Multistep model of carcinogenesis

• Cancers arise from the stepwise accumulation of mutations.
• Cancer development depends on predisposing and environmental factors, age, and other influences; furthermore, these variables have synergistic effects (for example, it appears that alcohol and tobacco act together to increase risk of certain cancers).

Genetic and acquired factors

Genetic mutations:

— Some mutations lead to loss-of-function in tumor suppressor genes; for example, a dysfunctional APC gene is associated with colon cancer.
— Mutations can also lead to oncogenic gain-of-function or gene amplification. For example, translocation and subsequent amplification of the MYC oncogene is associated with Burkitt Lymphoma.

Examples of acquired predisposing factors:

• Chronic infection and inflammation promotes increased cell turnover, metaplasia, and other pre-cancer events; for example, infection by Helicobacter pylori and the ensuing inflammation can lead to gastric cancer.
• Individuals with immunodeficiencies are more susceptible to cancers caused by oncogenic viruses; for example, lymphomas are associated with deficiencies in T-cell immunity.
• Some precursor lesions can progress to cancer. Such lesions may be detectable in screenings, and early treatment may reduce the risk that they progress to cancer.
  — Some histological samples of precursor lesions:
  • In our first sample, we see an example of inflammatory metaplasia in the bronchial mucosa; this can occur as the result of smoking.
  • In the next image, we see an example of non-inflammatory hyperplasia of the endometrium, which is result of continuous estrogen stimulation.
  • Finally, we see villous adenomas, which are benign neoplasms in the colon; unlike most other benign neoplasms, villous adenomas progress to cancer in about 50% of cases.

Carcinogenesis:
Three key steps: Initiation, Promotion, and Progression.

Initiation

• Occurs when the first driver mutation induces permanent non-lethal DNA damage to a cell.
  — Because the first driver mutation initiates the process of carcinogenesis, the agents responsible are called “initiators”:
• Initiating agents are carcinogens; they produce electrophiles (electron-deficient atoms that react with DNA, RNA, and proteins); they bind DNA to form adducts; they are mutagens; their initiating actions are irreversible; and, their activities are additive.
• Carcinogens can be chemical, microbial, or physical.

Chemical initiators

• Are by far the most common, comprising about 90% of all carcinogens; they can be indirect-acting or direct-acting.
• Indirect-acting carcinogens require metabolic activation.
  — Examples include: Polycyclic aromatic hydrocarbons (aka, PAHs), which are released by burning fossil fuels and tobacco; Aflatoxin B1, which is naturally produced by Aspergillus fungi; and, Benzidine, which is a synthetic chemical formerly used to produce dyes; because it is a known carcinogen, it is no longer sold in the U.S.
• Direct-acting chemical carcinogens do not require metabolic activation.
  — Paradoxically, some anticancer drugs fall into this category; Other examples include: dimethyl sulfate, which can be used as a methylating agent, and, diepoxybutane, which was formerly used in industrial settings.

Microbial initiators

• Some strains of the human papillomavirus (HPV) that are associated with oncogenic E6 and E7 proteins.
• Epstein-Barr Virus (EBV), which is associated with the African form of Burkitt Lymphoma.
• Hepatitis B and C, which are associated with liver cancer.
• Bacterium H. pylori, which is associated with gastric cancers.

Physical initiators

• Include UV rays, which are associated with squamous cell carcinoma.
• Other examples include electromagnetic and particulate radiations.

Promotion

• The promotion stage of carcinogenesis comprises clonal expansion, which occurs when promoters induce the proliferation of the DNA-damaged cell. Be aware that this can occur long after the initiation event.
• As a result, a tumor forms; it can be either benign or pre-neoplastic.
• Promoting agents do NOT produce electrophiles, nor do they bind DNA; they are not mutagens; and, their effects are usually reversible. Furthermore, their effects are modulated by diet, hormonal and environmental factors.
• Some agents can act as both initiators AND promoters.
• Promoters act as mitogens; that is, they promote cell division.
• Some examples of promoters include: hormones and growth factors; phorbol esters; and products of chronic inflammation.
• Continued proliferation exposes pre-neoplastic cells to additional driver mutations.
  — These 80+ mutations lead to the acquisition of defining cancer hallmarks, which include: Self-sufficiency and the ability to ignore growth suppressors; evasion of apoptosis and immortality; altered metabolism and angiogenesis to meet nutritional needs; invasion and metastasis into new niches; and, evasion of the host immune system.
  This process can take years, even decades, and helps account for the latent period it takes for some cancers to develop.

