Chapter 6 BLOOD, BONE MARROW, AND THE LYMPHOID SYSTEM
Bone marrow and the lymphoid system contribute cells to the circulating blood, and thus all three are traditionally included under the heading of hematology. The study of blood and blood-forming, or lymphoid, organs is an important aspect of internal medicine and many surgical specialties as illustrated by the following facts:
Some of the blood diseases, such as anemia or thrombosis, are extremely common and are encountered daily by medical specialists of any profile, including primary care physicians and general practitioners.
Blood abnormalities are found by means of laboratory testing not only in persons who have hematologic diseases per se, but also those who have many other systemic or organ-centered diseases. For example, hematologic abnormalities, such as high white blood cell (WBC) count, are encountered in the course of many infections. In such cases the hematologic abnormalities are more a symptom of other diseases than diseases in their own right.
Malignant tumors of the bone marrow and lymphoid organs are important causes of morbidity and mortality. Every year approximately 65,000 new cases of lymphoma and 25,000 cases of leukemia are diagnosed in the United States.
Basophil (basophilic leukocyte) Mononuclear white blood cell that contains abundant basophilic granules (stains blue with the Giemsa stain). Like mast cells, to which it is related, it participates in inflammatory reactions and atopic hypersensitivity reactions.
Bone marrow Central part of the bones, composed of trabecullar bone, fat cells or hematopoietic cells, and stroma. The hematopoietic bone marrow occupies the medullary part of most bones in neonates and infants, but in adults it is mostly limited to flat or short bones, such as the sternum, ilium, or vertebrae.
Clot (coagulum) Semisolid mass made up of a meshwork of polymerized fibrinogen (i.e., fibrin) and other coagulation proteins activated in the intrinsic or extrinsic coagulation pathway. In vivo formed clots also contain blood cells enmeshed in the strands of fibrin.
Cluster of differentiation (CD) antigens Cytoplasmic and cell surface molecules differentially expressed during the development and differentiation of various subsets of white blood cells and their bone marrow precursors. CD antigens are recognized immunohistochemically with monoclonal antibodies and named by consensus of an international committee. They carry numerical designations (e.g., CD4 as marker or helper T cells). CD antigens are also expressed on malignant cells and are important for the diagnosis of lymphomas and leukemias by immunohistochemistry and flow cytometry.
Coagulation factors Group of plasma proteins that participate in the coagulation cascade and the formation of the fibrin clot. Coagulation factors are numbered from 1 to 13, but most of them also have assigned names. Calcium is also a coagulation factor and is also known as factor IV. Protein coagulation factors are mostly synthesized by the liver. The activation of factors II, VII, IX, and X occurs only in the presence of the vitamin K-dependent carboxylase.
Eosinophil (eosinophilic leukocyte) Nucleated cell that in its mature form has a bilobed nucleus and numerous eosinophilic cytoplasmic granules. It participates in the defense of the body against infections and plays a prominent role in allergic reactions and the body’s response to parasites.
Erythrocyte precursor cells Nucleated ancestors of mature red blood cells that are derived in the bone marrow from erythroid progenitor cells in the erythroid burst-forming unit (BFU-E). As they differentiate sequentially, erythrocyte precursor cells can be recognized cytologically in bone marrow aspirates as pronormoblasts, normoblasts, and reticulocytes.
Fibrinolysis A physiologic process that leads to a controlled dissolution of the fibrin component of the clot. It is principally mediated by plasmin, a zymogen derived from the plasma protein plasminogen under the influence of tissue plasminogen activator (TPA). Plasmin acting on fibrin forms fibrin degradation products (FDPs), which may be found in plasma or urine during fibrinolysis.
Hematopoietic growth factors Polypeptides that act on hematopoietic progenitor and precursor cells, promoting their proliferation or differentiation (or both). This group of polypeptides includes erythropoietin; thrombopoietin; granulocyte colony-stimulating factor (G-CSF); macrophage colony-stimulating factor (M-CSF), interleukin-3; and related cytokines produced by stromal cells, macrophages, T lymphocytes, and many others.
Hemoglobin Main protein in erythrocytes enabling them to carry oxygen. Biochemically it is a heterodimeric tetramer composed of four globin polypeptide chains and four heme moieties linked to an iron. Several forms of hemoglobin are recognized on the basis of their globin composition. In adult red blood cells the most abundant is hemoglobin A, which is composed of two alpha and two beta globin chains (α2β2).
Lymphocyte Mononuclear white blood cell with a round nucleus and scant cytoplasm. Lymphocytes are subdivided into two major groups: T and B lymphocytes. They are derived from precursors located in the bone marrow, lymph nodes, spleen, thymus, and mucosa-associated lymphoid tissue. Lymphocytes participate in inflammatory reactions and are essential for immune reactions.
Monocyte Mononuclear cell containing only a few cytoplasmic granules that are not visible under light microscopy. It participates in inflammatory reactions and can differentiate into tissue macrophages.
Neutrophil (neutrophilic granulocyte or polymorphonuclear leukocyte (PMN) Motile phagocytic white blood cell (WBC) derived from precursors in the bone marrow. In its mature form it has a segmented nucleus and abundant cytoplasmic granules, which are neither acidophilic (as in eosinophils) nor basophilic (as in basophils). Neutrophils are the most numerous WBC, even though only 10% of neutrophils are found in circulating blood at any one time. Neutrophils can migrate through the vessel wall in response to chemotactic stimuli and participate in acute inflammatory reactions.
