA strands, the double helix.  Hydrogen bonding produces interstrand pairing of complementary bases (adenine with thymine; cytosine with guanine). Two hydrogen bonds exist between adenine and thymine; three hydrogen bonds exist between cytosine and guanine. The two strands of a DNA double helix run in opposite directions with the beginning 3′ carbon on one strand across from a free 5′ carbon on the opposing strand. This configuration is referred to as antiparallel. DNA synthesis in vivo and in vitro is unidirectional, proceeding from the 5′ to 3′ end with growth of the new strand only at the 3′ end. 3. The G2 phase is relatively short, lasting approximately 4 to 5 hours. This is the second period of growth, when the DNA can again function to its maximum in the synthesis of RNA and proteins in preparation for mitotic division. By the time a cell is ready to enter into mitotic division, proteins have been constructed in preparation for cell division, and both the DNA and the RNA are doubled. The centrioles have divided, forming a pair of new centrioles at right angles to each other. The Four Phases of Mitotic Division The M phase is the period of actual cell division, which lasts from 30 to 60 minutes; however, not all human body cells duplicate at this rate. The rate is most rapid in the early embryo, with a progressive slowing throughout the rest of the fetal life and childhood. In adults, most cells undergo mitotic division only fast enough to replace cells, with the eventual loss in old age of many types of cells. Abnormal conditions (malignancies) can alter the rate of mitosis of particular cell lines during any stage of growth and development. During mitosis, the replicated DNA and other cellular contents are equally distributed between the daughter cells. The four mitotic periods are prophase, metaphase, anaphase, and telophase (Box 3.2). Each state is visible in stained preparations by use of a conventional light microscope. BOX 3.2 Characteristics of the Four Mitotic Periods PROPHASE The chromatin becomes tightly coiled. Nucleolus and nuclear envelope disintegrate. Centrioles move to opposite poles of the cell. METAPHASE Sister chromatids move to the equatorial plate.  ANAPHASE Sister chromatids separate and move to opposite poles. TELOPHASE Chromosomes arrive at opposite poles. Nucleolus and nuclear membrane reappear. The chromatin pattern reappears. Prophase. In this stage of mitosis, the replicated strands of chromatin become tightly coiled, distinctive structures. The identical halves, referred to as chromatids, are joined at the centromere. The nucleolus and nuclear envelope disintegrate, with the fragments scattering in the cytoplasm. The centrioles, composed of microtubules, separate and migrate to the opposite poles of the cell. The microtubules aggregate to form the mitotic spindle that is attached to the centrioles. Metaphase. During metaphase, the identical sister chromatids move to the center of the spindle (the equatorial plate). Each of the chromatid pairs is attached to a spindle fiber and aligned along the equator of the cell. The point of attachment is the centromere, a constriction that divides the chromatid into an upper and a lower portion. Anaphase. This phase begins as soon as the chromatids are pulled apart and lasts until the newly formed chromosomes reach the opposite poles of the spindle. In this phase, the chromatid pairs are separated, with one half of each pair being pulled at their centromere by the spindle fibers toward each pole. Which half goes to which pole is random. Chromatids become chromosomes only after they have separated at the beginning of anaphase. Telophase. The chromosomes arrive at opposite poles of the cell in early telophase. One of each kind of chromosome arrives at each of the poles of the cell. The nucleolus and nuclear membrane reappear and the spindle fibers disappear during this phase. Because the chromosomes uncoil and become longer and thinner, the chromosome structural formations disappear. The DNA and proteins (nucleoproteins) now assume their distinctive chromatin arrangement. Following the stages that constitute nuclear division (karyokinesis), the cell undergoes cytokinesis. Cytokinesis is the division of cytoplasm. The cytoplasm around the two new nuclei becomes furrowed, and the cytoplasmic membrane pinches in. This pinching in is accomplished by the contraction of a ring of microfilaments that forms at the furrow. At the completion of cytokinesis, two new and identical daughter cells have been formed. G0 Phase Following the M phase, some cells continue through the mitotic cycle repeatedly, but others lose their mitotic ability and enter a protracted state of mitotic inactivity, the G0 phase. In some cases, cells will be stimulated by factors such as hormones (refer to Chapter 5 for a discussion of the hormone erythropoietin in the production of erythrocytes) to reenter the mitotic cycle. Abnormal proliferation of cells may result from overstimulation by extrinsic or intrinsic factors. Other nucleated cells, such as nerve cells, lose their ability to undergo mitosis and remain in the G0 (zero growth) phase permanently. NOTE: This is a good time to complete Review Questions related to the preceding content. APOPTOSIS In multicellular organisms, homeostasis is maintained through a balance between cell proliferation (mitosis) and programmed cell death, apoptosis. Cell death is generally classified into two major categories: apoptosis, representing “active” programmed cell death, and necrosis, representing “passive” cell death without known regulatory mechanisms. Cell death can be either physiologic or pathological. Physiologic cell death in animals generally occurs by apoptosis. Apoptosis is characterized by chromatin condensation and fragmentation, cell shrinkage, and elimination of dead cells by phagocytosis. In comparison, necrotic cell death is a pathologic form of cell death resulting from acute cellular injury, which produces rapid cell swelling and lysis (see Table 3.2). TABLE 3.2 A Comparison of the Characteristics of Necrosis versus Apoptosis Apoptosis Necrosis Stimuli Deprivation of survival factors such as Toxins, massive injury, severe growth factor or loss of extracellular hypoxia matrix Signals from death cytokines such as TNF Cell damaging stress Characteristic Physiological and pathological Conditions of ATP depletion conditions without ATP depletion Overall cell size Reduced by shrinkage Enlarged due to swelling Plasma membrane Intact but lost phospholipid Disrupted with the loss of integrity asymmetry Inflammation No Yes During embryonic development, excess numbers of developing cells die; in hormone-responsive tissues (e.g., uterus), cyclical depletion of a particular hormone leads to death. In both of these situations, cell death occurs by the process of apoptosis. Prevention of Apoptosis Most or all normal hematopoietic progenitor cells require specific growth factors to prevent apoptosis. Growth factors can prevent apoptosis by several mechanisms. One mechanism is increased synthesis of antiapoptotic proteins. These proteins differ for erythrocytes, leukocytes, megakaryocytes, and macrophages. The outcome of apoptosis in the normal mitotic process plays an important role in maintaining tissue homeostasis such as organ size in tissues that undergo continuous renewal by balancing cell proliferation with cell death. Beneficial Outcomes of Apoptosis in Hematopoietic and Lymphoid Systems Programmed cell death plays a key role in controlling the size of the lymphocyte pool at many stages of lymphocyte maturation and activation. If lymphocytes never encounter an antigen after cellular maturation, they die by apoptosis. Apoptosis protects us when it functions to remove lymphocytes with nonfunctional or autoreactive antigens, tumor cells, and virally infected lymphocytes. The cytotoxic T lymphocyte