Progression

• Progression comprises genetic evolution such that selection for aggressive cancer cell phenotypes ultimately produces a malignant tumor.
• Unlike the benign tumor we drew in the promotion stage, this malignant tumor is genetically heterogeneous.
• Common sites of cancer in men and women in the U.S. include: the female breast, the prostate, lungs and bronchi, colon and rectum, uterus, melanomas of the skin, urinary bladder, non-Hodgkin lymphoma, kidney, and thyroid gland.

Cancer cells are self-sufficient

• They promote their own self replication in the absence of the external signals non-cancer cells rely on.

Cancer cells ignore growth suppressors

• This means there’s no time for DNA repair, in part because cancer cells do not pause between the G1 and S phases.

Cancer cells evade apoptosis

• Thus, cells survive despite DNA damage.

Cancer cells are immortal

• They evade the mitotic crisis that results in the death of non-cancer cells.
• Thus, cancer cells continue replication and perpetuate the DNA damage.
• For example, the telomeres of cancer cells do not shorten over time.
  — Telomeres are protective end-caps, that, in normal somatic cells, shorten with each replication. Eventually, they are too short to protect the chromosomal DNA, and the cell dies.
  — However, telomeres are maintained in cancer cells by an enzyme called telomerase. Thus, the telomeres do not shorten and cell death is avoided.

Cancer cells exhibit altered metabolism

• Sometimes called the Warburg effect, this enables them to meet their unique metabolic needs.
  — Cancer cells use aerobic glycolysis to fuel biosynthesis of new organelles; thus, cancer cells are characterized by increased glucose and glutamine consumption.

EXTRACELLULAR EFFECTS

Cancer cells trigger angiogenesis

• Formation of new blood vessels from existing vasculature enables the tumor to meet its nutritional needs.
  — Interestingly, the “angiogenic switch” is triggered by tumors greater than 2 cm; below this threshold, simple diffusion suffices.

Cancer cells invade and metastasize

• This enables them to cross anatomical boundaries. In contrast, the growth of benign tumors is limited by anatomical boundaries.
• Steps:
  • Cancer cells break free from the primary tumor.
  • They invade the extracellular matrix and migrates to a nearby vessel.
  • Then, in a process called intravasation, the cancer cells enter circulation. Be aware that cancer cells can enter blood and/or lymphatic vessels, and that some cancers are more prone to a specific vessel type.
  • Cancer cells travel within the circulation, where they can form an embolus with T lymphocytes and platelets. This aggregation may protect the cancer cells from destruction.
  • Cancer cells can break free from the embolus and exit the vessel, a process called extravasation.
  • In their new environment, the cancer cells can proliferate to form a metastatic tumor; show that this tumor can also develop its own blood supply.
• Be aware that invasion and metastasis are major causes of morbidity and death from cancer.

Cancer cells evade the immune system

• Ensures their own survival.
  — They can downregulate expression of MHC proteins and presentation of antigens on their own cell surfaces. Thus, they “hide” from the immune system.
  — They can also suppress immune cell responses and release immunosuppressive cytokines, which dampens the ability of the immune system to defend the host.

Hemostasis is the process of platelet plug formation and coagulation to stop bleeding.

• Disorders occur as the results of imbalance in hemostatic forces.
  – Excessive bleeding is the result of vessel wall defects, platelet disorders, and coagulation disorders.
  – Excessive clotting occurs in hypercoagulation states, and leads to thrombosis of the arteries and veins.

Platelet Disorders

• von Willebrand disease is characterized by deficient levels of von Willebrand factor, which is responsible for platelet adhesion and acts as a binding protein for coagulation factor VIII.
  – Recall that von Willebrand factor is crucial in the early stages of hemostasis, in which it acts as a bridge between circulating platelets and exposed subendothelial collagen; after their activation, von Willebrand factor bridges adjacent platelets.
  – von Willebrand disease is inherited as a genetic mutation, with three types:
  Type 1 is the most common, and least severe; it is characterized by decreases in von Willebrand factor and coagulation factor VIII levels.
  Type 2 is intermediate, and comprises several subtypes that vary in severity.
  Type 3 is rare, but severe, and is characterized by nearly absent von Willebrand factor and factor VIII.
  – Be aware that von Willebrand disease can also be acquired, though this is rare.
  – Treatment includes desmopressin (ex: DDAVP), which induces release of von Willebrand factor and Factor VIII from the endothelium, and/or von Willebrand factor replacement therapy.