Neutrophil precursor cells Nucleated ancestors of mature neutrophils derived from the pluripotential stem cell, which gives rise to the myeloid stem cell. This multilineage precursor cell also gives rise to the precursors of monocytes, eosinophils, basophils, platelets, and erythrocytes. In the lineage giving rise to neutrophils it differentiates consecutively into myeloblasts, promyelocytes, metamyelocytes, and band neutrophils prior to becoming mature neutrophils.
Platelet (thrombocyte) Small (2–4 μm) anuclear cell, derived from the fragmentation of the cytoplasm of bone marrow megakaryocytes. It contains many biologically active substances and participates in blood coagulation and inflammation.
White blood cells (WBCs) Nucleated blood cells including neutrophils, eosinophils, basophils, monocytes, lymphocytes, and platelets. WBCs can be seen as a buffy coat on the interface between centrifuged red blood cells and plasma.
Activated partial thromboplastin time (aPTT) Laboratory test based on measuring the time needed for the in vitro formation of a clot under conditions most favorable for estimating the intrinsic and common coagulation pathway.
Agranulocytosis (granulocytopenia) Reduced number of granulated WBCs (neutrophils, eosinophils, and basophils) in peripheral blood. Typically it is caused by the reduced production of blood cells in the bone marrow and is a feature of aplastic anemia.
Anemia In laboratory medicine it is used as a designation for a reduced red blood cell mass. It is characterized by a reduced volume of RBCs (low hematocrit), a reduced RBC count, or a reduced concentration of hemoglobin.
Bone marrow biopsy Sampling of tissue from the bone marrow with a needle. The tissue may be prepared for histologic examination, or the aspirated cells can be smeared and stained for microscopic examination (bone marrow smear).
Coagulopathy Any disturbance of coagulation characterized by either increased coagulability of blood (hypercoagulability or thrombophilia), or a bleeding tendency (hemorrhagic diathesis) related to inadequate clotting of blood. Coagulopathies can be acquired (e.g., vitamin C and K deficiencies) or congenital (e.g., hemophilia).
Disseminated intravascular coagulation (DIC—consumption coagulopathy, or defibrination syndrome) Condition characterized by widespread clotting in the peripheral circulation. It is accompanied by the formation of microthrombi in arterioles, capillaries, and venules (microangiopathy), hemolytic anemia, and a tendency for uncontrolled bleeding due to the consumption of coagulation factors. It may be triggered by many diseases and is a common feature of shock.
Fibrin degradation products (FDPs or fibrin split products) Cleaved fragments of fibrin formed through the enzymatic action of fibrolytic enzymes such as plasmin. Since FDPs are relatively small, they pass in urine and can be detected there in DIC.
Hemolysis Lysis of red blood cells, which may occur in vivo in circulation or in tissues or in the test tube in vitro. A feature of various hemolytic anemias, lysis also occurs due to aging of RBCs or following bleeding. In vitro it may be induced by toxins, antibodies, or exposure to hypotonic fluid.
Lymphadenopathy Enlargement of lymph nodes of unknown origin. Most often it represents lymph node hyperplasia in response to infection, due to a neoplastic process involving the lymph nodes (e.g., lymphoma or metastatic carcinoma).
Monoclonal gammopathy Increased concentration of immunoglobulin gamma, presenting in serum electrophoresis as a sharp (“monoclonal”) peak. Typical of multiple myeloma, a neoplastic disorder characterized by monoclonal proliferation of malignant plasma cells that all secrete the same immunoglobulin. Monoclonal gammopathy is also found in monoclonal gammopathy of unknown significance (MNGUS), which may progress to multiple myeloma.
Peripheral blood smear Microscopic specimen prepared by spreading a thin film of peripheral blood on a glass slide and staining it with a metachromatic stain such as Giemsa stain. It used for microscopic examination of the morphology of RBCs and WBCs.
Prothrombin time Clinical test designed to measure the rate at which thrombin is formed in vitro under optimal conditions for estimating the function of factors II, V, VII, and X. It is thus used for assessing the extrinsic and common coagulation pathways. Clinical laboratories report it as standardized to external test values, such as the international normalized ratio (INR).
Anemia Group of diseases characterized by a decreased number of circulating RBCs or hemoglobin content of blood. It can be classified pathogenetically as anemia due to defective RBC production in the bone marrow or increased blood loss and hemolysis.
Hemophilia Congenital bleeding disorder characterized by a deficiency of factor VIII (hemophilia A) or factor IX (hemophilia B). Clinically it is characterized by uncontrollable bleeding following trauma or surgery.
Leukemia Group of clonal neoplastic disorders involving the stem cells and precursors of myeloid and lymphoid cells, in which the peripheral blood contains an increased number of neoplastic leukocytes. It can be classified as myelogenous or lymphocytic, and acute or chronic. Each of these groups comprises several distinct clinicopathologic subsets, which can be distinguished from one another by their unique cytogenetic, immunocytochemical, and molecular biologic features.
Lymphoma Malignancy involving the lymphoid system. It includes Hodgkin’s lymphoma and non-Hodgkin’s lymphoma, which in turn can be subclassified into several clinicopathologic entities. Each of these subtypes has unique histopathologic, immunocytochemical, cytogenetic, and often molecular biologic features, which must be taken into account when choosing proper chemotherapy.