and natural killer (NK) cells employ apoptosis as a body defense mechanism. Development of malignant tumors results from deregulated proliferation or an inability of cells to undergo apoptotic cell death. Another beneficial outcome of apoptosis occurs during cell cycle replication. When a cell replicates DNA during the S phase of mitosis, the process is not error- free. In the process of DNA replication, DNA repair systems are active and attempt to correct copying errors. If errors are not corrected, apoptosis may be activated. Uncorrected errors may produce ineffective hematopoiesis, such as the megaloblastic anemia. DNA repair mechanisms are capable of correcting some other errors that can occur in replication. Failure of DNA repair mechanisms can contribute to mutations that produce malignancies. Additional beneficial outcomes of apoptosis in hematopoiesis include activation of apoptotic caspases that are enzymes involved in platelet production and release from mature megakaryocytes. Regulatory Stimuli in Apoptosis A delicate balance between proapoptotic and antiapoptotic regulars of apoptosis pathways is at play on a continual basis, ensuring the survival of long-lived cells and the proper turnover of short-lived cells in various tissues, including the bone marrow, thymus, and peripheral lymphoid tissues. Apoptosis can be influenced by a wide variety of regulatory stimuli. Cell survival appears to depend on the constant supply of survival signals provided by neighboring cells and the extracellular matrix. Inducers of apoptosis include the cytokines (e.g., tumor necrosis factor [TNF] family). Bcl-2 was the first antideath gene discovered. Bcl-2 family proteins play a central role in controlling the mitochondrial pathway related to apoptosis. In humans, more than 20 members of the human Bcl-2 family of apoptosis-regulating proteins have been identified. Bcl-2 family proteins regulate all major types of cell death, including apoptosis and necrosis.  Apoptosis is caused by the activation of intracellular proteases, known as caspases. Two pathways of apoptosis exist: intrinsic and extrinsic. The intrinsic pathway focuses on mitochondria as initiators of cell death. In contrast, extrinsic apoptosis relies on TNF family death receptors for triggering apoptosis. In certain types of cells, these systems converge. Currently, more than 14 caspases have been cloned and partially characterized in mammals, some of which are not involved in apoptosis. Although proteolytic enzymes, such as caspases, are main effectors of apoptosis, the mechanisms involved in activation of the caspase system are less clear. Two distinct pathways upstream of the caspase cascade have been identified. Death receptors, for example, CD95, trigger caspase-8, and mitochondria release apoptogenic factors, for example, cytochrome c, leading to the activation of caspase- 9. Mutation of CD95 in humans results in a lymphoproliferative syndrome caused by the inability to delete long-term activated T cells. Stressed ER contributes to apoptosis. JNK signaling has been implicated in various, often opposing cellular responses, including proliferation, differentiation, and cellular stress–induced apoptosis. Multiple other stress-inducible molecules, such as p53, JNK, AP-1, NF-kB, PKC/MAPK/ERK, and members of the sphingomyelin pathway, have a profound influence on apoptosis. Various stress stimuli such as cytotoxic drugs, γ-irradiation, heat shock, hypoxia, osmotic shock, and DNA-damaging agents stabilize the tumor suppressor protein p53, which promotes cell cycle arrest to enable DNA repair or apoptosis to eliminate defective cells. It continues to be largely unknown how p53 selects the pathways of G1 arrest or apoptosis. It is known that phosphorylation and acetylation play important roles for regulating biological activities of p53. Other negative regulatory mechanisms involve binding of JNK to p53, which mediates ubiquitination and proteolytic removal of p53 and the retinoblastoma gene product (Rb), which prevents the apoptotic function of p53. The p53 inhibitors are cleaved by caspases during apoptosis, which suggests a positive self-regulation of programmed cell death and close connection to key cell cycle regulators. Disorders Related to Decreased or Increased Apoptosis Alterations in cell survival contribute to the pathogenesis of a number of human diseases. Intracellular protein aggregates can stimulate apoptosis. A normally functioning apoptotic condition is necessary to remove cellular membrane debris. The lack of proper removal of cellular debris has been implicated in autoimmune disorders. Certain diseases are associated with a decrease of apoptosis; other diseases are associated with increased apoptosis. Decreased apoptosis is associated with leukemias such as chronic lymphocytic leukemia, systemic lupus erythematosus, follicular lymphomas, and cancers with p53 mutations. Anticancer treatment using cytotoxic drugs is considered to increase cell death by activating apoptosis. Diseases associated with increased apoptosis include acquired immunodeficiency disorder (AIDS) and aplastic anemia. Increased apoptosis of hematopoietic progenitor cells has been implicated in the pathophysiology of cytopenias associated with myelodysplastic syndromes. Meiosis Meiosis (Fig. 3.10) is the process of cell division unique to gametes (ova and sperm). In contrast to mitosis, the process of meiosis produces four gametes with genetic variability. Gametes have only one of the homologues of each of the 23 pairs of chromosomes (the haploid [1n] number). Other nucleated human body cells contain 23 homologous pairs of chromosomes (the diploid [2n] number).  FIGURE 3.10 First and second meiotic divisions. A. Homologous chromosomes approach each other. B. Homologous chromosomes pair, and each member of the pair consists of two chromatids. C. Intimately paired homologous chromosomes interchange chromatid fragments (crossover). Note the chiasma. D. Double- structured chromosomes pull apart. E. Anaphase of the first meiotic division. F, G. During the second meiotic division, the double-structured chromosomes split at the centromere. At completion of division, chromosomes in each of the four daughter cells are different from each other. (Reprinted with permission from Sadler T. Langman’s Medical Embryology, 9th ed, Baltimore, MD: Lippincott Williams & Wilkins, 2003.) The phases of meiosis differ from mitosis in several important ways. During phase I of meiosis, the homologous sister chromatids in a tetrad formation undergo the process of synapsis, lining up end to end. Synapsis allows for the easy exchange of genetic material through crossing over. In phase II of meiosis, reduction division occurs, producing the haploid number in the resulting gametes. Foundations of Genetic Interactions Genomics is the study of the entire genome of an organism. The study of actual gene expression or gene profile of a specific cell at an exact stage of cellular differentiation or functional activity is called functional genomics. The term, proteomics, refers to the study of the composition, structure, function, and interaction of proteins produced by a cell. Both gene profiles and proteins produced by a cell are factors of importance in the laboratory studies of a various disorders or diseases. During the past 30 years, a revolution has occurred in our understanding of genetic diseases. The identification of single-gene disorders is proceeding at an exponential rate. More than 200 human genes have been cloned, and the chromosomal map location is known for more than 140 of these genes. More than 100 genes are known to be associated with one or more diseases. The hemoglobinopathies and thalassemias (discussed in Chapter 17) have been extensively studied at the DNA level. Today, leukemias are being classified and treated at the molecular level. Information