• Thrombocytopenia is characterized by platelet counts below 150 thousand per microliter of blood; normal platelet count is between 150-450 thousand per microliter of blood.
  – The causes of thrombocytopenia include:
  Decreased platelet production in the bone marrow
  Increased sequestration (particularly in the spleen)
  Increased platelet destruction
  – Severity and treatment for thrombocytopenia depends on its causes.
  – A notable characteristic of mild to severe thrombocytopenia is the appearance of petechiae, which are small reddish purple spots caused by blood leakage from capillaries. Recall that thousands of small nicks occur in the walls of the blood vessels each day; without sufficient platelet populations and plug formation, bleeding ensues.

Coagulation Disorders
Coagulation disorders occur when coagulation factors are insufficient or absent and clot formation is impaired.

• Hemophilia is a congenital disorder that, because it is x-linked, primarily affects males.
  – There are two main types of hemophilia: A and B.
  – Hemophilia A is characterized by absent or deficient factor VIII; this is the most common form and results from a mutation of the F8 gene.
  – Hemophilia B is characterized by absent or deficient factor IX; it results from a mutation of the F9 gene.
  – To understand their pathogenesis, quickly review a portion of the coagulation cascade:
  Activated factor IX, complexed with activated co-factor VIII and calcium ions, activates factor X. In turn, activated factor X complexes with activated cofactor V and calcium ions to form prothrombinase. Prothrombinase then converts prothrombin to thrombin, which converts fibrinogen to fibrin, which creates a mesh-like clot surrounding blood cells and other circulating molecules.
  – Treatment for hemophilia typically involves replacement of the deficient factor.
  – Common bleeding sites in hemophilia include the oral mucosa and musculoskeletal sites; hemophilia is characterized by hemarthrosis, which is bleeding into the joint spaces. Intracerebral and gastrointestinal bleeding can be fatal.

• Vitamin K Deficiency
  – Vitamin K is necessary for hepatic synthesis of prothrombin, factors VII, IX, and X.
  – Two key sources of vitamin K are the diet (especially green, leafy vegetables) and bacterial production (in the proximal intestine).
  – From the GI tract, vitamin K is absorbed and transported to the liver, where it is used to synthesize four of the coagulation factors.
  – These factors are then carried in the blood, ready to participate in the coagulation cascade and production of thrombin.
  – Thus, if the quantity of vitamin K present in GI tract is insufficient, or it is not properly absorbed and transported to the liver, thrombin production and clot formation will be impaired.
  – Be aware that liver diseases can also inhibit the production of coagulation factors, independent of vitamin K deficiencies.

Thrombosis
Excessive clot formation obstructs blood flow, which can lead to ischemia and organ failure.
– When clots have formed and acute treatment is necessary, fibrinolytic (aka, thrombolytic) drugs, such as streptokinase, tissue-type plasmin activator, or urokinase are administered. Thus, fibrinolytic drugs can be life-saving when clots obstruct blood flow in the lungs, myocardium, or the brain.
– Risk factors for arterial and venous thrombosis are similar, and include age, surgery, trauma, cancer, and pregnancy (the risk of thromboembolism increases 4-5 fold in pregnant women, and accounts for approximately 15% of maternal death during pregnancy).
– Arterial and venous thrombosis are similar in many ways, but can have different mechanisms of pathogenesis.

• Arterial thrombosis tends to result from excessive platelet aggregation.
  – Because arterial thrombosis blocks delivery of oxygenated blood to the organs, including the heart and brain, it can be deadly.
  – Prevention of arterial thrombosis typically relies upon antiplatelet treatments, such as aspirin or ADP receptor inhibitors that prevent platelet aggregation.

• Deep vein thrombosis tends to result from clot formation in the valvular cusps.
  – Pulmonary embolism, which can block blood flow to the lungs, is a major clinical concern in patients with deep vein thrombosis.
  – Prevention typically relies on anticoagulants, such as exogenous heparin and warfarin, etc.

