Hematology Lecture Notes PDF - New Era University

New Era University HEMATOLOGY 1 College of Medical...

New Era University HEMATOLOGY 1 College of Medical Technology LECTURE | Prof. Olivia Victoria Bondoc S.Y. 2023 – 2024 By: Andrea Ysabel Cortuna OVERVIEW OF CLINICAL HEMATOLOGY LESSON 1: HISTORY Since before 1900, physicians and medical laboratory professionals counted RBCs in HISTORY measured volumes to detect anemia or polycythemia. Athanasius Kircher in 1657, described “worms” Anemia in the blood o loss of oxygen-carrying capacity Anton van Leeuwenhoek in 1674 gave an account o often reflected in a reduced RBC count or of red blood cells decreased RBC hemoglobin Late 1800s – Giulio Bizzozero described platelets concentration as “petites plaques” Polycythemia 1902 – development of the Wright stain by James o increased RBC count reflecting increased Homer Wright circulating RBC mass, a condition that o For visualizing blood film examination leads to hyperviscosity through the microscope Diluted blood was transferred to a glass counting Wright’s Romanowsky-type stain chamber called a hemacytometer. (polychromatic, a mixture of acidic and basic The microscopist observed and counted RBCs in dyes), and refinements thereof, remain the selected areas of the hemacytometer, applied a foundation of blood cell identification. mathematical formula based on the dilution and At present, RBC, WBC, and platelet appearance on the area of the hemacytometer counted. are analyzed through automation or visually Report RBC count in cells per microliter (mL, using 500x to 1000x light microscopy mcL, also called cubic millimeter, mm3), examination of cells fixed to a glass microscope milliliter (mL, also called cubic centimeter, or slide and stained with Wright or Wright-Giemsa cc), or liter (L) stain. The scientific term for cell appearance is morphology o Encompasses cell color, size, shape, cytoplasmic inclusions, and nuclear condensation LESSON 2: RED BLOOD CELLS RED BLOOD CELLS (RBC) Anucleate, biconcave, discoid cells filled with a Visual RBC counting was developed before 1900 reddish protein (hemoglobin), which transports and was the only way to count RBCs oxygen and carbon dioxide In 1958, automated particle counters became Appear salmon pink and measure 7 to 8 mm in available in the clinical laboratory. diameter with a zone of pallor that occupies 1/3 The 1st electronic counter was patented in 1953 of their center (biconcavity) by Joseph and Wallace Coulter of Chicago, Illinois. The Coulter principle of direct current electrical impedance is still used to count RBCs in many automated blood cell analyzers. Visual counting skills remain useful where automated counters are unavailable. 1  TRANS: HGB, HCT, AND RBC INDICES o Mean cell hemoglobin concentration (MCHC) RBCs also are assayed for hemoglobin (HGB) MCV is recorded in femtoliters (fL), and reflects concentration and hematocrit (HCT). RBC diameter on a Wright-stained blood film. Hemoglobin measurement relies on a weak MCHC, expressed in grams per deciliter (g/dL), solution of potassium cyanide and potassium reflects RBC staining intensity and amount of ferricyanide – Drabkin reagent central pallor. An aliquot of whole blood is mixed with a MCH, in pictograms (pg) expresses the mass of measured volume of Drabkin reagent hemoglobin per cell and parallels the MCHC. Hgb is converted to stable cyanmethemoglobin RBC distribution width (RDW), expresses the (hemiglobincyanide) degree of variation in RBC volume Absorbance or color intensity of the solution is o Extreme RBC volume variability is measured in a spectrophotometer at 540 nm visible on the Wright-stained blood film wavelength as variation in diameter and is called Modifications of cyanmethemoglobin method are anisocytosis. used in most automated applications o In addition to aiding in the diagnosis of o Some automated blood cell analyzers anemia, RBC indices provide stable replace it with a formulation of the ionic measurements for internal quality control surfactant (detergent) sodium lauryl of automated blood cell analyzers. sulfate. Hematocrit is the ratio of the volume of packed RETICULOCYTES RBCs to the volume of whole blood. o Manually determined by transferring Polychromatic (polychromatophilic) erythrocytes are blood to a plastic tube with a uniform newly released from the RBC production site: the bore, centrifuging, measuring the column bone marrow of RBCs, and dividing by the total length o In Wright-stained blood film, 0.5% to 2.5% of the column of RBCs plus plasma exceed the 7- to 8-mm average diameter and Hematocrit is also called packed cell volume stain slightly blue-gray. (PCV) o Are closely observed because they indicate The light-colored layer between the RBCs and the ability of the bone marrow to increase plasma is known as the buffy coat RBC production in anemia caused by blood o WBCs and platelets loss or excessive RBC destruction Methylene blue dyes (nucleic acid stains or vital stains) are used to differentiate and count these young RBCs. Vital (or “supravital”) stains are dyes absorbed by live cells. Young RBCs contain ribonucleic acid (RNA) and are called reticulocytes when the RNA is visualized using vital stains. All fully automated blood cell analyzers provide: o Relative reticulocyte percentage o Absolute reticulocyte count o Immature reticulocyte fraction WHITE BLOOD CELLS (WBC) WBCs, or leukocytes, are a loosely related The medical laboratory professional may use the category of cell types dedicated to protecting three numerical results, RBC count, HGB, and their host from infection and injury. HCT, to compute the RBC indices: WBCs are transported in the blood from their o Mean cell volume (MCV) source (bone marrow or lymphoid tissue) to their o Mean cell hemoglobin (MCH) tissue or body cavity destination. 