is also rapidly emerging about alterations in genes for factors VIII:C and IX of the coagulation system. Unfortunately, many disorders are multiple gene disorders present a more challenging genetic profile. Composition of DNA In 1953, Watson and Crick described the double-helix model of DNA in which genetic information is encoded into linear arrays in the form of the deoxyribonucleotide bases adenine (A), thymine (T), cytosine (C), and guanine (G). The two strands of DNA have antiparallel complementary sequences that pair by hydrogen bonding between the bases; thymine pairs with adenine and cytosine with guanine. The genetic code, which stores hereditary information, is stored as triplets of nucleotides that encode for various amino acids. Genomes of different organisms are unique and distinguishable from one another. The human genome consists of double-stranded DNA (ds-DNA) molecules organized into chromosomes with cell nuclei. Most human DNA is in the right-handed beta configuration having 10.5 bp per helical turn. A gene is a segment of DNA that is arranged along the chromosome at a specific position called a locus. Genes at a specific locus that differ in their nucleotide sequence are called alleles. Thus, in each somatic cell, one of the members of a set of alleles is maternally derived and the other paternally derived. Genes that lie close to each other in the linear array along the chromosomes have less opportunity for crossing over; genes that recombine once in every 100 meiotic opportunities are said to be 1 centimorgan (cM) apart. The relationship between the linear proximity of genetic loci and the recombinational frequency between them provides the basis for linkage mapping. However, this relationship is not always linear. Particular segments of DNA seem to be recombination hotspots and are predisposed to crossing over much more often than would be predicted from their DNA lengths. Gene Expression and Translation Control of gene expression is regulated during certain developmental stages and locations in the body. Transcription factors are proteins that regulate express of that gene. Some genes have enhancer elements or silencer elements, which are nucleotide sequences that can amplify or suppress gene expression. Many signals that regulate genes come from outside the cell, such as cytokine control of hematopoiesis (see Chapter 4). When an external molecule binds to its specific receptor on the surface of a cell, it activates the receptor and initiates a cell signaling pathway that conveys the activation signal from the receptor to the nucleus. The end result is an interaction with DNA that either activates or represses the target gene(s). Each gene has a unique sequence of nucleotides that is transcribed into mRNA. It is the sequence of nucleotides that determines gene function. Most genes are not composed of continuous stretches of nucleotides. In most cases, the coding sequences, or exons, are interrupted by intervening sequences, or introns. The entire gene, including both exons and introns, is transcribed in a pre-mRNA. The exon sequences are ultimately translated on the ribosomes into protein, but the intron sequences are spliced out as the pre-mRNA is processed into mature RNA. The sequences at the intron-exon junctions, called splices, are critical for mRNA processing and are important potential sites of mutation. Some inherited hematologic mutations, such as some of the thalassemias, are the consequence of mutations that derange mRNA splicing. Multiple exons can participate in alternative transcription of a gene. This produces several different mRNAs and proteins from a single gene. Alternative transcription and splicing allow for even more genetic diversity than the recognized genes in the human genome. Two forms of RNA are involved in regulating translation of mRNA, micro-RNA (m- RNA) and small interfering RNA (siRNA). Abnormalities in the translation into protein by mature mRNA do occur. The 5′ and 3′ end of mature mRNA encoding for protein contain untranslated regions (UTRs). UTRs affect the stability of mRNA and the efficiency of translation protein. An important function of UTRs is the regulation of many cellular proteins, such as proteins involved in the iron metabolism of maturing erythrocytes. Genetic Alterations A gene, as the functional unit of a chromosome, is responsible for determining the structure of a single protein or polypeptide. Variations in a nucleotide sequence of a gene that seen in different individuals are called alleles. Not every change in DNA produces an abnormality. Many of the alternate alleles identified in human globin chains do not result in a functional abnormality. Normally, a gene is a very stable unit that undergoes thousands of replications, with perfect copies resulting each time. On rare occasions, a copy may be produced that varies slightly and leads to an alteration in transcription from the long DNA molecule, with far-reaching consequences. A change in the gene is caused by mutation producing a change in the actual structure of DNA. A single nucleotide change among the thousands of base pairs in a gene may have crucial consequences to the gene product. If miscopied DNA base pairs are not corrected by the DNA repair systems, a new polymorphism or mutation can occur. If the change in the DNA sequence does not result in a functional abnormality, the change is called a polymorphism. A region of DNA that differs in only a single DNA nucleotide is called a single nucleotide polymorphism (SNP). If an SNP is a true polymorphism, it must occur with a frequency of than 1% in the general population. In comparison, if a miscopied DNA base pair is identified as the cause of a disorder or disease, the nucleotide change is considered to be a mutation rather than an SNP. An example of such a gene alteration has been traced to Queen Victoria or one of her immediate ancestors; the alteration led to classic hemophilia (discussed in Chapter 28) that spread throughout the royal families of Europe. Mutations usually affect a single base in the DNA. The sequence of nucleotide bases in the DNA is altered by the substitution of a single different base at one point along the DNA molecule. These mutations may act by affecting transcription of the gene, RNA processing to produce the mature mRNA, or translation of the mRNA into protein, or they may act by altering an important amino acid in the protein products. The sickle cell mutation (Fig. 3.11) is the best known example of a single nucleotide alteration. Human hemoglobin was one of the first proteins for which the genetic code was worked out and is a good example of the relationship between genes and proteins. The normal adult hemoglobin molecule (discussed in Chapter 6) consists in part of the protein globin. Globin is arranged into four chains in the form of two identical pairs. In each pair, the alpha chain consists of 141 amino acids and the beta chain contains 146 amino acids. The laboratory procedures of electrophoresis and chromatography allow determination of the exact sequence of amino acids on each of these two chains. In the case of sickle cell disease, hemoglobin S has a difference in one amino acid on the beta chain (Fig. 3.12). On this chain, valine is substituted for glutamic acid at the sixth position on the chain because an A in the sixth codon is changed to a T; this changes the codon GAG (glutamic acid) to GTG (valine). In hemoglobin C disorder, a substitution of lysine for glutamic acid at the same position in the beta chain occurs.  FIGURE 3.11 Hemoglobin S amino acid sequence. Hemoglobin S differs from hemoglobin A in one amino acid residue on the beta chain of the hemoglobin molecule. On this chain, valine (Val) is substituted for glutamic acid (Glu) at the sixth position of the chain.  