HEMOSTASIS

Hemostasis is the process by which platelets, clotting factors, and endothelial cells produce clots to stop bleeding.
The body is constantly working to maintain a balance between coagulation, which is necessary to prevent vascular leakage, and excessive clotting, which presents hazards to blood flow and tissue perfusion.

3 key mechanisms of hemostasis:

1. Vascular spasm occurs immediately after vessel damage and serves to slow blood flow and bleeding. It is not usually sufficient to stop vessel leakage, but it allows time for more extensive interventions.
2. Platelet plugs form as the result of platelet activation and aggregation; this happens quickly. Thousands of time each day, small tears form in the vasculature and are effectively closed by platelet plugs.
3. Coagulation reinforces the plug by forming a clot comprised of platelets and fibrin mesh with trapped blood cells.

Platelet Plug Formation via Adhesion, Activation, and Aggregation

Adhesion

• Disc-shaped inactive platelets with granules are pulled to the edges of the vessel.
• Adhesion occurs mainly via bridging by von Willebrand Factor
  • Platelet receptor glycoprotein Ib binds to von Willebrand Factor, which binds to exposed collagen fibers.
• Platelets can also adhere to collagen directly via alpha-2-beta-1 integrins; other platelet integrins bind to subendothelial fibronectin and laminin (we’ve omitted this for simplicity).
• Adhesion triggers platelet activation.

Activation

• Platelets transform form disc-shaped structures to “spiky”structures with filopodia; this morphological change increases platelet surface area.
• Upon activation, platelets release hundreds of protein types:
  • Thrombin, ADP, and thromboxane A2 promote additional platelet recruitment and activation; as we’ll see, thrombin also plays a key role in coagulation.
  • Other important molecules released include co-factor V, which we’ll see again in the coagulation cascade, and, Von Willebrand Factor, which, as we’ve seen, is vital for platelet adhesion and subsequent activation.

Aggregation

• ADP upregulates expression of the alpha-IIb beta-3 integrin on activated platelets; this receptor binds with fibrinogen, which acts as a bridge between adjacent platelets.
• Pharmacological correlation: Aspirin limits platelet production of thromboxane A2, thus limiting platelet recruitment and formation of platelet plugs.

Coagulation Cascade

After the plug is formed, and if vascular injury warrants clot formation, the coagulation cascade begins within seconds to minutes. The coagulation cascade comprises a series of amplifying enzymatic reactions that result in the deposition of an insoluble fibrin clot; the production of thrombin, which converts fibrinogen to fibrin, is integral to this process.

3 key components of the coagulation cascade:

• Calcium ions, which are required for several key reaction steps.
• Coagulation factors, which are the driving forces of the cascade; they comprise proenzyme factors are transformed to active enzymes, and are signified by the lower case “a.” Many coagulation factors are synthesized by the liver; thus, coagulation disorders are associated with liver disease.
• Co-factors are reaction accelerators; they form complexes with activated coagulation factors and calcium ions.

2 Pathways:

Traditionally, these pathways have been categorized as “intrinsic” and “extrinsic,” but modern models emphasize their cooperation in thrombin formation.

• Endothelial rupture exposes tissue factor to the blood; because tissue factor is exogenous to the blood, it is the start of the so-called “extrinsic” pathway (tissue factor is also known as thromboplastin or CD142).
• Tissue factor activates coagulation factor VII, and, with calcium, forms a complex that activates factor IX (this complex is sometimes called extrinsic tenase).
• Activated factor IX, with calcium and co-factor VIIIa, activates factor X.
• Activated factor X combines with calcium and co-factor Va to form prothrombinase, which is the complex that transforms prothrombin (aka, factor II) to thrombin (factor IIa).
• Thrombin converts fibrinogen to fibrin, which forms a meshwork that surrounds the aggregated platelets and traps other materials, including red and white blood cells and serum.

Return to the aggregated platelets to show how they influence thrombin production; because all the elements in this pathway are inherent to the blood, this is traditionally called the “intrinsic” pathway.

• The negative surface of activated platelets activates Factor XII(aka, Hageman factor).
• Factor XI Ia activates factor XI.
• Factor XIa activates factor IX, which, as we’ve seen, forms a complex that ultimately leads to the production of thrombin and conversion of fibrinogen to fibrin.

Thrombin has other, indirect ways of promoting clotting:

• It activates co-factors VIII and V, and factor XI; thus, it promotes its own production.
• Thrombin also ensures the stability of the clot: show that it activates factor XIII, aka, fibrin stabilizing factor, which, as its name implies, strengthens the bonds of the fibrin mesh of the clot.