Myelodysplastic syndrome Group of clonal hematologic disorders affecting maturation of erythroid, myeloid, and megakaryocytic precursors, with consequent trilineage cytopenia (pancytopenia) in the peripheral blood. It may be hereditary (genetic), primary (idiopathic), or secondary (treatment-related). This group of disorders includes several variants of refractory anemia (e.g., with ring sideroblasts or with an excess of blasts) and chronic myelomonocytic leukemia.
Myeloproliferative disorders Group of neoplastic hematopoietic stem cell disorders characterized by clonal expansion of the bone marrow or ineffective hematopoiesis and the appearance of neoplastic cells and their descendants in the peripheral blood. This group of disorders includes clinicopathologic entities such as chronic myelogenous leukemia, polycythemia vera, essential thrombocythemia, and agnogenic myeloid metaplasia (myelofibrosis).
Polycythemia vera Form of neoplastic clonal myeloproliferative disorder characterized by panmyelocytic hypercellularity of the bone marrow and an increased red blood cell mass. It must be distinguished from secondary polycythemia, in which the increased production of red blood cells is related to exogenous factors evoking oversecretion of erythropoietin.
Purpura Group of bleeding disorders characterized by widespread bleeding into the skin and internal organs. It results from diseases affecting the small blood vessels, platelets, or the entire coagulation system. It comprises a variety of diseases such as vascular, thrombotic, thrombocytopenic, or idiopathic purpura. Etiologically it may be classified as genetic, immune, drug-induced, viral, or idiopathic.
The blood consists of plasma and cells suspended in it. The red blood cells (RBCs) and most of the white blood cells (WBCs) are formed in the bone marrow. The lymphoid system, including the lymph nodes, the thymus, the spleen, and the mucosa-associated lymphoid tissue (MALT), contribute some of the lymphocytes.
During prenatal life, the first signs of embryonic hematopoiesis are found in the yolk sac 4 weeks after conception. During the second trimester the liver and the spleen become the primary sites of fetal hematopoiesis, to be gradually relocated to the bone marrow inside the developing skeleton. In adults, the hematopoietic bone marrow is confined to the axial skeleton, including the sternum, ribs, vertebrae, and pelvic bones (Fig. 6-2).
The hematopoietic bone marrow comprises hematopoietic cells and bone marrow stroma, which form the microenvironment essential for the growth and differentiation of the hematopoietic cells (Fig. 6-3). It also contains blood vessels, mostly in the form of sinusoids. These sinusoids have fenestrated walls so that the newly formed blood cells can easily enter the circulation. The bone marrow also contains thin bone trabeculae, which provide mechanical support and protection, and fat cells. With aging the hematopoietic elements decrease in number, and the fat cells become more numerous, replacing up to 70% of the total hematopoietic bone marrow in the elderly.
Figure 6-3 Bone marrow. The hematopoietic cells are located between the bone trabeculae. Cells of a single lineage tend to form groups, most prominent of which are the myeloid and erythroid nests. Megakaryocytes stand out because of their large size and multilobed nuclei. Blood vessels occur mostly in the form of sinusoids, which have fenestrated walls. RBC, red blood cell; WBC, white blood cell.
Blood cells develop from pluripotent hematopoietic stem cells under the influence of growth and differentiation factors.
The formation of blood cells is a highly regulated process that occurs in several steps involving both cell multiplication and differentiation. Several cell lineages are formed, ultimately giving rise to mature RBCs, platelets, and several distinct types of WBCs: neutrophils, eosinophils, basophils, monocytes, and lymphocytes (Fig. 6-4).
Figure 6-4 Cell lineages of hematopoiesis. Baso, basophil; BFU, burst-forming unit; CFU, colony-forming unit; E, erythrocyte; Eo, eosinophil; GM, granulocyte/macrophage; M, monocyte; Meg, megakaryocyte; NK, natural killer.
All hematopoietic cell lineages can be traced to a common ancestor—the pluripotent hematopoietic stem cell. Even though these cells account for less than 1% of all hematopoietic bone marrow cells, the adult bone marrow contains 25 to 500 million pluripotent stem cells. Some of these stem cells also enter into the circulating blood, from where they can be harvested for bone marrow transplantation. The pluripotent hematopoietic stem cells resemble small lymphocytes and can be identified only immunocytologically or by flow cytometry using labeled antibodies to their cell membrane marker—CD34.
Like all stem cells the totipotent hematopoeitic stem cells have a capacity for self-renewal but can also differentiate into two types of multilineage progenitor cells: lymphoid and myeloid progenitor cells. These progenitor cells give rise to unilineage progenitor cells, which in turn differentiate into committed precursor cells of mature lymphocytes, RBCs, neutrophils, monocytes, eosinophils, basophils, and platelets.
The replication of stem cells and progenitor cells and the differentiation of precursor cells depend on the action of growth and differentiation factors produced by the stromal cells, macrophages, and T lymphocytes. Numerous growth and differentiation factors have been isolated and characterized; many of these have been synthesized using recombinant DNA technology and are used in clinical practice to stimulate and or regulate hematopoiesis. Erythropoietin acts on the precursors of RBCs, thrombopoietin stimulates platelet production, and granulocyte colony–stimulating factor (G-CSF) acts on the differentiation of neutrophil precursors. Interleukin 3 (IL-3) behaves as a nonlineage-specific growth factor, acting on both myeloid and lymphoid lineages. Note that the terminal stages of lymphocyte maturation and differentiation are antigen-dependent and can be significantly amplified during immune reactions.