2  TRANS: WBCs are so named because they are nearly Basophils (BASOs) colorless in an unstained cell suspension. o Cells with dark purple, irregular WBCs may be counted visually using a cytoplasmic granules that obscure the microscope and hemacytometer. nucleus. The technique is the same as RBC counting, but o Basophil granules contain histamines and the typical dilution is 1:20, and the diluent is a various other proteins. dilute acid solution. o An elevated basophil count is called Visual WBC counting has been largely replaced basophilia – hematologic disease by automated blood cell analyzers. Lymphocytes (LYMPHs) Medical laboratory professionals who analyze o Lymphocytes comprise a complex body fluids such as cerebrospinal fluid or pleural system of cells that provide for host fluid may employ visual WBC counting. immunity. A decreased WBC count is called leukopenia, and o They recognize foreign antigens and an increased WBC count is called leukocytosis. mount humoral (antibodies) and cell- mediated antagonistic responses. o On a Wright-stained blood film, most TYPES OF WHITE BLOOD CELLS lymphocytes are nearly round, are slightly larger than RBCs, and have Neutrophils (NEUTs, segmented neutrophils round nuclei and a thin rim of [SEGs], polymorphonuclear neutrophils nongranular cytoplasm. [PMNs]). o An increase in the lymphocyte count is o These are phagocytic cells whose major called lymphocytosis – viral infections purpose is to engulf and destroy o An abnormally low lymphocyte count is microorganisms and foreign material. called lymphopenia or lymphocytopenia – drug therapy or immunodeficiency Monocytes (MONOs) o It is an immature macrophage passing through the blood from its point of origin (bone marrow) to a targeted tissue location. o Macrophages are the most abundant cell type in the body. o Macrophages occupy every body cavity o The term segmented refers to their – some motile and some are multilobed nuclei. immobilized. o The cytoplasm of neutrophils contains o Benign monocytosis may be found in pink- or lavender-staining granules filled certain infections or in inflammation. with bactericidal substances. Leukemia o An increase in neutrophils is called o It is an uncontrolled proliferation of a neutrophilia – bacterial infection clone of malignant WBCs. o A decrease is called neutropenia – o It may be chronic or acute. medications or viral infections o It may involve any of the cell lines and are categorized by their respective Bands (band neutrophils, BANDs) immunophenotypes and genetic o Slightly less mature neutrophils with a aberrations nonsegmented nucleus in a U or S shape. o Some leukemias are more common in a o An increase in bands also signals specific age group bacterial infection – left shift ▪ Chronic lymphocytic leukemia Eosinophils (EOs) is more prevalent in people >65 o Cells with round, bright orange-red years old cytoplasmic granules filled with proteins ▪ Acute lymphoblastic leukemia is involved in immune system regulation. the most common form of o An elevated eosinophil count is called childhood leukemia eosinophilia – allergy or parasitic infection 3  TRANS: o Medical laboratory scientists participate platelet counts and uncontrolled platelet in the characterization of leukemias production using Wright-stained bone marrow o Life-threatening hematologic disorder smears, flow cytometric A low platelet count is called thrombocytopenia immunophenotyping, molecular – a common consequence of drug treatment diagnostic technology, cytogenetics, and, o It is usually accompanied by easy occasionally, cytochemical staining. bruising and uncontrolled hemorrhage. Accurate platelet counting contributes to patient safety. PLATELETS OR THROMBOCYTES Platelets, or thrombocytes, are true blood cells COMPLETE BLOOD COUNT that maintain blood vessel integrity by initiating vessel wall repairs. A complete blood count (CBC) is performed on They rapidly adhere to the surfaces of damaged automated blood cell analyzers and includes the blood vessels, form aggregates with neighboring RBC, WBC, and platelet measurements platelets, and secrete proteins and small The medical laboratory professional is molecules that trigger thrombosis, or clot responsible for the integrity of the specimen and formation. ensures that it is submitted in the appropriate Platelets are the major cells that control anticoagulant and tube and is free of clots and hemostasis – a series of cellular and plasma- hemolysis. based mechanisms that seal wounds, repair vessel The specimen must be of sufficient volume, walls, and maintain vascular patency. because “short draws” result in incorrect They are only 2 to 4 mm in diameter, round or anticoagulant-to-blood ratios. ▹ The specimen oval, anucleate, and slightly granular. must be tested or prepared for storage w/in the Uncontrolled platelet and hemostatic activation is appropriate time frame and must be accurately responsible for: registered in the work list – specimen accession o Deep vein thrombosis Most laboratories employ automated blood cell o Pulmonary emboli analyzers to generate the CBC. o Acute myocardial infarctions o When one of the results