Retraction

Soon after the clot is formed, retraction occurs: the platelets contract and expel serum.

• Serum lacks fibrinogen and clotting factors, so it cannot coagulate.

Thrombin also acts outside of the coagulation cascade via protease activation receptors (PARs):

• Thrombin promotes platelet activation and release of thromboxane A2 for platelet plug formation.
• It triggers endothelial activation and release of anticoagulants.
• Thrombin also up-regulates pro-inflammatory mediators, such as cytokines and chemokines, implicating it in inflammatory conditions.

Endogenous anti-coagulation mechanisms:

• Fibrin fibers absorb thrombin, which prevents excessive clotting.
• Unabsorbed thrombin combines with anti-thrombin III, which neutralizes it.
• Heparin, which is normally circulating in small concentrations but can be administered in higher doses, increases the effectiveness of anti-thrombin III and removes factors IX, X, XI, XII. Thus, it is prescribed to prevent thrombosis.
• Healthy, intact endothelial cells produce nitric oxide, prostacyclin, and tissue plasminogen activator (t-PA) to prevent platelet adhesion and clot formation.

Fibrinolysis

Once the vessel wall heals, the clot must be removed via fibrinolysis, the breakdown of the fibrin mesh.

• Plasminogen binds to the fibrin; plasminogen is a proenzyme produced by the liver.
• Endothelial cells release tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), which bind to fibrin and cleave plasminogen to form active plasmin. Recall that thrombin itself is a stimulus for t-PA release.
• Plasmin breaks down fibrin, fibrinogen, and some coagulation factors to dissolve the clot.
  • In a positive feedback cycle, plasmin also converts t-PA and u-PA into their more active forms, which promotes further fibrinolysis.
• Fibrinolysis produces fibrin degradation products (FDPs), most notably D-dimer, which is measured to assess thrombotic states (an elevated result may indicate excessive thrombotic states).
• When not bound to fibrin, plasminogen and plasmin activity is inhibited:
  • Unbound t-PA and u-PA are inhibited by plasminogen activator inhibitor 1 (PAI-1).
  • Alpha-2 antiplasmin inhibits unbound plasmin.

Vitamin K

• Vitamin K is critical to hepatic synthesis of prothrombin and factors VII, IX, and X.
  • Thus, vitamin K antagonists, such as warfarin, are prescribed to inhibit clot formation in patients at risk of thrombosis.

EARLY ORIGINS

Hemopoietic stem cell

• Gives rise to two types of progenitor cells:
  • The common myeloid progenitor
  • The common lymphoid progenitor

Common myeloid progenitor (CMP, aka, CFU-GEMM) gives rise to the following leukocytes:

• Neutrophil
• Eosinophil
• Basophil
• Monocyte

As we learn elsewhere, the common myeloid progenitor also gives rise to red blood cells and platelets.

Common lymphoid progenitor (CLP)

• Gives rise to lymphocytes.

HISTOLOGICAL DETAILS

CMP Lineage:

Neutrophils

• Comprise 60-70% of circulating white blood cells; they are the most common leukocyte.
• Have multi-lobed nuclei.
  • As they age, the number of lobes increases: immature band neutrophils have no formed lobes, older neutrophils may have 5 lobes.
• Various types of cytoplasmic granules lend the cytoplasm a pale pink hue.
• Neutrophils phagocytose bacteria by engulfing them with pseudopodia; the bacteria are trapped within a phagosome, where granular digestive enzymes destroy them.

Eosinophils

• Comprise 2-4% of circulating white blood cells.
• Nuclei are multi-lobed.
• Granules stain a bright orange-red color, which helps to differentiate them from neutrophils under the microscope.
• Eosinophilic granules contain parasite-destroying enzymes and polypeptides, including major basic protein.
• Eosinophils also participate in the immune response, and play a role in triggering bronchial asthma.

Basophils

• Comprise 1% of circulating white blood cells.
• Bi-lobed or bean-shaped nucleus that is obscured by an abundance of large metachromatic granules (metachromatic means that the granules stain a different color from that of the dye).
• Basophils participate in hypersensitivity, such as bronchial asthma and dermatological reactions.
• Their granules contain heparin, which prevents blood coagulation, and histamine, which causes inflammation.