The differentiation of precursor cells is accompanied by the appearance and disappearance of specific cell surface molecules. Cell surface molecules known as clusters of differentiation (CD) have been especially useful for monitoring the maturation and differentiation of myeloid and lymphoid cells. For example CD34 is a marker of stem cells, CD4 is a marker for T helper cells, and CD8 is a marker for T cytotoxic cells, whereas CD20 is marker of B cells. Over 200 CD markers have been identified today, and many of them are used for diagnostic purposes in clinical hematopathology and immunology.
Formation and maturation of hematopoietic stem cells are tightly controlled by growth and differentiation factors. This process is also critically dependent on the normal supply of energy, nutrients, and vitamins. The most important among these are as follows:
Proteins. Proteins are essential ingredients of food, and a protein-deficient diet adversely affects hematopoiesis. This is most prominent in persons who have unusual dietary habits, suffer from eating disorders, or have intestinal malabsorption syndromes. Increased demand for proteins during pregnancy or childhood also may affect hematopoiesis, especially if combined with other nutritional deficiencies, such as an iron or vitamin deficiency.
Iron. A normal diet contains adequate amounts of iron, which is essential for the formation of hemoglobin (Hb). Inadequate intake or abnormal absorption in malabsorption syndromes, or an increased loss of RBCs (e.g., during heavy menstruation), may cause iron deficiency.
Vitamin B12. This vitamin complex includes several cobalamines, which are normally present in meat and animal products. Vitamin B12 binds to the intrinsic factor secreted by gastric parietal cells and is absorbed in the small intestines. Dietary deficiency or abnormal absorption may adversely affect the synthesis of DNA during hematopoietic cell growth and maturation. Vitamin B12 is crucial for the formation of tetrahydrofolate, which plays the role of an essential coenzyme in the synthesis of DNA (Fig. 6-5). A lack of vitamin B12 typically results in megaloblastic anemia.
Folic acid. Normally present in green leafy vegetables, folic acid together with cobalamin is essential for normal DNA synthesis. Deficiency may result from inadequate intake, absorption, or utilization (i.e., as in patients treated with folic acid antagonists). Clinically it also manifests as megaloblastic anemia.
The blood cells can be divided into three groups: RBCs (erythrocytes), WBCs (leukocytes), and platelets, or thrombocytes. The RBCs and platelets do not have nuclei, whereas the WBCs do. On the basis of the shape of their nuclei the WBCs can be further divided into segmented cells (neutrophils and eosinophils) and nonsegmented, or mononuclear, cells (monocytes and lymphocytes).
The normal life span of blood cells varies. The process of formation of blood cells and their maturation inside the bone marrow varies, and the duration the mature cells remain in the bone marrow is also cell lineage-specific. Likewise, the half-life of cells in the circulation varies. Remember that mature RBCs spend all their life inside the blood vessels, whereas WBCs can exit into the tissues and live there as well.
Red blood cells. Erythrocytes live in the circulation the longest—on average 120 days. This is important because RBCs can be removed from the blood and stored a few days or even a week or two for transfusion. Old and damaged RBCs are mostly lyzed by the splenic macrophages, but a small portion of them are hemolyzed inside the blood vessels. Thus we can distinguish between extravascular and intravascular hemolysis.
Extravascular hemolysis. Lysis of RBCs in the splenic macrophages results in an efflux of bilirubin that is bound to albumin (“unconjugated bilirubin”) and transferred for further processing and excretion into the liver (Fig. 6-6). In the liver bilirubin is conjugated and made water-soluble and excreted into the intestine. Most of the intestinal bilirubin is recirculated and reused, but part of it is excreted in the urine as urobilinogen.
Intravascular hemolysis. Under normal circumstances intravascular hemolysis affects only a small percentage of RBCs, but under certain abnormal conditions it may take major proportions. The fragmented RBCs release free Hb, mostly in the form of Hb dimers, which bind to a plasma protein called haptoglobin. It serves as a carrier for free Hb dimers and transports them to the liver. A part of Hb is oxidized into methemoglobin, which is degraded into globin and oxidized heme (ferriheme). Ferriheme binds to hemopexin, which carries it to the liver. After hemopexin is exhausted, excess oxidized heme can also bind to albumin and thus form methemalbumin, which is also taken up by the liver. Hemoglobin dimers that are not bound to plasma proteins are excreted in the urine. Hence, intravascular hemolysis is accompanied by hemoglobinuria, hemosiderinuria, and urobilinuria.
Figure 6-6 Lysis of senescent red blood cells (RBCs). Extravascular hemolysis occurs in the spleen and results in the formation of bilirubin, which is processed in the liver. Intravascular hemolysis leads to the formation of hemoglobin degradation products, which bind to specific plasma proteins carrying them into the liver. Unbound hemoglobin dimers are excreted in the urine, accounting for hemoglobinuria and hemosiderinuria. Both forms of hemolysis are associated with an increased excretion of urobilinogen in urine. Met Hb, methemoglobin.
Granulocytes. White blood cell precursors in the bone marrow form two compartments: a proliferative compartment and a maturation-storage compartment. In the first the cells remain for less than one day. It has been estimated that the maturing neutrophils remain in the maturation-storage department of the bone marrow for 7 to 10 days. Eosinophils remain there 2.5 days and basophils for only 12 hours.