from the blood o Cerebrovascular accidents cell analyzer is abnormal, the instrument o Peripheral artery disease provides an indication of this – flag o Repeated spontaneous abortions ▪ Blood film examination should (miscarriages) be performed Platelet count uses the same technique used in counting WBCs on a hemacytometer but uses a BLOOD FILM EXAMINATION different counting area, diluent, and dilution. o Phase microscopy provides for easier Preparation of a “wedge-prep” blood film on a identification glass microscope slide, and staining it using Automated blood cell analyzers have largely Wright or Wright-Giemsa stain replaced visual platelet counting and provide Visually estimate: greater accuracy. o WBC count (with the 40x or 50x One advantage of automated blood cell analyzers objective at 400x or 500x magnification) is their ability to generate a mean platelet volume o Platelet count (with the 100x OIO at (MPV) 1000x magnification) o Presence of predominantly larger Next, systematically review, identify, and platelets generates an elevated MPV tabulate 100 WBCs to determine their percent value – regenerative bone marrow distribution – WBC differential (“diff”) response to platelet consumption Lastly, examination of the morphology of WBCs, Elevated platelet counts, called thrombocytosis – RBCs, and platelets by light microscopy for inflammation or trauma abnormalities of shape, diameter, color, or Essential thrombocythemia is a rare malignant inclusions (1000x magnification) condition characterized by extremely high 4  TRANS: o Erythroid series and myeloid series ENDOTHELIAL CELLS Medical laboratory scientists, clinical pathologists, and hematologists review Wright- Endothelial cells are the endodermal cells that stained aspirate smears for morphologic form the inner surface of the blood vessel. abnormalities, high or low bone marrow cell They are important in maintaining normal blood concentration, and inappropriate cell line flow, decelerating platelets during times of distributions. injury, and enabling WBCs to escape from the The biopsy specimen, enhanced by hematoxylin vessel to the surrounding tissue when needed. and eosin (H&E) staining, may reveal COAGULATION abnormalities in bone marrow architecture indicating leukemia, bone marrow failure, or one Most hematology laboratories include a blood of a host of additional hematologic disorders. coagulation testing department. Cytochemical stains may be used to differentiate Platelets are a key component of hemostasis, and abnormal myeloid, erythroid, and lymphoid cells. plasma coagulation is the second component. o These stains include: The coagulation system employs a complex ▪ Myeloperoxidase sequence of plasma proteins, some enzymes, and ▪ Sudan black B some enzyme cofactors – clot formation ▪ Nonspecific and specific Another system of enzymes and cofactors digests esterase clots to restore vessel patency – Fibrinolysis ▪ Periodic acid-Schiff The coagulation section of the hematology laboratory provides a series of laboratory assays o These stains include: that assess plasma proteins and their interactions ▪ Phosphatase with hematologic cells. ▪ Alkaline phosphatase Focuses on blood specimen integrity for the In most laboratories, cytochemical staining has coagulation laboratory, because minor blood been replaced by flow cytometry specimen defects, including clots, hemolysis, immunophenotyping, molecular diagnostic, and lipemia, plasma bilirubin, and short draws, render cytogenetic techniques. the specimen useless Immunostaining methods have enabled the High-volume coagulation tests for acute care identification of cell lines by detecting lineage- facility includes: specific antigens on the surface or in the o Platelet count and MPV cytoplasm of leukemia and lymphoma cells. o Prothrombin time o E.g. CD42b (diagnostic for o Partial thromboplastin time/APTT megakaryoblastic leukemia) o Thrombin time (or thrombin clotting Flow cytometers may be quantitative, or time) qualitative. o Fibrinogen assay The former devices are automated clinical blood o D-dimer assay cell analyzers that generate the quantitative The prothrombin time and partial thromboplastin parameters of the CBC through the application of time assess each portion of the coagulation electrical impedance and laser or light beam pathway for deficiencies and are used to monitor interruption. anticoagulant therapy. Both qualitative and quantitative flow cytometers are employed to analyze cell populations by measuring the effects of individual cells on laser ADVANCED HEMATOLOGY PROCEDURES light, and by immunophenotyping for cell membrane epitopes using monoclonal antibodies The hematology laboratory provides: labeled with fluorescent dyes. o Bone marrow examinations Cytogenetics, a form of chromosome analysis, is o Flow cytometry immunophenotyping employed in bone marrow aspirate examination o Cytogenetic analysis to find gross genetic errors such as the o Molecular diagnosis assays Philadelphia chromosome (chronic myeloid Bone marrow aspirates and biopsy specimens are leukemia) collected and stained to analyze nucleated cells Cytogenetic analysis remains essential to the that are the immature precursors to blood cells. diagnosis and treatment of leukemia. 