Monocytes

• Comprise 5% of circulating white blood cells.
• Their bean-shaped nucleus is visible against light blue/gray staining cytoplasm; lack visible granules.
• In the connective tissues, monocytes become macrophages, and can phagocytose bacteria and other particles (including red blood cells), clean up cellular debris, and present antigens.
• In the bones, they become osteoclasts, which resorb bone tissue as part of growth and remodeling.

CLP Lineage:

Lymphocytes

• Comprise 20-40% of circulating white blood cells.
• Large, round nucleus leaves just a thin rim of cytoplasm.
• Lymphocytes diverge to form B cells, which mature within the bone and participate in humoral immunity, and T cells, which mature in the thymus and participate in cell-mediated immunity.

Granulocytes vs Agranulocytes

Granulocytes:

Neutrophils, eosinophils, and basophils are histologically classified as granulocytes, because of the presence of primary, secondary, and, sometimes, tertiary granules in their cytoplasm.

Agranulocytes:

Monocytes and lymphocytes are called “agranulocytes,” because they have only primary granulocytes in their cytoplasm; these can be difficult to see.

ERYTHROPOIESIS

• The process of erythrocyte (red blood cell) development, which, in adults, primarily occurs in the spongy bone marrow.
• Takes place in red marrow of spongy bone, aka, trabecular or cancellous bone.

Stages:

• Common myeloid progenitor cell
  Arises within the red bone marrow*
• Colony-forming unit – erythroid cells (CFU-E)
  • EPO (hormone released from the kidneys) induces entry into the erythroid series.
• Proerythroblast
  Has a large nucleus, so that only a basophilic rim of agranular cytoplasm can be seen.
  Golgi apparatus appears as a white “ghost” in the histological sample; though not shown here, the proerythroblast has an abundance of RNA and ribosome content.
  The nuclear chromatin is fine and granular, with 1 – 2 nucleoli.
• Basophilic erythroblast
  Smaller (~8-16 µm)
  Cytoplasm appears even darker, more basophilic, because the polyribosomes are active in hemoglobin (Hb) synthesis.
  The nuclear chromatin begins to form clumps, which, against the lighter-staining surrounding matter, can create a so-called “checkerboard” pattern.
• Polychromatophilic erythroblast
  Bluish-gray hue – the presence of hemoglobin increases and the cytoplasm becomes more acidophilic (“polychromatophilic” = cell contains both basophilic and acidophilic matter).
  Nucleus decreases in size, and the chromatin condenses; “checkerboard” pattern becomes more visible.
• Orthocromatic erythroblast
  More acidophilic (hence, it is also referred to as an acidophilic erythrobast), due to the high concentration of hemoglobin
  Pyknotic nucleus: as the chromatin degenerates, the nucleus shrivels to form a dense basophilic mass
  • In transition to the next stage, the nucleus is extruded from the cell.
• Reticulocyte
  Light pink/blue staining
  Has no nucleus
  Basophilic remnants of the reticulum and other organelles can still be seen.
  • Eventually, these remnants are also lost, and the mature erythrocyte stains red; however, upon close inspection, we can see that its biconcave center stains lighter – this is referred to as the central pallor.

Additional Information:

• As the erythropoid series progresses, the cells become smaller: the proerythroblast is approximately 15-20 micrometers in diameter; the erythrocyte is only 7-8.5 micrometers.
• Be aware that a great deal of intertextual variation in nomenclature exists; in this tutorial, we’ve used the nomenclature that reflects changes in cytoplasmic staining as it goes from basophilic (dark purple/blue) to acidophilic (bright pinkish).
• Be aware that many texts use “erythroblast” and “normoblast” interchangeably.

Erythropoiesis is the process of erythrocyte development

• Erythrocyte (aka, red blood cell) formation occurs primarily in the red marrow of spongy bone, in close association with macrophages.

Key Stages

Pluripotent hematopoieticstem cell (HSC)

Pluripotent means it has the potential to form any blood cell type.
– Under stimulation from PU.1 transcription factor, gives rise to the common myeloid progenitor (CMU)

CMU

– Also called the “CFU-GEMM,” because it can give rise to any of the following lineages: Granulocytes, Erythrocytes, Monocytes, and Megakaryocytes

CFU-MegE (Colony Forming Units – Megakaryocytes and Erythrocytes)

Blast-Forming Units – Erythrocytes (BFU-E)

– BFU-E requires the following for proper development: Stem Cell Factors, Interleukins 3 and 6, and IGF-1.
BFU-E cells give rise to Colony-Forming Units – Erythrocytes (CFU-E).