Neutrophils enter the circulation from the bone marrow pool maturation-storage compartment, which contains the equivalent of neutrophils in reserve for 4 to 8 days. The half-life of neutrophilic granulocytes in circulation is 6 to 12 days. Neutrophils enter the blood in the form of segmented neutrophils or band cells. In the circulation they form two distinct sets: the marginating pool (storage portion) and the circulating pool. Neutrophils also can enter into the tissue, where they live, on average, for 2 to 3 days (Fig. 6-7).
Figure 6-7 Typical life span of neutrophils, platelets, and red blood cells and their bone marrow precursors. As shown in the diagram, all these cells originate from stem cells, but their life span in the bone marrow and outside of it varies. In the bone marrow the neutrophils are found in a proliferation–maturation compartment and a storage compartment. Platelets and erythrocytes are released as soon as they are formed and do not form a storage compartment. In peripheral blood the neutrophils and platelets form two compartments: a storage pool and a functional compartment. All red blood cells are in circulation and there is no red blood cell storage compartment. Under normal circumstances only neutrophils (and other leukocytes) emigrate into tissues.
Approximately 6% of all circulating WBCs are nonsegmented (band cells), whereas all others undergo nuclear segmentation. As they age their nuclei become more segmented, and the oldest ones have five segments. This segmentation can be used to estimate the average age of neutrophils in the Arneth index, which is a curve based on the number of segments of neutrophilic nuclei (Fig. 6-8). Left shift indicates that fewer nuclei are segmented; that is, there is a prevalence of young PMNs, whereas right shift indicates that there are more aging PMNs.
Figure 6-8 Arneth index. The blood contains young neutrophils (band cells) and old neutrophils (cells with five segmented nuclei), but most neutrophils have nuclei with three segments. Left shift of the curve indicates more young neutrophils in the circulation, and right shift more aging neutrophils.
Platelets. The half-life of platelets in circulation is 8 to 10 days. Platelets leaving the bone marrow initially enter the spleen where they remain for approximately 2 days. Two thirds of platelets enter the circulation, whereas one third remain as the active reserve pool of the spleen. Aging platelets are phagocytosed by splenic or hepatic phagocytic cells but may also be consumed at sites of minor endothelial cell injury, thereby activating intravascular coagulation.
Red blood cells—Oxygen transport. Red blood cells also serve as the primary transport vehicle for carbon dioxide, carrying it from the tissues into the lungs, where it is excreted. They also bind other gases and some other chemicals and act as part of the buffer system of the blood.
Granulocytes—Defense against infectious pathogens. Neutrophils, eosinophils, and basophils form the primary line of defense against bacteria, and eosinophils act against parasites. All WBCs secrete cytokines and other biologically active substances that are important for the inflammatory reactions and also may contribute to the metabolic response to infections.
Monocytes—Defense against pathogens and particulate matter. Monocytes are precursors of some tissue macrophages, and together they form the body’s phagocytic system. Tissue derivatives of monocytes act as antigen-presenting cells and participate in antigen uptake and presentation to lymphocytes; hence, they are an important part of the immune response. Macrophages produce cytokines and many other biologically active substances. They also participate in tissue repair, as in wound healing or healing of fractures.
Lymphocytes—Immune response. B lymphocytes differentiate into antibody-secreting plasma cells, whereas the T lymphocytes participate in the cell-mediated immune reactions. These cells also participate in the defense against viral infections. Lymphocytes are a major source of cytokines, which regulate many body functions in health and disease.
Erythrocytes, or red blood cells (RBCs), are highly specialized cells that do not have nuclei. In contrast to RBCs in the peripheral blood, their precursors in the bone marrow are nucleated. As the erythroid precursors mature, their nuclei become smaller and smaller and are finally extruded from the cytoplasm. The anucleated RBCs that still have residual RNA and a few cytoplasmic organelles may be also released into the circulation. These cells, called reticulocytes, account for less than 1% of all RBCs under normal circumstances.
The primary function of RBCs is transport of oxygen from the lungs into the peripheral tissues, but they also can bind other gases and also act as buffers. These functions are accomplished primarily through Hb, which accounts for 98% of their total weight.
Hemoglobin is a complex protein composed of globin and heme (Fig. 6-9). Globin is a tetramer composed of two α chains and two β chains or their equivalents γ and δ. The most abundant form of Hb is Hb A composed of two α and two β chains (α2β2). During fetal life the most predominant Hb is fetal hemoglobin (Hb F), which contains two α chains and two γ chains (α2γ2), but its level drops precipitously after birth, and by the age of 6 months it accounts for only 1% of the total Hb. Adult blood also contains Hb A2, which is composed of two α and two δ chains (α2δ2), accounting for less than 3.5% of total Hb. The concentration of Hb in the blood is lower in females than in males and also lower in adults than in newborns (Table 6-1).
Figure 6-9 Hemoglobin structure.
(Reproduced with permission from Damjanov I: Pathology for Health Professions, 3rd ed. Philadelphia, Elsevier, 2005.)
|Newborns 16.5–21.5 g/dL (165–215 g/L)|
Hemoglobin has a high affinity for oxygen, but it also binds carbon dioxide and carbon monoxide. The affinity of Hb for oxygen depends on the pH of the blood, temperature, and concentration of 2,3-biphosphoglycerate (2,3-BPG), and the presence of variant hemoglobins that have a higher affinity for oxygen. The affinity for oxygen can be determined by measuring the oxygen pressure needed to achieve 50% saturation of Hb (P50) (Fig. 6-10). In a normal person at pH 7.35 this saturation can be achieved at a PO2 of 27 mm Hg. The oxygen saturation curve can be left-shifted, meaning that a P50 can be achieved at lower partial pressure by changing the following variables:
Figure 6-10 Oxygen dissociation curve. Alkalization of the blood, low temperature, low partial pressure of CO2 (PCO2), and a low concentration or availability of 2,3-biphosphoglycerate (2,3-BPG) cause a left shift, whereas acidity of the blood, high temperature, and a high 2,3-BPG cause a right shift. Fetal hemoglobin (Hb F) has a higher affinity for oxygen than adult hemoglobin, thus causing a left shift. Hb S found in sickle cell disease shifts the curve to the right.