5  TRANS: Molecular diagnostic techniques enhance and even replace some of the advanced hematologic methods. o Real-time polymerase chain reaction o Microarray analysis o Fluorescence in-situ hybridization o DNA sequencing systems o Detect various chromosome translocations and gene mutations that confirm specific types of leukemia and lymphoma, establish their therapeutic profile and prognosis, and monitor the effectiveness of treatment ADDITIONAL HEMATOLOGY PROCEDURES The G6PD assay phenotypically detects an inherited RBC enzyme deficiency causing episodic hemolytic anemia. The sickle cell solubility screening assay, Hgb electrophoresis,and HPLC, are used to detect and diagnose sickle cell anemia and other inherited qualitative hgb abnormalities and thalassemias. Erythrocyte sedimentation rate (ESR) detects inflammation and roughly estimates its intensity. Review of cellular counts, distribution, and morphology in body fluids other than blood o CSF, synovial (joint) fluid, pericardial fluid, pleural fluid, and peritoneal fluid HEMATOLOGY QUALITY ASSURANCE AND QUALITY CONTROL Medical laboratory professionals employ particularly complex quality control systems in the hematology laboratory. The measurement of cells and biological systems requires elaborate calibration, validation, matrix effect examination, linearity, and reference interval determinations. An internal standard methodology known as the moving average also supports hematology laboratory applications. Medical laboratory professionals in all disciplines compare methods through clinical efficacy calculations that produce clinical sensitivity, specificity, and positive and negative predictive values for each assay. Should monitor specimen integrity and test ordering patterns Ensure the integrity and delivery of reports 6  TRANS: Hematopoiesis spleen, placenta, and ultimately the bone marrow HEMATOPOIETIC DEVELOPMENT space in the final medullary phase. Developing erythroblasts signal the beginning of Hematopoiesis is the continuous, regulated definitive hematopoiesis with a decline in process of renewal, proliferation, differentiation, primitive hematopoiesis of the yolk sac. and maturation of all blood cell lines. In addition, lymphoid cells begin to appear. These processes result in the formation, Hematopoiesis during this phase occurs development, and specialization of all functional extravascularly, with the liver remaining the blood cells that are released from the bone major site of hematopoiesis during the second marrow into the circulation. trimester of fetal life. A hematopoietic stem cell (HSC) is capable of Hematopoiesis in the AGM region and the yolk self-renewal and directed differentiation into all sac disappear during this stage. required cell lineages. Hematopoiesis in the fetal liver reaches its peak During fetal development, the sequential by the third month of fetal development, then distribution of cells is initiated in the yolk sac and gradually declines after the sixth month, retaining then progresses in the aorta-gonad-mesonephros minimal activity until 1 to 2 weeks after birth. (AGM) region (mesoblastic phase), then to the The developing spleen, kidney, thymus, and fetal liver (hepatic phase), and finally resides in lymph nodes contribute to the hematopoietic the bone marrow (medullary phase). process during this phase. The thymus, the first fully developed organ in the MESOBLASTIC PHASE fetus, becomes the major site of T cell production, whereas the kidney and spleen produce B cells. Hematopoiesis begins around the 19th day of Production of megakaryocytes begins during the embryonic development after fertilization. hepatic phase. Early in embryonic development, cells from the The spleen gradually decreases granulocytic mesoderm migrate to the yolk sac, forming: production and subsequently contributes solely to o Primitive erythroblasts in the central lymphopoiesis. cavity of the yolk sax During the hepatic phase, fetal hemoglobin (Hb Produce hemoglobin (Gower-1, F) is the predominant hemoglobin. Gower-2, and Portland) needed for delivery of oxygen to rapidly MEDULLARY (MYELOID) PHASE developing embryonic tissues. Angioblasts surround the cavity Medullary hematopoiesis occurs in the medulla of the yolk sac and eventually or inner part of the bone cavity. form blood vessels It begins between the fourth and fifth month of Yolk sac hematopoiesis differs from fetal development. hematopoiesis that occurs later in the fetus and During the myeloid phase, HSCs and adult in that it occurs intravascularly. mesenchymal cells migrate into the core of the Cells of mesodermal origin migrate to the AGM bone. region and give rise to HSCs for definitive or o Mesenchymal cells differentiate into permanent adult hematopoiesis. structural elements that support Yolk sac was the major site of adult blood developing hematopoietic elements. formation in the embryo. Hematopoietic activity is apparent during this stage of development, and the myeloid-to- HEPATIC PHASE erythroid ratio gradually approaches 3:1 to 4:1 (normal adult levels). The hepatic phase of hematopoiesis begins at 5 to By the end of 24 weeks’ gestation, the bone 7 gestational weeks marrow becomes the primary site of It is characterized by recognizable clusters of hematopoiesis. developing erythroblasts, granulocytes, and Measurable levels of erythropoietin (EPO), monocytes colonizing the fetal liver, thymus, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating 7  TRANS: factor (GM-CSF), and hemoglobins F and A can o Yellow marrow: hematopoietically be detected. inactive marrow composed primarily of In addition, cells at various stages of maturation adipocytes with undifferentiated can be seen in all blood