CFU-E

– By default, the fate of the CFU-E is apoptosis

• EPO
  – Hormone produced by the kidneys, blocks CFU-E cell death
  – Allows initiation of the erythroid series

Erythroid series

– The differentiation and maturation of erythroblasts, aka, normoblasts; this process appears to require an intimate relationship with the macrophage, which forms the basis of the macrophage-erythroid island.
– Requires:
– Fibronectin, which is an extracellular glycoprotein synthesized by fibroblasts, and other adhesion molecules that secure the macrophage-erythroblast island.
KLF-1 (Kruppel-Like Factor) transcription factor is necessary for the production of adult hemoglobin; abnormally low levels are associated with higher levels of fetal hemoglobin (Hereditary Persistence of Fetal Hemoglobin). (incidentally, this transcription factor has been suggested to play additional essential roles in erythropoiesis).
– Erythrocyte maturation factors, including Vitamin B12 and folic acid, are necessary for healthy red blood cell development; deficiencies produce malformed cells and, consequently, anemias.

• Proerythroblast
  – The first stage in the erythroid series, comprises a relatively large basophilic cell inhabited by a large nucleus; we’ve labeled the fine, granular nuclear chromatin.
  – Basophilic erythroblast
  – Hemoglobin synthesis is indicated by condensation of the nuclear chromatin.
• Polychromatophilic erythroblast
  – Increasing presence of hemoglobin renders the cell both basophilic and acidophilic — hence, “poly” chromatophilic.
  – Polychromatophilic erythroblastis the last stage in which cell division via mitosis is possible (intertextual variation on this timing exists).
• Orthochromatic (aka, acidophilic) erythroblast
  – The abundant hemoglobin renders the cytoplasm acidophilic.
  – As the chromatin degenerates, it leaves a dense, basophilic mass in the pyknotic nucleus (pyknotic = nucleus undergoing necrosis).
• Reticulocyte
  – Nucleus is extruded from the orthochromatic erythroblast as it transitions to a reticulocyte; is engulfed by the macrophage and phagocytosed.
  – Other organelles, including the mitochondria, suffer the same fate; recognize that this has functional consequences for the mature red blood cell – without mitochondria, they rely on anaerobic respiration – and, thus, consume none of the oxygen as they transport it to body tissues.
  – Some organelle remnants, particularly of the endoplasmic reticulum, may still be visible in the reticulocyte; However, these, too, will be extruded and phagocytosed by the macrophage.
  – Reticulocyte exits the red marrow via the medullar venous sinus, travels in the bloodstream to the spleen for the final stages of maturation.
• Erythrocyte
  – Emerges from the spleen to perform its gas transport duties in the circulatory system.
  – The lifespan of the average erythrocyte is 120 days; After this period, defects in the cell membrane render it susceptible to rupture or phagocytosis by splenic macrophages.

HEMATOPOIETIC STEM CELL:

• Resides in adult red bone marrow. Directed by chemical signals, this stem cell will give rise to all of the blood cells types.
  • When PU.1 levels surpass a given threshold, the hematopoietic stem cell gives rise to the common myeloid progenitor (CMP); this progenitor gives rise to 6 of the 7 cell types.
  • When levels of PU.1 transcription factor below that threshold, the hematopoietic stem cell gives rise to the common lymphoid progenitor (CPL), which ultimately produces lymphocytes.

CMP:

Megakaryocytes:

• In the presence of interleukin 11, the descendants of the CMP gives rise to megakaryoblasts, which are large basophilic cells with a bean-shaped nucleus;
• Under the influence of thrombopoietin, the megakaryoblast matures to form the megakaryocyte (be aware that thrombopoietin is also referred to as THPO and megakaryocyte growth and development factor (MGDF), and c-Mpl ligand).
• Megakaryoctes are large cells with multilobed, irregular nuclei;
  • The plasma membrane of mature cells invaginates to form demarcation membranes that fragment the cytoplasm.
  • The fragments are shed into the blood as platelets, aka, thrombocytes, which perform key hemostatic functions.

Erythrocyte:

• Erythropoietin (EPO) initiates the erythroid series, which begins with formation of the proerythroblast and ends with the mature erythrocyte (aka, red blood cell).