Fetal hemoglobin (Hb F) binds weakly with 2,3-BPG, thus shifting the curve to the left. In fetal or neonatal blood a P50 can be achieved at a PO2 of 19 to 21 mm Hg. This has an advantage before birth because it enables the fetus to “steal” oxygen from the mother. However, since the release of oxygen is also slower, in postnatal life the persistence of Hb F does not serve the affected person well.
Methemoglobin. Instead of the ferrous iron (Fe2+) present in the normal Hb, methemoglobin contains ferric iron (Fe3+). Normal blood contains less than 1% of Hb in this form, which is produced due to spontaneous oxidation of Hb. Methemoglobin has an increased affinity for oxygen, and it cannot release it in tissues as efficiently as normal Hb. Increased amounts of methemoglobin, caused by drugs and toxins may result in cyanosis, especially in infants whose normal methemoglobin-reducing capacity has not been fully developed. It leads to a left shift of the oxygen dissociation curve.
Carboxyhemoglobin. Carbon monoxide (CO) is formed in small amounts in the healthy body. It binds to Hb, forming carboxyhemoglobin, which in normal persons accounts for 0.2 to 0.8% of total Hb in blood. In smokers it may be elevated from 4% to 15%. Since Hb has approximately 200 times higher affinity for CO than for oxygen, large amounts of carboxyhemoglobin are formed in CO poisoning, as after suicide by inhaling car exhaust gases or kitchen oven gas. Death results from CO preventing oxygenation of Hb, which ultimately leads to lethal hypoxia.
Granulocytes respond to infection by entering the circulation and moving toward the site of infection (Fig. 6-11). At the site of infection the neutrophils marginate, adhere to the endothelial cells of capillaries and venules, and undergo activation. In addition to surface changes and changes in motility, activated neutrophils secrete biologically active substances that act on other cells in the tissues, most notably endothelial cells, macrophages, and lymphocytes. In concert with the activated neutrophils these cells secrete cytokines and growth factors, such as interleukins, tumor necrosis factor (TNF), platelet-activating factor (PAF), and many others. These substances act on the bone marrow, stimulating it to release new neutrophils into circulation and to proliferate stem cells and the precursors of granulocytes. All these events lead to neutrophilia—an increase in the number of neutrophils in circulation.
Figure 6-11 Reaction of neutrophils to infection. The neutrophils become marginated, adhere to the endothelium, and are activated in that process. Neutrophils respond to chemotactic stimuli and migrate toward the bacteria in the tissue. Cytokines and other biologically active substances produced at the site of inflammation act on the bone marrow, causing a release of neutrophils into the circulation (neutrophilia) and increased production of neutrophils in the bone marrow.
Neutrophils (also known as polymorphonuclear leukocytes—PMNs) form the first line of defense against bacterial infection. PMNs are so efficient at fighting bacteria because of the following properties:
Phagocytic capacity. PMNs readily form phagocytic vacuoles and are thus capable of ingesting bacteria and any other fragments found at the site of infection. Bacterial ingestion is facilitated by opsonins, such as complement fragment C3a and immunoglobulin G.
Figure 6-12 Phagocytosis of bacteria. Several phases can be recognized. (1) The bacterium acts chemotactically, attracting the neutrophil. (2) Adherence of bacteria to the surface of leukocytes is facilitated by opsonins such as complement fragments (C3) or IgG. (3) The bacterium is engulfed in the pseudopodia formed by the elongation of the cytoplasm of the neutrophil. (4) Phagocytosis of the bacterium includes formation of a phagosome from the phagocytic vacuoles and lysosomes. (5) Phagocytosed bacteria are killed by oxygen-dependent and oxygen-independent mechanisms. (6) Killed bacteria are digested. (7) During the interaction with the bacterium the leukocyte may be killed, releasing its cytoplasmic contents into the extracellular space. The exudate composed of dead and dying leukocytes and lyzed tissue components is called pus.
Loss of phagocytic cells, such as occurs in agranulocytosis caused by cytotoxic drugs and various disorders affecting the basic functions of the neutrophils, results in increased susceptibility to bacterial infections. These disorders can be congenital or acquired. The most important examples of deficient function of neutrophils are listed in Table 6-2.
|Defective adhesion||Leukocyte adhesion deficiency I and II|
|Decreased motility||Lazy leukocyte syndrome|
|Decreased phagocytosis||Chediak-Higashi syndrome|
|Decreased bacterial killing|
Neutrophil dysfunction may be induced by drugs and alcohol and is typically found in many metabolic diseases, such as diabetes or end-stage kidney disease. Autoimmune diseases and HIV infection also cause neutrophil dysfunctions.
Eosinophils participate in the body’s defense against bacteria. However, eosinophils are less numerous and migrate much slower than neutrophils, and thus they contribute significantly less in the fight against bacteria than PMNs. If the infection is long-lasting eosinophils become more prominent. Eosinophils are most prominently involved in the reaction to parasites. Eosinophilia is also found in patients who have allergies, autoimmune diseases, and skin diseases and is present in response to certain malignancies, such as Hodgkin’s disease.