cell lineages. mesenchymal cells and macrophages. During infancy and early childhood, all the bones in the body contain primarily red (active) marrow. Between 5 and 7 years of age, adipocytes become more abundant and begin to occupy the spaces in the long bones. Retrogression: is the process of replacing the active marrow by adipocytes (yellow marrow) during development o Results in restriction of the active marrow in the adult to the sternum, vertebrae, scapulae, pelvis, ribs, skull, and proximal portion of the long bones. ADULT HEMATOPOIETIC TISSUE In adults, hematopoietic tissue is located in: o Bone marrow: contains developing erythroid, myeloid, megakaryocytic, and lymphoid cells o Lymph nodes ▪ Primary lymphoid tissue consists of the bone marrow and thymus and is where T and B lymphocytes are derived. ▪ Secondary lymphoid tissue consists of the spleen, lymph nodes, and mucosa-associated lymphoid tissue. Spleen Liver Thymus BONE MARROW Bone marrow is located within the cavities of the cortical bones. In adults, there is approximately equal Projections of calcified bone, called trabeculae, amounts of red and yellow marrow in provide structural support for the developing these areas. blood cells that mature within a sea of interposed The ratio of the red marrow to the yellow mature adipocytes. marrow, often termed marrow cellularity, Normal bone marrow contains two major typically decreases with age. components: Bone marrow contains hematopoietic o Red marrow: hematopoietically active cells, stromal cells, and blood vessels. marrow that is composed of developing blood cells and their progenitors 8  TRANS: Stromal cells originate from BONE MARROW: RED MARROW mesenchymal cells that migrate into the The red marrow is composed of hematopoietic central cavity of the bone. cells arranged in extravascular cords. o Include endothelial cells, Hematopoietic cells develop in specific niches adipocytes (fat cells), within the cords. macrophages and lymphocytes, Erythroid precursors or erythroblasts osteoblasts, osteoclasts, and develop in small clusters, and the more reticular adventitial cells mature forms are located adjacent to the (fibroblasts). outer surfaces of the vascular sinuses and Endothelial cells are broad, flat cells that are found surrounding ironladen form a single continuous layer along the macrophages. inner surface of the arteries, veins, and Megakaryocytes are located adjacent to the walls vascular sinuses. of the vascular sinuses, which facilitates the o Regulate the flow of particles release of platelets into the lumen of the sinus. entering and leaving Immature myeloid (granulocytic) cells through hematopoietic spaces in the the metamyelocyte stage are located deep within vascular sinuses the cords. Adipocytes are large cells with a single As they mature, proceed along fat vacuole. differentiation, they move closer to the o Regulates the volume of the vascular sinuses marrow in which active The mature blood cells of the bone marrow hematopoiesis occurs eventually enter the peripheral circulation. o Secrete cytokines or growth factors that positively stimulate MARROW CIRCULATION HSC numbers and bone The nutrient and oxygen requirements of the homeostasis marrow are fulfilled by the nutrient and periosteal Macrophages function in phagocytosis, arteries, which enter via the bone foramina. and both macrophages and lymphocytes The nutrient artery supplies blood only to the secrete various cytokines that regulate marrow. hematopoiesis and are located The periosteal arteries provide nutrients for the throughout the marrow space. osseous bone and the marrow. Osteoblasts are bone-forming cells Hematopoietic cells located in the endosteal bed Osteoclasts are bone-resorbing cells. receive their nutrients from the nutrient artery. Reticular adventitial cells form an incomplete layer of cells on the luminal HEMATOPOIETIC MICROENVIRONMENT surface of the vascular sinuses. o Forms a supporting lattice for the The hematopoietic inductive microenvironment, developing hematopoietic cells or niche, plays an important role in nurturing and Stromal cells secrete a semifluid protecting HSCs and regulating their quiescence, extracellular matrix that serves to anchor self-renewal, and differentiation. developing hematopoietic cells in the As the site of hematopoiesis transitions from yolk bone cavity. sac to liver to bone marrow, so must the o Regulates hematopoietic stem microenvironmental niche for HSCs. and progenitor cell survival and Stromal cells form an extracellular matrix in the differentiation niche to promote cell adhesion and regulate HSCs through complex signaling networks involving cytokines, adhesion molecules, and maintenance proteins. Key stromal cells that support HSCs in bone marrow niches include: Osteoblasts Endothelial cells Mesenchymal stem cells CXCL12-abundant reticular cells 9  TRANS: Perivascular stromal cells Approx. 