FIGURE 3.12 Sickle cell trait and anemia. When two persons with sickle cell trait (genotype: A/S) produce offspring, the expected genotypic ratio is 1:2:1, or a 25% chance of offspring with a normal hemoglobin (A/A), a 50% chance of offspring with sickle cell trait (A/S), and a 25% chance of offspring with sickle cell anemia (S/S). Hgb, hemoglobin. Through meiosis, a parent with the trait may pass the mutation to another generation. In the case of sickle cell disease (anemia), an individual is homozygous for the trait. Because in the genetic expression of this disorder a lack of dominance exists, both genes of an allelic pair are partially and about equally expressed. Those individuals who are heterozygous for the trait are designated as suffering from sickle cell trait. The mode of inheritance of hemoglobin S is depicted in Figure 3.13. A further discussion of abnormal hemoglobins is presented in Chapter 17. FIGURE 3.13 Inheritance of hemoglobin S. Linkage studies can be used for those families in which the precise mutation is unknown but the locus of the mutation is known, such as in the hemoglobinopathies. Linkage analysis has proved highly useful as an indirect method of distinguishing between chromosomes carrying normal and mutant alleles. These polymorphisms represent so-called neutral mutations. Indirect analysis of this type has been used in the prenatal diagnosis of β-thalassemia (see Chapter 17) and is available for hemophilia A. At the present time, prenatal diagnosis by DNA analysis is available for several hematological disorders including hemophilia A, hemophilia B, sickle cell disease, α-thalassemia, and β-thalassemia. Oncogenes Cancer including leukemias and lymphomas is caused by genetic alterations oncogenes, as well as alterations in tumor suppressor genes, and microRNA genes. These alterations are usually somatic cell events, but germ-line mutations can predispose a person to inherited or familial cancer. A single genetic change is rarely enough for the development of a malignancy. Most evidence suggests a multistep process of sequential alteration in several, often many, oncogenes, tumor suppressor genes, or microRNA genes in the affected cells. The first evidence that cancer arises from somatic genetic alterations came from studies of Burkitt’s lymphoma where one of three different translocations juxtaposes an oncogene, MYC, on chromosome 8q24 to one of the loci for immunoglobulin (Ig) genes. In CML, which is initiated by a reciprocal to t(9;22) chromosomal translocation that fuses the ABL protooncogene to the BCR gene. The fusion gene encodes an oncogenic ABL fusion protein with enhanced tyrosine kinase activity. All leukemic cells in CML carry this chromosomal alteration. Many viral oncogenes have normal counterparts in the human genome, called protooncogenes. Identification of protooncogenes established the fact that the human genome carries genes with the potential to dramatically alter cell growth and to cause malignancy when altered or activated to an oncogene. Protooncogene activity in normal growth has the following functions: Growth factors Growth factor receptors Signal transducers Transcription factors Activation of a protooncogene to an oncogene disrupts the growth control mechanisms of a cell. Activation of protooncogenes results from mutation, gene rearrangement, or gene amplification. Cancer Stem Cells When a normal hematopoietic precursor cell, a stem cell or more differentiated progenitor cell, acquires a cancer-inducing mutation, it is called a hematopoietic neoplasm. The mutated cell of origin is referred to as the cancer-initiating cell. In some cases, already differentiated cells that have lost the ability to self-renew under normal circumstances, acquire a mutation(s) that reactivates self-renewal and produces a cancer-initiating cell. A cancer-initiating cell produces a cancer stem cell. Signaling pathways that regulate normal stem cell development have been implicated in cancer cell proliferation. This influence promotes overproduction of defective offspring. In the study of malignancies in tumors, it has been demonstrated that most malignancies do not arise from just one mutated predecessor but consist of diverse abnormal cell populations. The hematopoietic equivalent of the cancer stem cell is called the leukemic stem cell. Malignant transformation can produce abnormal karyotypes (discussed earlier) and abnormalities in DNA. Resultant genomic changes in cancer cells produce a survival or proliferation advance over normal cells. In acute leukemia, unregulated proliferation is accompanied by arrested cell development at the blast stage of development. Tumor Protein, p53 Specific tumor-suppressing genes, such as p53 gene, inhibit cell growth in normal cells. p53, also known as protein 53 or tumor protein 53, is a transcription factor, encoded by the TP53 gene. p53 is described as the guardian of the genome because it conserves stability by preventing genome mutations. p53 gene is important in regulation of the cell cycle and functions as a tumor suppressor. p53 can activate the repair of DNA when damaged, can hold the cell cycle at the G1/S regulation point until DNA can be repaired and continue in the cell cycle, and can initiate apoptosis if DNA damage is beyond repair. Hematologic malignancies demonstrating a specific genetic alteration of p53 include acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, and non-Hodgkin’s lymphoma. Minimal Residual Disease Minimal residual disease (MRD) is defined as the low level of disease, for example, leukemic cells, in a patient who appears to be in a state of clinical remission. In leukemia, the cells resistant to therapy remain in the bone marrow and/or peripheral blood. Following treatment, one million or more leukemic cells may persist, even when the residual leukemic cells are undetectable, and the patient appears to be in complete molecular remission (CMR). CMR can be further defined as the failure to detect cancer cells by the most sensitive molecular methodology available and by being valid only when leukemic cells are undetectable in three sequential samples 1 month apart. Molecular techniques are more sensitive to a low number of cells than morphologic appearance in the peripheral blood. Molecular techniques permit early detection of leukemia relapse at subclinical levels. These techniques; allow for early clinical intervention, perhaps before early progenitor cells, including CD34+ cells; and acquire genetic lesions that increase the aggressiveness of the clone. In the past, molecular detection and monitoring of patients with chronic myeloid leukemia patients have been successful. Now, the current state of the art and development of molecular techniques in other leukemias, for example, childhood ALL, are of growing interest. Tumor load, type of leukemia, whether disease-specific marker is identifiable, and technological limits will determine the optimum methodology for monitoring MRD (Box 3.3; Table 3.3). TABLE 3.3 Examples of Hematologic Disorders That Are Detectable Using Molecular Diagnostics Disorder Hemoglobinopathies Sickle cell anemia β-Thalassemias α-Thalassemias α-Globin Erythrocyte disorders Hereditary spherocytosis Hereditary elliptocytosis Leukocyte disorders Chronic granulomatous disease Neutrophil NADPH oxidase Lipid storage disorders Gaucher’s disease Niemann-Pick disease Coagulopathies Factor V Leiden (inherited resistance to activated protein C [APC]) BOX 3.3 Examples of Inherited Molecular Hematologic Disorders HEMOGLOBINOPATHIES Sickle cell anemia Hemoglobin C, SC, E, or D disease Thalassemias (α-thalassemia, β-thalassemia) COAGULOPATHIES Hemophilia (factor VIII, factor IX deficiencies) Factor V Leiden NOTE: This is a good time to complete the end of chapter Review Questions. CHAPTER HIGHLIGHTS Cellular Morphology: Ultrastructure and Organization Cells, as the smallest organized units of living tissues, have the ability to