Like all other WBCs and RBCs, lymphocytes originate from a common hematopoietic stem cell, which gives rise to developmentally restricted stem cells, populating the bone marrow and the peripheral lymphoid tissues. Three cell lines develop from this common lymphocytic precursor: T lymphocytes, B lymphocytes, and natural killer (NK) cells. In the circulating blood approximately 70% to 80% lymphocytes are B cells, 10% to 15% T cells, and 10% to 15% NK cells.
T cells mature by passing through the thymus, which typically occurs during fetal life and childhood. Inside the thymus the T cells differentiate into CD4+ T helper cells and CD8+ T cytotoxic cells. T cells are primarily involved in cell-mediated immune reactions, but they also regulate the functions of B cells.
B cells also differentiate stepwise and finally give rise to plasma cells—terminally differentiated cells involved in the production of immunoglobulins. Like T cells, the B cells interact with antigen-presenting cells, such as macrophages, Langerhans cells, or dendritic cells, but also among themselves.
Platelets are derived from the fragmentation of the cytoplasm of megakaryocytes in the bone marrow. This process is primarily regulated by thrombopoietin, a cytokine produced by the liver. The platelets released from the bone marrow are carried to the spleen, which serves as their primary reservoir. From the spleen the platelets periodically enter the circulation and are dispatched to areas of endothelial injury.
Platelets are small membrane-bounded particles measuring 2 to 4 μm. They do not have nuclei and are barely visible under light microscopy. The cytoplasm of platelets contains several components that are essential for their function (Fig. 6-13). Thus, it contains the following organelles:
Granules. The platelets contain several forms of granules. The most abundant are the alpha granules, which contain numerous proinflammatory and procoagulant and even anticoagulant proteins. Among other substances, alpha granules contain von Willebrand’s factor, fibrinogen, factor V, platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and the anticoagulant protein S. Dense granules contain energy-rich compounds such as adenosine diphosphate (ADP) and vasoactive substances such as serotonin. Lysosomes are important for lytic functions.
Cytoskeleton. The principal components of the cytoskeleton are the microtubules, composed of tubulin, and microfilaments, composed of actin and myosin. These fibrils are important for the maintenance of the shape of the platelets, their contraction, and the movement of other organelles and extrusion of granules.
Plasma membrane. This complex membrane is important for maintaining the integrity and the shape of platelets. It invaginates into the cytoplasm in the form of open canaliculi; it also contains numerous cell surface receptors and adhesion molecules. These complex glycoproteins are part of the cell membrane and are essential for the adhesion of platelets to surfaces and the initiation of the coagulation process. For example, the adhesion molecule GP IIb-IIIa binds to fibrinogen, and GP Ib binds to von Willebrand’s factor and collagen. Surface receptors also bind activators of platelets such as ADP or thromboxane A2 (TXA2).
Figure 6-13 Ultrastructure of the platelet. The external plasma membrane is studded with receptors and adhesion molecules (cannot be seen by electron microscopy). The plasma membrane invaginates, forming a canaliculus. Cytoskeletal fibers (microtubules and microfilaments) are important for the changes in the shape of platelets and the excretion of preformed substances sorted in various granules (alpha granules, dense granules, dense tubular system, lysosomes). Glycogen forms pools and is an important source of energy, which is produced primarily by the mitochondria.
Circulating platelets readily adhere to damaged endothelial cells and fill the endothelial defects in disrupted or damaged blood vessels. The hemostatic platelet plug that forms under such conditions is a hallmark of primary hemostasis (Fig. 6-14). It leads to the activation of the coagulation cascade and formation of the fibrin clot known as secondary hemostasis (Fig. 6-15).
Figure 6-14 Formation of the platelet plug. A, Adhesion of platelets to the collagen in the vessel wall denuded of endothelial cells is mediated by von Willebrand’s factor (vWF), which binds to collagen and the platelet adhesion molecule GPIb. B, Adhesion is followed by a change in the shape of platelets accompanied by a release of various procoagulants, most notably adenosine diphosphate (ADP) and thromboxane A2 (TXA2), and fibrinogen. C, The procoagulants released from the activated platelets recruit additional platelets to the site of endothelial injury and also promote their aggregation, leading to the formation of the primary hemostatic plug.
Figure 6-15 Primary and secondary hemostasis. As shown in Figure 6-14, vessel injury leads to the formation of the platelet adhesion, followed by the release of procoagulants and platelet aggregation. These events, leading to the formation of the primary hemostatic plug, are known as primary hemostasis. Substances released from platelets also cause vasoconstriction (thereby reducing blood flow to the damaged vessels) and activate the coagulation cascade and the formation of thrombin. Thrombin acts on platelets, promoting platelet aggregation, consolidation of the primary hemostatic plug, and its retraction. It also promotes polymerization of fibrin and thus contributes to the formation of a stable fibrin clot. These events are called secondary hemostasis. The fibrin clot is the substrate for several anticoagulants and fibrinolytic proteins, the most important of which is plasmin. ADP, adenosine diphosphate.
Defective primary hemostasis manifests as bleeding from capillaries and venules. Clinically skin bruises (petechiae and ecchymoses) or bleeding from mucosae of the mouth (gingival bleeding), nose (epistaxis), or intestine (hematochezia) may be evident. Bleeding usually results from vascular defects or platelet abnormalities, such as thrombocytopenia. The bleeding time is prolonged.