30% of the total platelet count is Glial cells sequestered in the spleen Macrophages LYMPH NODES LIVER Lymph nodes are organs of the lymphatic system The liver serves as the major site of blood cell located along the lymphatic capillaries that are production during the second trimester of fetal parallel to, but not part of, the circulatory system. development. These bean-shaped structures (1 to 5 mm in In adults, hepatocytes have many functions, diameter) are typically present in groups or including protein synthesis and degradation, chains at various intervals along lymphatic coagulation factor synthesis, iron recycling and vessels. storage, and hemoglobin degradation. Lymph is the fluid portion of blood that escapes The lumen of the sinusoids contains Kupffer cells into the connective tissue and is characterized by that maintain contact with the endothelial cell a low protein concentration and the absence of lining. RBCs. Are macrophages that remove senescent Afferent lymphatic vessels carry circulating cells and foreign debris from the blood lymph to the lymph nodes. that circulates through the liver Lymph is filtered by the lymph nodes and exits The liver can maintain hematopoietic stem and via the efferent lymphatic vessels located in the progenitor cells to produce various blood cells as hilus of the lymph node. a response to infectious agents or in pathologic Lymph nodes have three main functions: myelofibrosis of the bone marrow. Site of lymphocyte proliferation In severe hemolytic anemias the liver increases Involved in the initiation of the specific the conjugation of bilirubin and the storage of immune response to foreign antigens iron and sequesters membrane-damaged RBCs Filter particulate matter, debris, and and removes them from the circulation. bacteria entering the lymph node via the lymph SPLEEN The spleen is vital but not essential for life and HEMATOPOIETIC STEM CELLS AND CYTOKINES functions as an indiscriminate filter of the circulating blood. STEM CELL THEORY In a healthy individual the spleen contains about Hematopoietic progenitor cells can be divided 350 mL of blood. into two major types: The red pulp is composed primarily of vascular Noncommitted or undifferentiated HSCs sinuses separated by cords of reticular cell Committed progenitor cells meshwork (cords of Billroth) containing loosely Theories describing the origin of hematopoietic connected specialized macrophages. progenitor cells: Creates a sponge-like matrix that Monophyletic theory suggests that all functions as a filter for blood passing blood cells are derived from a single through the region. progenitor stem cell called a pluripotent As RBCs pass through the cords of hematopoietic stem cell Billroth, there is a decrease in the flow of Polyphyletic theory suggests that each of blood - leads to stagnation and depletion the blood cell lineages is derived from its of the RBCs’ glucose supply own unique stem cell. It uses two methods for removing senescent or HSCs are capable of self-renewal, are pluripotent abnormal RBCs from the circulation: and give rise to differentiated progeny, and are Culling: cells are phagocytized with able to reconstitute the hematopoietic system. subsequent degradation of cell organelles The undifferentiated HSCs can differentiate into Pitting: splenic macrophages remove progenitor cells committed to either lymphoid or inclusions or damaged surface membrane myeloid lineages. from the circulating RBCs Lineage-specific progenitor cells: The spleen also serves as a storage site for Common lymphoid progenitor platelets. proliferates and differentiates into T, B, 10  TRANS: and natural killer lymphocyte and dendritic lineages Common myeloid progenitor proliferates and differentiates into individual granulocytic, erythrocytic, monocytic, and megakaryocytic lineages Most of the cells in normal bone marrow are precursor cells at various stages of maturation. HSCs are directed to one of three possible fates: Self-renewal Differentiation Apoptosis 11  TRANS: Erythrocyte Production and Destruction CRITERIA USED IN ID OF ERYTHROID PRECURSORS NORMOBLASTIC MATURATION Morphologic identification of blood cells depends on a well-stained peripheral blood film TERMINOLOGY or bone marrow smear. The stage of maturation of any blood cell is Erythrocytes: Red blood cells determined by careful examination of the nucleus Erythroblasts: Nucleated RBC precursors, and the cytoplasm. normally restricted to the bone marrow The most important features in the identification o Normoblasts of RBCs are: o Nuclear chromatin pattern (texture, density, homogeneity) o Nuclear diameter o Nucleus-to-cytoplasm (N:C) ratio o Presence or absence of nucleoli o Cytoplasmic color MATURATION PROCESS: ERYTHROID PROGENITORS The morphologically identifiable erythrocyte precursors develop from two progenitors: o Burst-forming unit-erythroid (BFU-E) o Colony-forming unit-erythroid (CFU-E) Both are committed to the erythroid cell line. BFU-E gives rise to large colonies since they are capable of multi-subunit colonies (bursts) CFU-E gives rise to smaller colonies Pronormoblast is the first morphologically identifiable RBC precursor. Approximately 18 to 21 days are required to produce a mature RBC from the BFU-E MATURATION SEQUENCE Normoblastic proliferation is a process encompassing replication (division) to increase cell numbers and development from immature to mature cell stages. 