individually perform all the functions essential for life processes. Cellular membranes provide a semipermeable separation between the various cellular components, the organelles, and the surrounding environment. The cytoplasmic membrane, or outer membrane, defines the boundaries of the cell. Chemically, membranes consist of proteins, phospholipids, cholesterol, and traces of polysaccharide. The most popular hypothesis to explain the arrangement of these molecular components is the fluid mosaic model. Membrane-bound carbohydrates act as surface antigens, which function in the process of cellular recognition and interaction between cells. The term osmosis is used to describe the net movement of water molecules through a semipermeable membrane. Diffusion is an important process in overall cellular physiology, such as the physiological activities of the erythrocyte. This passive process through a semipermeable membrane may also be referred to as dialysis. Active transport is another essential membrane function. Because the cellular membrane also functions as a metabolic regulator, enzyme molecules are incorporated into the membrane. Two processes, pinocytosis (the engulfment of fluids) and phagocytosis (the engulfment and destruction of particles), are forms of endocytosis. Cytoplasmic Organelles and Metabolites Organelles are functional units of a cell. Cells contain a variety of inclusions. Some of these structures are vacuoles with ingested fluids or particles, stored fats, and granules of glycogen and other substances. The nucleus is the largest organelle. It functions as the control center of the cell and is essential for its long-term survival. The genetic material is composed of nucleic acids and protein (nucleoprotein), which is referred to as chromatin. The total genetic material stored in an organism’s chromosomes constitute its genome. This exists as diffuse elongated chromatin fibers during cellular interphase. However, during cellular division (mitosis), the individual strands condense into short visible structures, the chromosomes. Mitosis is the process of replication in nucleated body cells (except ova and sperm cells). Apoptosis Cell death is generally classified into two major categories: apoptosis, representing “active” programmed cell death, and necrosis, representing “passive” cell death without (known) underlying regulatory mechanisms. Meiosis Meiosis is the process of cell division unique to gametes (ova and sperm). The Foundations of Genetic Interactions Genomics is the study of the entire genome of an organism. The term proteomics refers to the study of the composition, structure, function, and interaction of proteins produced by a cell. A gene is a segment of DNA that is arranged along the chromosome at a specific position called a locus. Genes at a specific locus that differ in their nucleotide sequence are called alleles. Each gene has a unique sequence of nucleotides that is transcribed into mRNA. It is the sequence of nucleotides that determines gene function. In most cases, the coding sequences, or exons, are interrupted by intervening sequences, or introns. The entire gene, including both exons and introns, is transcribed in a pre-mRNA. A gene, as the functional unit of a chromosome, is responsible for determining the structure of a single protein or polypeptide. Cancer including leukemias and lymphomas is caused by alteration in oncogenes, tumor suppressor genes, and microRNA genes. Specific tumor-suppressing genes, such as p53 gene, inhibit cell growth in normal cells. Minimal residual disease is defined as the low level of disease, for example, leukemic cells, in a patient who appears to be in a state of clinical remission. Molecular techniques permit early detection of leukemia relapse at subclinical levels; allow for early clinical intervention, perhaps before early progenitor cells, including CD34+ cells; and acquire genetic lesions that increase the aggressiveness of the clone. Gene rearrangement studies are important in diagnostic hematopathology as indicators of clonality and as aids in determining the cellular lineage of a particular malignant proliferation. ONTOGENY OF HEMATOPOIESIS Hematopoiesis is the collective term used to describe the processes involved in the production of blood cells from human stem cells (HSCs) with subsequent cellular differentiation and development. Hematopoiesis occurs in several different locations during human development. The major locations include the yolk sac, aorta-gonad-mesonephros (AGM) region, fetal liver, bone marrow, and thymus. Further differentiation of lymphocytes also occurs in the spleen and lymph nodes of the secondary lymphoid tissues. The Embryonic Phase Embryonic blood cells, excluding the lymphocyte type of white blood cell, originate from the mesenchymal tissue that arises from the embryonic germ layer, the mesoderm (Fig. 4.1). The major anatomical sites of hematopoiesis progress from the yolk sac to the hepatic (liver) phase to the bone marrow (see Fig. 4.2). FIGURE 4.1 Cross-sectional view of the embryo at the time of mesoderm migration. The mesoderm cells coalesce into three distinct clumps, or colonies. The paraxial mesoderm tracks the path of the notochord. The intermediate mesoderm hovers just beside it for a short stretch of the embryo’s length. The lateral plate mesoderm fills the rest of the space and forms an important contact with the ectoderm above (dorsally), the endoderm below (ventrally), and the extraembryonic shell to the outside. (Reprinted with permission from Hartwig W. Fundamental Anatomy, Baltimore, MD: Lippincott Williams & Wilkins, 2008.)  Description FIGURE 4.2 Hemopoiesis in various organs before and after birth. (Reprinted with permission from Rubin E, Farber JL. Pathology, 3rd ed, Philadelphia, PA: Lippincott Williams & Wilkins, 1999.) Description Hematopoiesis begins as erythrocyte precursors appear in the yolk sac at 2 weeks gestation. The sites of hematopoiesis change several times. In humans, primitive hematopoiesis begins in the yolk sac in structures called blood islands. This process begins on day 19 and continues until week 8 of gestation. Primitive hematopoiesis generates erythrocytes, macrophages, and platelets but does not generate lymphocytes or granulocytes. Primitive erythrocytes are large, nucleated cells. These cells contain embryonic hemoglobins, Gower and Portland. Definitive erythropoiesis begins 1 to 2 days later than does primitive hematopoiesis. This process starts with the formation of self-renewing HSCs in the mesodermally derived intraembryonic region known as the AGM. The HSCs are the common precursor for all blood cells. HSCs are characterized by their ability to proliferate without differentiation. This process is called self-renewal. The mammalian embryo contains at least two spatially separated sources of hematopoietic cells. Fetal Hepatic Phase Hematopoiesis is the main function of the liver during a considerable period of prenatal development. It begins at 5 to 7 weeks of gestation and is characterized by recognizable clusters of erythroblasts, granulocytes, and monocytes that colonize the fetal liver, spleen, thymus, placenta, and ultimately the bone marrow in the final medullary phase. Hematopoiesis in the fetal liver reaches its peak by the third month of fetal life. Hematopoietic cells of the fetal liver exist in a specific microenvironment that controls their proliferation and differentiation. This microenvironment is created by different cell populations. Various cell types produce cytokines and chemoattractants and directly interact with hematopoietic cells, which provides for the functioning of the liver as a hematopoietic organ. The predominant type of hemoglobin during this phase is hemoglobin F (fetal hemoglobin). Hematopoiesis in the liver gradually declines after the sixth month in utero. At birth and continuing