Defective secondary hemostasis is associated with bleeding from small arteries and arterioles and is often related to trauma, surgical procedures, or tooth extraction. It may result in the formation of large intramuscular or retroperitoneal hematomas, or hemarthrosis. Bleeding is usually related to a congenital deficiency of clotting factors, as occurs in hemophilia, or acquired abnormalities of the coagulation pathway, as occurs in chronic liver disease. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are prolonged.
|PARAMETER||PRIMARY HEMOSTASIS||SECONDARY HEMOSTASIS|
|Cause||Vessel wall, platelets||Coagulation factors|
|Source of bleeding||Capillaries, venules||Arteries and arterioles|
|Mode of bleeding||Spontaneous||Trauma/surgery-related|
|Site of bleeding||Skin and mucosae||Intramuscular hematomas, hemarthrosis, surgical wounds|
|Screening tests||Platelet count||Prothrombin time|
|Bleeding time||Activated partial thromboplastin time|
Family and personal history can point to some important risk factors that play a role in the pathogenesis of hematologic diseases. A few examples of such links and associations are given in Table 6-4.
|TYPE OF RISK FACTOR||SPECIFIC DISEASES–RISK FACTOR ASSOCIATIONS|
|Nutritional factors||Folate or vitamin B12 deficient diet: Megaloblastic anemia|
|Gastrointestinal bleeding||Iron deficiency: Microcytic anemia|
|Cirrhosis||Bleeding tendency: Prolonged PTT or thrombocytopenia|
|Medical and surgical procedures|
|External mechanical factors|
DIC, disseminated intravascular coagulation; PTT, prothrombin time
Symptoms and signs of hematologic diseases usually result from a loss or disturbance in the formation and function of RBCs, WBCs, or platelets and coagulation proteins. In some patients the hematologic problems dominate the clinical picture, whereas in others they are of secondary importance. Most manifestations of hematologic disorders are nonspecific and linked to a specific disturbance of blood-forming organs only with the judicious use of laboratory examinations. The following list includes some signs and symptoms that illustrate how these findings could point to a hematologic disease.
Easy fatigability. Fatigue is a common symptom of anemia and is related to the reduced capacity of blood to carry oxygen. It may also manifest as shortness of breath, drowsiness, or inability to concentrate.
Pallor. A reduced concentration of Hb in anemia causes paleness of the mucosae best seen by examining the conjunctiva or oral mucosa. Previously when leukemia could not be treated, excess of WBCs in circulation also caused a white complexion.
Ruddy red face. In contrast to anemia, which causes pallor, polycythemia is typically associated with a ruddy red face. Sluggish flow of the hyperviscous blood in these patients also leads to prominent dilatation of the retinal blood vessels, which can be seen with the ophthalmoscope.
Excessive bleeding. Bleeding from the gums or nose or into the skin are signs of thrombocytopenia caused by bone marrow failure, which occurs in aplastic anemia or in various forms of leukemia. Hemophilia presents with post-traumatic bleeding, hemarthrosis, or prolonged bleeding after surgical interventions.
Lymphadenopathy. Lymph node enlargement may be the first sign of lymphoma, but it could also be related to infections or autoimmune diseases. Clinically it is best to consider lymphadenopathy as either localized or generalized (Table 6-5).
Splenomegaly. Enlargement of the spleen may be a sign of increased hemolysis in some forms of anemia, such as hereditary spherocytosis. Splenic enlargement is found in many forms of leukemia and lymphoma. Other causes of splenomegaly are listed in Table 6-6.
|TYPE OF DISEASE||EXAMPLES|
|Acute infection||Strep throat, viral pharyngitis, syphilis, cat scratch disease|
|Chronic infection||Tuberculosis, histoplasmosis, chronic dermatitis|
|Autoimmune disease/unknown origin||SLE, sarcoidosis, erythema nodosum|
|Lymphoma||Non-Hodgkin’s lymphoma, Hodgkin’s lymphoma|
SLE, systemic lupus erythematosus.
|TYPE OF DISEASE/MECHANISM||EXAMPLES|
|Hemolytic anemia||Spherocytosis, thalassemia|
|Infections||Sepsis, endocarditis, malaria|
|Immune disorders||Rheumatoid arthritis, SLE, sarcoidosis|
|Storage diseases||Gaucher’s disease, Niemann-Pick disease|
|Portal hypertension||Cirrhosis, cardiac failure|
CLL, chronic lymphocytic leukemia; CML, chronic myelogenous leukemia; SLE, systemic lupus erythematosus.
Laboratory studies used in the work-up of hematologic disorders range from routine tests, such as complete blood count (CBC), to specialized tests for assessing the function of WBCs or platelets, to cytogenetic analysis of bone marrow cells and molecular analysis of specific genes known to be mutated in certain diseases. These tests are essential for the proper diagnosis of all hematologic diseases.
We discuss only the most commonly performed tests, however, as they are used for evaluating RBC, WBC, and coagulation disorders. The most important hematologic values used in general clinical practice are listed in Tables 6-7 to 6-9.
|Red blood cell (RBC) count|
|Mean corpuscular hemoglobin||27–33 pg/cell|
|Mean corpuscular hemoglobin concentration||33–35%|
|Mean corpuscular volume||80–96 m3 (fL)|
|Red cell distribution width||11.5–14.5%|
|Reticulocyte count||0.5–2.5% of all RBCs|
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