12  TRANS: PRONORMOBLAST (RUBRIBLAST) CYTOPLASM Stained cytoplasm may be a deeper, richer blue NUCLEUS DIVISION N:C ratio of 8:1 It undergoes mitosis – two daughter cells Round to oval, containing one or two nucleoli More than one division is possible before the Purple red chromatin is open and contains few daughter cells mature into polychromatic fine clumps normoblasts. CYTOPLASM LOCATION Dark blue – concentration of ribosomes and RNA It is present only in the bone marrow in healthy Golgi complex may be visible next to the nucleus states. as a pale, unstained area CELLULAR ACTIVITY May show small tufts of irregular cytoplasm Detectable hemoglobin synthesis occurs, but the along the periphery of the membrane many cytoplasmic organelles, completely mask DIVISION the minute amount of hemoglobin pigmentation It undergoes mitosis and gives rise to two LENGTH OF TIME IN THIS STAGE daughter pronormoblasts. This stage lasts slightly more than 24 hours More than one division is possible before maturation into basophilic normoblasts. LOCATION It is present only in the bone marrow in healthy states. CELLULAR ACTIVITY It begins to accumulate the components necessary for Hgb production. Proteins and enzymes necessary for iron uptake and protoporphyrin synthesis are produced. Globin production begins. LENGTH OF TIME IN THIS STAGE This stage lasts slightly more than 24 hours. POLYCHROMATIC (POLYCHROMATOPHILIC) NORMOBLAST (RUBRICYTE) NUCLEUS Variation in chromatin pattern, showing some openness early in the stage but becoming condensed by the end N:C ratio decreases from 4:1 to about 1:1 by the end of the stage No nucleoli are present. CYTOPLASM 1st stage in which the pink color associated with stained hemoglobin can be seen BASOPHILIC NORMOBLAST (PRORUBRICYTE) o Reflects the accumulation of Hgb pigmentation over time and concurrent NUCLEUS decreasing amounts of RNA Chromatin begins to condense, revealing clumps o Mixture of pink and blue – murky gray- along the periphery of the nuclear membrane and blue a few in the interior The stage’s name refers to this combination of o Parachromatin areas become larger and multiple colors – “many color loving” sharper, and N:C ratio decreases (6:1) DIVISION The chromatin stains deep purple-red. Last stage in which the cell is capable of Nucleoli may be present early in the stage but undergoing mitosis disappear later. LOCATION It is present only in the BM in healthy states. 13  TRANS: CELLULAR ACTIVITY CELLULAR ACTIVITY Hgb synthesis increases, and the accumulation Hgb production continues on the remaining begins to be visible as a pinkish color in the ribosomes using mRNA produced earlier. cytoplasm. Late in this stage, the nucleus is ejected from the Cellular RNA and organelles are still present, cell. ribosomes which contribute a blue color to the o Loss of vimentin, a protein responsible cytoplasm for holding organelles in proper location The progressive condensation of the nucleus and in the cytoplasm disappearance of nucleoli are evidence of o Nonmuscle myosin of the membrane progressive decline in transcription of LENGTH OF TIME IN THIS STAGE deoxyribonucleic acid (DNA). This stage lasts approximately 48 hours. LENGTH OF TIME IN THIS STAGE This stage lasts approximately 30 hours. POLYCHROMATIC (POLYCHROMATOPHILIC) ERYTHROCYTE OR RETICULOCYTE ORTHOCHROMIC NORMOBLAST (METARUBRICYTE) NUCLEUS NUCLEUS Beginning at this stage, there is no nucleus. It is completely condensed (i.e., pyknotic) or When a cell loses its nucleus, regardless of nearly so. cytoplasmic appearance, it is a polychromatic N:C ratio is low or approximately 1:2. erythrocyte. CYTOPLASM CYTOPLASM Increase in the salmon pink color of the Predominant color is that of hemoglobin with a cytoplasm reflects nearly complete hemoglobin bluish tinge (residual ribosomes and RNA) production By the end of this stage, the cell is the same The residual ribosomes and RNA react with the color as a mature RBC, salmon pink. basic component of the stain – slightly bluish It remains larger than a mature cell. hue to the cell DIVISION o Fades toward the end of the stage as the It cannot divide. RNA and organelles are degraded LOCATION DIVISION It resides in the BM for about 1 to 2 days and It is not capable of division because of the then moves into the PB for about 1 day before condensation of the chromatin. reaching maturity. LOCATION During the first several days after exiting the It is present only in the BM in healthy states. marrow, it is retained in the spleen for pitting of 14  TRANS: inclusions and membrane polishing by splenic ERYTHROCYTE macrophages – biconcave discoid mature RBC. CELLULAR ACTIVITY NUCLEUS It completes production of hemoglobin from a No nucleus is present in mature RBCs. small amount of residual mRNA using the CYTOPLASM remaining ribosomes. It is a biconcave disc measuring 7 to 8 mm in Endoribonuclease, in particular, digests the diameter, with a thickness of about 1.5 to 2.5 ribosomes. mm. A small amount of residual ribosomal RNA is It appears as a salmon-pink stained cell with a present – visualized with a vital stain central pale area LENGTH OF TIME IN THIS STAGE DIVISION The cell typically remains a polychromatic The erythrocyte cannot divide. erythrocyte for about 3 days LOCATION AND LENGTH OF TIME IN THIS STAGE o 1st 2 days in the BM Mature RBCs remain active in the circulation for o 3rd day in the PB 120 days. CELLULAR ACTIVITY The mature erythrocyte delivers oxygen to tissues, releases it, and returns to the lung to be reoxygenated. The interior of the erythrocyte contains mostly hemoglobin. With the biconcave shape, even Hgb molecules that are toward the center of the cell are not distant from the membrane and are able to exchange oxygen. The cell’s main function of oxygen delivery throughout the body requires a membrane that is flexible and deformable, able to flex but return to its original shape. Deformability is crucial for RBCs to enter and subsequently remain in the circulation. 