for a month or more, minimal hematopoiesis can still be found in the liver. The developing spleen, thymus, and lymph nodes contribute to hematopoiesis during this phase. The thymus is the first fully developed organ in the fetus. The spleen gradually decreases granulocytic production but remains active in lymphopoiesis. Production of megakaryocytes also begins during the hepatic phase. Early Medullary Hematopoiesis Beginning in the fourth month of gestation, the bones become large enough to have marrow cavities. After the fifth fetal month, the bone marrow begins to assume its ultimate role as the primary site of hematopoiesis (medullary hematopoiesis). Hemoglobin F and hemoglobin A are present at this stage of hematopoiesis. Under condition of fetal and neonatal stress, hematopoiesis can shift outside of the marrow (extramedullary hematopoiesis) to other organs. Cells of various cell lineages in all stages of development can be seen. Erythropoietin, granulocyte-stimulating factor, and granulocyte-monocyte–stimulating factor are present. Myeloid activity is evident during this phase with the myeloid:erythroid ratio gradually reaching the adult level of a 3:1 ratio. NOTE: This is a good time to review the definitions of Key Terms in the Glossary and flash cards on the Navigate 2 Advantage course. HEMATOPOIETIC ORGANS AND TISSUES The mature organs and tissues of the hematopoietic system consist of the bone marrow, thymus, liver, spleen, and lymph nodes. Before considering the general maturational characteristics of cells, knowledge of factors influencing the overall blood cell development is essential. The important characteristics of hematopoietic organs include the following: 1. The anatomic structure consists of different tissue types and component cells. 2. The stroma consists of various cells and extracellular macromolecules that occupy the hematopoietic tissue with hematopoietic cells. Stromal cells include specialized cells such as macrophages and lymphocytes, derived from HSCs, as well as epithelial cells of sinuses. The stroma constitutes the microenvironment where hematopoietic progenitor cells (HPCs) grow and differentiate. Various stromal cells and extracellular matrix molecules are believed to play a critical role in hematopoiesis. 3. The HPCs are the HSCs and their offspring that become blood cells of multiple specific lineages. Primary Hematopoietic Organs and Tissues Bone Marrow Sites and Function Bone marrow is found within the cavities of all bones and may be present in two forms: yellow marrow, which is normally inactive and composed mostly of fat (adipose) tissue, and red marrow, which is normally active in the production of most types of leukocytes, erythrocytes, and thrombocytes (Figs. 4.3–4.5). FIGURE 4.3 Normal bone marrow biopsy. Showing distribution of hematopoietic cells, fat, and trabecular bone: erythroid precursors (E), neutrophil precursors (N), eosinophil precursors (Eo), megakaryocyte (M). Giemsa: biopsies ×250 (A) and ×1,000 (B). (Reprinted with permission from Handin RI, Lux SE, Stossel TP. Blood: Principles and Practice of Hematology, 2nd ed, Philadelphia, PA: Lippincott Williams and Wilkins, 2003.) Description FIGURE 4.4 Bone marrow biopsy sections demonstrate normal cellularity. Approximately 40% to 50% cellularity in an otherwise healthy 60-year-old man. (Reprinted with permission from McClatchey KD. Clinical Laboratory Medicine, 2nd ed, Philadelphia, PA: Lippincott Williams & Wilkins, 2002.) Description FIGURE 4.5 Bone marrow biopsy sections demonstrate normal cellularity. Virtually 100% cellular marrow from a newborn boy. (Reprinted with permission from McClatchey KD. Clinical Laboratory Medicine, 2nd ed, Philadelphia, PA: Lippincott Williams & Wilkins, 2002.) Description The bone marrow is one of the body’s largest organs. It represents approximately 3.5% to 6% of total body weight and averages around 1,500 g in adults, with the hematopoietic marrow being organized around the bone vasculature (see Fig. 4.6). The bone marrow consists of hematopoietic cells (erythroid, myeloid, lymphoid, and megakaryocyte), fat (adipose) tissue, osteoblasts and osteoclasts, and stroma. Hematopoietic cell colonies are compartmentalized in the cords. Following maturation in the hematopoietic cords, hematopoietic cells cross the walls of the sinuses, specialized vascular spaces, and enter the circulating blood (Fig. 4.7). FIGURE 4.6 The development of blood cells: humerus bone, cortical bone, red bone marrow, and yellow bone marrow. (Asset provided by Anatomical Chart Co.)  FIGURE 4.7 Normal peripheral blood cells. A. Lymphocytes. B. Basophils. C. Eosinophils. D. Segmented neutrophils. E. Monocytes. F. Band form neutrophil. Description During the first few years of life, the marrow of all bones is red and cellular. The red bone marrow is initially found in both the appendicular and the axial skeleton (Fig. 4.8A) in young persons but progressively becomes confined to the axial skeleton and proximal ends of the long bones in adults (Fig. 4.8B). By age 18, red marrow is found only in the vertebrae, ribs, sternum, skull bones, pelvis, and, to some extent, the proximal epiphyses of the femur and humerus.  FIGURE 4.8 Sites of red bone marrow activity. A. Child: Red bone marrow (red-shaded areas) is located throughout the skeletal system in children. B. Adult: Yellow marrow replaces red marrow (dark-shaded areas) in the adult skeletal system. Red marrow activity occurs in the central portion of the skeleton. (Reprinted with permission from Dzierzak E. Ontogenic emergence of definitive hematopoietic stem cells, Curr Opin Hematol, 10(3):230, 2003.) Description In certain abnormal circumstances, the spleen and liver revert back to producing immature blood cells as extramedullary sites. In these cases, enlargement of the spleen and liver, hepatosplenomegaly, is frequently noted on physical examination. This situation suggests that undifferentiated primitive blood cells are present in these areas and are able to proliferate if an appropriate stimulus is present. This situation occurs under the following conditions: 1. When the bone marrow becomes dysfunctional in cases such as aplastic anemia, infiltration by malignant cells, or overproliferation of a cell line (e.g., leukemia). 2. When the bone marrow is unable to meet the demands placed on it, as in the hemolytic anemias (full discussions of the erythrocyte disorders are presented in Chapters 12 through 17.) Bone Marrow Structure and Function The bone marrow is found within the central cavities of axial and long bones. It consists of hematopoietic tissue islands and adipose cells surrounded by vascular sinuses interspersed within a meshwork of trabecular bone, approximately 5% in humans. Bone marrow is the major hematopoietic organ, and a primary lymphoid tissue, responsible for the production of erythrocytes, granulocytes, monocytes, lymphocytes, and platelets. Hematopoiesis takes place in a unique microenvironment in the marrow consisting of stromal cells and extracellular matrix. For hematopoiesis to occur, it must be supported by a microenvironment that is able to recognize and retain hematopoietic stem cells and provide the factors (e.g., cytokines) required to support proliferation, differentiation, and maturation of stem cells along committed lineages. The hematopoietic microenvironment consists of adventitial reticular cells, endothelial cells, macrophages, adipocytes, and, possibly, bone lining cells (e.g., osteoblasts) and elements of the extracellular matrix. Hematopoiesis is a compartmentalized process within the hematopoietic tissue with erythropoiesis taking place in distinct anatomical units (erythroblastic islands); granulopoiesis occurs in less distinct foci, and megakaryopoiesis occurs adjacent to the sinus endothelium. Upon maturation, the hematopoietic cells, regulated by the barrier cells, traverse