Granulocytes:

• Granulocyte-macrophage colony stimulating factor (GM-CSF) triggers formation of the myeloblast, which will give rise to 4 of the 5 white blood cell lines:

Eosinophils

• Interleukin 5 triggers production of eosinophils
• These white blood cells are characterized by multi-lobed nuclei and bright orange/red staining granules.

Basophils

• Interleukin 3 triggers production of basophils, which, as their name implies, are highly basophilic.
  (be aware that interleukin 3 has widespread influence over the other cell lines, too.)

Neutrophils

• Presence of granulocyte – colony stimulating factor (G-CSF), myeloblasts become neutrophils.
  • These white blood cells comprise lobulated nuclei and a pale pink cytoplasm.

Agranulocytes:

Monocyte

• In the presence of monoblast – colony stimulating factor (M-CSF), the monoblast gives rise to the monocyte
• This white blood cell has a bean-shaped nucleus, with visible chromatin, and blue-gray cytoplasm.
• Outside of the red marrow, the monocyte differentiates:
  • In the connective tissues, it becomes a macrophage, which can phagocytose dozens of particles, even damaged red blood cells.
  • In the bone, the macrophage becomes an osteoclast, which resorbs bone tissue during growth and remodeling.

CLP

Lymphoblast

• Interleukin 7 facilitates the formation of the lymphoblast, which is a large, spherical cell with a large basophilic nucleus.
• The end product of this developmental line is the lymphocyte.
  • T cells mature in the thymus and participate in cell-mediated immunity.
  • B cells mature in the bone; upon antigen activation, the B cells become plasma cells and participate in the humoral immune response.

Be aware that there is tremendous intertextual variation regarding the specifics of the hematopoietic pathways. Thus, we will omit details of the contentious relationships between cell lines, and focus only on those chemical signals that are most widely agreed upon.

ABO Blood Groups

Comprises A and B antigens, which are oligosaccharide molecules produced on the surfaces of red blood cells (aka, erythrocytes).

• These antigens are genetically determined by the alleles A, B, and O.
• A and B are codominant, and O is recessive; so, from 6 possible genotypes, we get 4 phenotypic blood types.

Antibodies

A unique feature of the ABO blood group is that individuals produce antibodies against antigens absent in their blood. These antibodies attack the red blood cells displaying the corresponding antigens, causing agglutination and hemolysis.

Type A blood

• Blood type A is characterized by red blood cells with the A antigen on their surfaces
• Anti-B antibodies, aka, agglutinins, circulate in the plasma
• Addition of Type B blood/B antigens will cause agglutination.

Type B blood

• Blood type B is characterized by B antigens on the surfaces of red blood cells
• Anti-A antibodies circulate in the plasma
• Addition of Type A blood/A antigens will cause agglutination.

Type AB blood

• Type AB blood cells have both A and B antigens on their surfaces
• Neither anti-A nor anti-B antibodies circulate in the plasma, which make sense: Anti -A or Anti-B antibodies would attack a person’s own red blood cells.
• Addition of A or B antigens does not cause agglutination.

Type O blood

• Type O has neither A nor B antigens on its red blood cells
• Both anti-A and anti-B antibodies circulate in the plasma.
• Addition of A or B antigens causes agglutination.

Rh Blood Group

• There are several Rh antigens, but the D antigen is most prevalent and most cross-reactive; thus, it is most clinically relevant.
• D antigen is either present on the surface of red blood cells or not
• It is coded for by two alleles: D and d.
• Unlike the ABO blood types, antibodies against the D antigen are not pre-produced in Rh negative individuals.
• Rh negative individuals produce anti-Rh antibodies in response to exposure to D antigens.
• Thus, if Rh+ blood is added to Rh- blood that happens to have anti-Rh+ antibodies, agglutination will occur.

Clinical Correlations:

• Blood transfusion recipients and donors must be matched to avoid agglutination.
• When an Rh negative woman gives birth to an Rh positive infant; invariably, there will be some mixture of maternal and fetal blood. Consequently, the mother’s body will produce anti-Rh antibodies, which will have negligible, if any, immediate effects. But, the circulating anti-Rh antibodies will attack the red blood cells of any subsequent Rh positive fetus. Preventative assessment of maternal Rh status and immunization protects against this reaction.