15  TRANS: ERYTHROKINETICS A second mechanism by which EPO increases the number of circulating RBCs is by increasing Erythrokinetics is the term describing the the number of cells that will be able to mature dynamics of RBC production and destruction. into circulating erythrocytes. Erythron is the name given to the collection of o By decreasing apoptosis of RBC all stages of erythrocytes throughout the body: progenitors the developing precursors in the BM and the Another effect of EPO is to increase the rate at circulating RBCs in the PB and vascular spaces which the surviving precursors can enter the within organs. circulation When the term erythron is used, it conveys the o This is accomplished by two means: concept of a unified functional tissue. ▪ Increased rate of cellular o Erythron is the entirety of erythroid cells processes in the body o RBC mass refers only to the cells in ▪ Decreased cell cycle times circulation o Erythrokinetics begins by looking at RBCs in MEASUREMENT OF ERYTHROPOIETIN the BM and the factors that affect their numbers, Quantitative measurements of EPO are their progressive development, and their performed on plasma and other body fluids. ultimate release into the peripheral blood. EPO can be measured by chemiluminescence. Reference interval: 10 to 30 U/L HYPOXIA THERAPEUTIC USES OF ERYTHROPOIETIN o RBCs carry oxygen to tissues. Recombinant EPO is used as therapy in certain o To regulate the production of RBCs for that anemias such as those associated with chronic purpose, the body requires a sensing mechanism kidney disease and chemotherapy. to check if adequate oxygen is being carried to It is also used to stimulate RBC production the tissues. before autologous blood donation and after bone o The primary oxygen-sensing system of the body marrow transplantation. is located in peritubular fibroblasts of the kidney. MICROENVIRONMENT OF THE BONE MARROW o Hypoxia is detected by the peritubular fibroblasts – produce erythropoietin (EPO), Erythropoiesis typically occurs in what are major stimulatory cytokine for RBCs. called erythroid islands within the bone marrow. These islands consist of a central macrophage ERYTHROPOIETIN surrounded by erythroid precursors in various stages of development. EPO is a thermostable, nondialyzable, Anchoring system that holds developing cells glycoprotein hormone with a molecular weight within the marrow of 34 kD. There are three components to the anchoring It consists of a carbohydrate unit and a terminal system: sialic acid unit, both of which play a role in the o A stable matrix of accessory and stromal biologic activity of the hormone. cells – normoblasts attach EPO is a hormone, being produced at one o Bridging (adhesive) molecules location (kidney) and acting at a distant location attachment (bone marrow). o Receptors on the normoblast membrane It is a growth factor that initiates an intracellular Macrophage is the major cellular anchor for the message to the developing erythroid cells – developing normoblasts. signal transduction Several systems of adhesive molecules and EPO must bind to its receptor (EPOR) on the normoblast receptors tie the developing surface of EPO-responsive immature erythroid normoblasts to the macrophages. cells to initiate the signal At the same time, normoblasts are anchored to EPO promotes early release of developing the extracellular matrix of the bone marrow, erythroid precursors from the bone marrow. chiefly by fibronectin. 16  TRANS: When it comes time for RBCs to leave the When an RBC lyses within a macrophage, the marrow, they cease production of the receptors major components are catabolized. for adhesive molecules. o Iron is removed from the heme and stored in the macrophage as ferritin until transported out. o The globin of Hgb is degraded and returned to the metabolic amino acid pool. o The protoporphyrin component of heme is degraded through several intermediaries to bilirubin, which is released into the blood and ultimately excreted by the liver in bile. Most natural RBC deaths occur in the spleen, and a small portion of RBCs rupture intravascularly. The intravascular rupture of RBCs from purely mechanical or traumatic stress results in fragmentation and release of the cell contents into the blood – fragmentation or intravascular hemolysis. ERYTHROCYTE DESTRUCTION A system of plasma proteins, including haptoglobin and hemopexin, salvage the released As a nonnucleated cell, the mature erythrocyte is hemoglobin so that its iron is not lost in the urine. unable to generate new proteins, so as its cellular functions decline, the cell ultimately approaches death. The average RBC has sufficient enzyme function to live 120 days. Because RBCs lack mitochondria, they rely on glycolysis for production of ATP. The loss of glycolytic enzymes is central to this process of cellular aging (senescence), which culminates in phagocytosis by macrophages. MACROPHAGE – MEDIATED HEMOLYSIS (EXTRAVASCULAR HEMOLYSIS) Some researchers view erythrocyte death as a nonnucleated cell version of apoptosis, termed eryptosis, which is precipitated by: o Oxidative stress o Energy depletion o Other mechanisms that create membrane signals that stimulate phagocytosis Examples of the signals include: o Binding of autologous immunoglobulin G (IgG) to band-3 membrane protein clusters o Exposure of phosphatidylserine on the exterior (plasma side) of the membrane o Inability to maintain cation balance Senescent changes to leukocyte surface antigen CD47 (integrin-associated protein) may also be involved by binding thrombospondin-1, which then provides an “eat me” signal to macrophages. 17