the wall of the venous sinuses to enter the bloodstream; platelets are released directly into the blood from cytoplasmic processes of megakaryocytes penetrating through the sinus wall into the sinus lumen. The production, differentiation, and maturation of blood cells are regulated by humoral factors. The sources of hematopoietic factors vary. An example of a factor that stimulates hematopoiesis is erythropoietin (Epo). Erythropoietin is produced primarily in the kidney with minor amounts from the liver and stimulates proliferation of committed erythrocytic progenitors and release of immature red cells; high levels increase the rate of differentiation into erythrocyte progenitors. Hormones of the pituitary, adrenals, thyroid, and gonads appear to participate in erythropoiesis by altering erythropoietin production and erythroid progenitor response to other factors. For example, androgens, thyroxine, and growth hormone increase erythropoietin production; estrogen has an inhibitory erythropoietic effect. Hematopoietic tissue is also sensitive to external influences and can become suppressed in response to dietary restriction, malnutrition, chronic inflammation, toxicity, and proliferative or neoplastic disorders. Thymus Early in embryonic development, the stroma and nonlymphoid epithelium of the thymus are derived from the third and fourth pharyngeal pouches. This structure, located in the mediastinum, exercises control over the entire immune system. It is believed that the development of diversity occurs mainly in the thymus and bone marrow, although clonal expansion can occur anywhere in the peripheral lymphoid tissue. Initially, the thymus is populated by primitive lymphoid cells from the yolk sac and liver. An increased population of lymphoid cells physically push the epithelial cells of the thymus apart. In adults, T-cell progenitors migrate to the thymus from the bone marrow for further differentiation. T lymphocytes arise in the thymus from fetal liver or bone marrow precursors that seed the thymus during embryonic development. These CD34+ progenitor cells develop in the thymic cortex. Lymphocytes in the thymus, thymocytes, are T lymphocytes at various states of maturation (Fig. 4.9). T lymphocytes mature in the thymus, an organ found in the anterior mediastinum, and function in immune responses such as allergic reactions (delayed hypersensitivity), graft versus host transplantation reactions, and graft rejection. Progenitor cells that migrate to the thymus proliferate and differentiate under the influence of the humoral factor, thymosin. These lymphocyte precursors with acquired surface membrane antigens are referred to as thymocytes. The reticular structure of the thymus allows a significant number of lymphocytes to pass through it to become fully immunocompetent (able to function in the immune response), thymus-derived T cells. The thymus also regulates immune function by secretion of multiple soluble hormones.  FIGURE 4.9 Histology of thymus showing lobules (L) consisting of cortex (C) and medulla (M). H. Hassall corpuscles, S. septa (trabeculae). (Greer JP (ed). Wintrobes Clinical Hematology, 12th ed, Vol. 1, Philadelphia, PA: Lippincott, Williams & Wilkins, 2009:305.) Description The cortex of the thymus is characterized by a blood supply system; this is unique because it consists only of capillaries. Its apparent function is to act as a densely populated “waiting zone” of progenitor T cells. When these progenitor T cells migrate from the bone marrow and first enter the thymus, they have no identifiable CD4 or CD8 surface markers (double negative) and are located in the cortex- medullary junction. Cytokines and other soluble mediators influence these cells to move to the cortex and express both CD4 and CD8 surface membrane markers (double positive). As they move toward the medulla, the cells will progress to mature T cells that express either CD4 or CD8 surface antigens (markers). T cells make up the majority of the lymphocytes circulating in the peripheral blood. In the periphery of the thymus, they further differentiate into multiple different T-cell subpopulations with different functions, including cytotoxicity and the secretion of soluble factors, termed cytokines. Many different cytokines have been identified. Their functions include growth promotion, differentiation, chemotaxis, and cell stimulation. Many cells die in the thymus and apparently are phagocytized, a mechanism to eliminate lymphocyte clones reactive against self. Viable cells migrate to the secondary tissues. The absence or abnormal development of the thymus results in a T-lymphocyte deficiency. For example, patients with DiGeorge syndrome suffer from T-cell deficiency because of a chromosomal deletion that eliminates genes required for thymus development. Eventually, mature T cells leave the thymus to populate specific regions of other lymphoid tissue such as T cell–dependent areas of the spleen, lymph nodes, and other lymphoid tissues. All stages in the maturation process are gradual, and it is often impossible to identify an exact stage with certainty. The most immature forms of all cell types appear to be very similar morphologically, and their identification is often based on surrounding cell types. Lymphoid cells that do not express the appropriate antigens or are self-reactive die in the cortex or medulla as a result of apoptosis and are phagocytized by macrophages. The medulla contains only 15% mature T cells and apparently acts as a holding zone for mature T cells. A subset of epithelial cells found only in the medulla, called medullary thymic epithelial cells, plays a special role in presenting self-antigen to developing T cells and causing their deletion. This is one mechanism to ensure that the immune system remains tolerant to self (see Chapter 19 Disorders of Lymphocytes). The thymus contains other cell types including B cells, eosinophils, neutrophils, and other myeloid cells. Involution of the thymus is the first age-related change occurring in the immune system of humans. The thymus gradually atrophies and loses up to 95% of its mass during the first 50 years of life (Fig. 4.10). However, earlier thymic function has produced a pool of T lymphocytes that is maintained for a lifetime.  FIGURE 4.10 Thymic development. Description The accompanying functional changes of decreased synthesis of thymic hormones and the loss of ability to differentiate immature lymphocytes are reflected in an increased number of immature lymphocytes both within the thymus and as circulating peripheral blood T cells. Most of the changes in immune function, such as dysfunction of T and B lymphocytes, elevated levels of circulating immune (antigen- antibody) complexes, increases in autoantibodies, and monoclonal gammopathies, are correlated to involution of the thymus. Immune senescence may account for the increased susceptibility of older adults to infections, autoimmune disease, and neoplasms. SECONDARY LYMPHOID TISSUES The secondary lymphoid tissues include lymph nodes, spleen, gut-associated lymphoid tissue (GALT), thoracic duct, bronchus-associated lymphoid tissue (BALT), skin-associated lymphoid tissue, and blood. Lymphocytes circulate in the peripheral blood and lymphatic tissues and through secondary lymphoid organs (e.g., lymph nodes, spleen). Mature lymphocytes and accessory cells (e.g., antigen-presenting cells) are found throughout the body, although the relative percentages of T and B cells vary in different locations (see Table 9.1). Proliferation of the T and B lymphocytes in the secondary or peripheral lymphoid tissues is primarily dependent on antigenic stimulation. Spleen The spleen is a highly vascular organ with the major functions of removing aging and damaged blood cells and particles, such as antigen-antibody complexes and opsonized microbes from the circulation and initiate adaptive (antibody) immune response to blood-borne antigens. In addition to the function of filtering foreign substances and old erythrocytes from the circulation, the spleen stores platelets and participates in immune defense. The spleen contains the largest collection of lymphocytes and macrophages in the body. These cells with a reticular meshwork are organized into three zones: white pulp, red pulp, and the marginal zone (Fig. 4.11). FIGURE 4.11 Section of the spleen. White pulp (WP); lymphocytes (L) packed around an arteriole (A); red pulp (RP) surrounds white pulp and consists mainly of sinuses, the cords, and cordal spaces. (Greer JP (ed). Wintrobes Clinical Hematology, 12th ed, Vol. 1, Philadelphia, PA: Lippincott, Williams & Wilkins, 2009:305, FIG 14.7.) Description The sinusoids of the red pulp are lined by discontinuous epithelium, allowing passage of cells between the cords and the sinuses. These sinuses are lined with macrophages, which are loosely connected, creating a filter through which the blood can seep. These sinuses trap red cell inclusions, older red blood cells (greater than 120 days) for recycling, and platelets. In a normal adult, up to 2 L of blood per minute will filter through the spleen. Pitting refers to the spleen’s ability to pluck out particles from intact erythrocytes without destroying them. Blood cells coated with antibody are susceptible to pitting by splenic macrophages. Red blood cell membrane can reseal itself, but the cell can no longer synthesize proteins and lipids for new membrane because of its lack of cellular organelles. Pitting reduces the surface to volume ratio and results in formation of abnormally shaped spherocytes (see Chapter 7). Of all the lymphoid organs in the body, the spleen is the only one with a majority of B lymphocytes rather than T cells. The white pulp in the spleen acts in the same way as the lymphoid tissues in lymph nodes in initiating the immune reactions involving both cellular and humoral (antibody) immunity. The lymphoid cells of the white pulp form a cylindrical cuff around splenic arterioles, and these are mainly T cells. At branch points of the arterioles, there may be lymphoid nodules that contain B cells. The spleen also has a storage function. About 1/3 of the body pool of platelets is sequestered in the spleen. When the spleen enlarges (splenomegaly), too many platelets may be sequestered, leading to thrombocytopenia. Red pulp is the reservoir for platelets, sequestering approximately one third of circulating platelet mass. Removal of the spleen produces a transient thrombocytosis, but platelets return to normal concentrations in about 10 days postoperatively. Hypersplenism or splenomegaly is an enlargement of the overall size of the spleen. This enlargement causes some degree of extreme reduction of erythrocytes, leukocytes, and platelets, pancytopenia, in the circulating blood. Splenomegaly can result in pooling of 80% to 90% of platelets and produces peripheral blood thrombocytopenia. Infiltration of the spleen with additional cells or metabolic by- products can be referred to as hypersplenism. One of the disorders in which macrophages accumulate large quantities of undigestible substances is Gaucher’s disorder. Neoplasms in which malignant cells occupy much of the splenic volume can cause splenomegaly. If both the liver and spleen become congested with abnormal cells, a condition of hepatosplenomegaly can result. If tumor cells incapacitate the spleen, the peripheral blood will show evidence of hyposplenism. Acquired hypersplenism is a complication of sickle cell anemia. Acidosis, hypoxia, and hypoglycemia can lead to red blood cell sickling. This produces blockage of smaller blood vessels and blood clots that create necrosis of the tissue due to the lack of oxygen or RBCs in the spleen. Cumulative tissue damage leads to a condition called functional splenectomy or autosplenectomy. Lymph Nodes Lymph nodes are located all along the lymphatic vessels, and the lymph fluid circulates through the nodes as it progresses through the lymphatic system. Lymph nodes have three primary functions: 1. Site of lymphocyte proliferation in the germinal centers 2. Filter particulate matter, bacteria, or debris that enters the node via the lymph 3. Participate in the immune response to foreign antigens Anatomically, an outer capsule of the node forms trabeculae that radiate through the cortex and provide support for the macrophages and lymphocytes located in the node. Many of the lymphocytes from the lymph nodes circulate back and forth between the blood, the organs, and the lymphatic tissues. Functionally, there are two major subsets of lymphocytes, T cells, or T lymphocytes, and B cells, or B lymphocytes. Lymphoid progenitors give rise to natural killer (NK) cells, T lymphocytes, and B lymphocytes. B lymphocytes differentiate further in the bone marrow and further still on encountering an antigen. T lymphocytes differentiate and acquire antigen specificity, principally in the thymus and in some cases other lymphoid organs (e.g., gut). If productive gene rearrangement does not occur as the result of antigen exposure, the lymphocytes will die. T cells develop in the thymus and extrathymic tissues from a lymphoid precursor. Maturation of lymphocytes in the bone marrow or thymus results in cells that are immunocompetent. The cells are able to respond to antigenic challenges by directing the immune responses of the host defense. They migrate to various sites in the body to await antigenic stimulus and activation. As lymphocytes mature, their identity and function are specified by the antigenic structures on their external membrane surface, clusters of differentiation, CDs. When cell surface marker studies are performed, lymphocytes can be identified as belonging to specific subsets of lymphocytes. B lymphocytes are derived from hematopoietic stem cells by a series of differentiation events that occur in the fetal liver and, in adult life, in the bone marrow. B-lymphocyte differentiation is complex and proceeds through both an antigen- independent state and an antigen-dependent state, culminating in the generation of mature, end-stage, nonmotile cells called plasma cells. Some activated B cells differentiate into memory B cells, long-lived cells that circulate in the blood. B cells develop from lymphoid progenitors in the bone marrow and at sites at which the B cell encounters antigen (e.g., secondary lymphoid organs). B lymphocytes most likely mature in the bone marrow and function primarily in antibody production or the formation of immunoglobulins. B cells constitute about 10% to 30% of the blood lymphocytes. Memory B cells may live for years, but mature B cells that are not activated only live for days. If increased numbers of microorganisms flow through the lymph nodes, the resident macrophages can be overwhelmed and an infection of the node, adenitis, can result. Lymphadenopathy represents enlargement of the lymph node due to lymphoproliferation. If malignant cells from tumors break loose, they can enter the lymph nodes. Malignant cells have the potential of growing and metastasizing to other nearby nodes. Blood Blood is an important lymphoid organ and immunologic effector tissue. Circulating blood has enough mature T cells to produce a graft versus host reaction in transplantation. In addition, blood transfusions have been responsible for inducing acquired immunologic tolerance in kidney allograft patients. Blood is the most frequently sampled lymphoid organ. It is assumed that what is found in blood samples represents what is present in other lymphoid tissues. Although this may be a true representation, it is not always accurate. NOTE: This is a good time to complete Review Questions related to preceding content. CELLULAR ELEMENTS OF BONE MARROW Mature, terminally differentiated blood cells are derived from